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Cloning and Characterization of p70S6KβDefines a Novel Family of p70 S6 Kinases
Abstract
The human cDNA encoding a novel protein serine/threonine kinase most closely related to p70 S6 kinase was isolated from the human erythroleukemia cDNA library and termed p70(S6Kbeta). p70(S6Kbeta) has 67% amino acid identity in overall sequence with human p70(S6K), and the potential phosphorylation sites of p70(S6K) are conserved in p70(S6Kbeta). Northern blot analysis identified two major transcripts of p70(S6Kbeta) that are ubiquitously expressed in human adult tissues. Similar to p70(S6K), p70(S6Kbeta) was activated by serum stimulation, and the serum-induced activation was inhibited by wortmannin and rapamycin. These findings suggest that p70(S6Kbeta) is an isoform of p70(S6K) with similar regulatory mechanisms.
Supplementary resource (1)
Nucleotide Sequence
October 1998
... However, S6 phosphorylation in such animals was still sensitive to the bacterial macrolide rapamycin (51), which inhibits the mammalian target of rapamycin (mTOR) (1,7,16,48), the upstream S6K1 kinase (4,8,18), suggesting the existence of a second S6K. Subsequent searches of expressed sequence tag databases and biochemical studies led to the identification of S6K2, which exhibited overall homology of over 80% with S6K1 in the highly conserved kinase and linker domains (17,47,51). In all tissues examined from S6K1-deficient mice, S6K2 transcripts were upregulated (51). ...
... Given the large relative abundance of S6 in the cytoplasm compared to the nucleus, the quantitation of S6 phosphorylation by 2D PAGE would largely reflect the status of S6 phosphorylation in the cytoplasm. However, as the p85 S6K isoform of S6K1 is targeted to the nucleus (46) and S6K2 also contains a nuclear targeting sequence (17,47,51), it is possible that one of the two kinases is responsible for mediating selective phosphorylation of S6 in the nucleolus. Consistent with this idea, previous studies demonstrated that S6K1 and S6K2 are differentially targeted within the cell (26,46,57). ...
... 24,2004 S6K1 Ϫ/Ϫ /S6K2 Ϫ/Ϫ MICE 3121 lated S6 (24,53). However, in tissues derived from S6K1 Ϫ/Ϫ mice, S6 phosphorylation was shown to be intact but still rapamycin sensitive (51), leading to the discovery of S6K2 (17,47,51). Although studies with ES cells have been difficult to rationalize, some investigators recently demonstrated that growth factor stimulation of these same cells leads to S6K2 activation and increased S6 phosphorylation in a rapamycinsensitive manner (31,60). ...
... These data suggest that there may exist additional kinase(s) that can substitute for S6K1. This has led to identification and cloning of ribosomal S6 kinase 2 (S6K2) [11][12][13][14][15]. ...
... S6K2 was cloned based on its nucleotide sequence homology to S6K1, and its cellular function and in vivo substrate(s) are not yet established. Initial studies on S6K2 showed that it can phosphorylate S6 in vitro, and that upstream kinases that have been shown to regulate the catalytic activity of S6K1, such as phosphoinositide 3-kinase (PI3K), phosphoinositide-dependent protein kinase (PDK1), mTOR, Cdc42, Rac and protein kinase C ζ , may also play a role in S6K2 regulation [11][12][13][14][15][16]. The current hypothesis is that S6K1 and S6K2 may have common as well as distinct functions in cells. ...
... The nuclear localization signal for S6K1 resides in this 23 amino acid N-terminal portion, and therefore the αII isoform is found mainly in the cytoplasm whereas the longer αI isoform resides in the nucleus [19,20]. S6K2 also has two isoforms with alternative translational start sites (βI and βII), but the nuclear localization signal for S6K2 is in the C-terminal portion of the protein and therefore both isoforms are found in the nucleus [12][13][14][15]. A recent study suggests that mTOR shuttling to and from the nucleus is important for S6K2 activation [21]. ...
Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.
... However, S6 phosphorylation in such animals was still sensitive to the bacterial macrolide rapamycin (51), which inhibits the mammalian target of rapamycin (mTOR) (1,7,16,48), the upstream S6K1 kinase (4,8,18), suggesting the existence of a second S6K. Subsequent searches of expressed sequence tag databases and biochemical studies led to the identification of S6K2, which exhibited overall homology of over 80% with S6K1 in the highly conserved kinase and linker domains (17,47,51). In all tissues examined from S6K1-deficient mice, S6K2 transcripts were upregulated (51). ...
... Given the large relative abundance of S6 in the cytoplasm compared to the nucleus, the quantitation of S6 phosphorylation by 2D PAGE would largely reflect the status of S6 phosphorylation in the cytoplasm. However, as the p85 S6K isoform of S6K1 is targeted to the nucleus (46) and S6K2 also contains a nuclear targeting sequence (17,47,51), it is possible that one of the two kinases is responsible for mediating selective phosphorylation of S6 in the nucleolus. Consistent with this idea, previous studies demonstrated that S6K1 and S6K2 are differentially targeted within the cell (26,46,57). ...
... (24,53). However, in tissues derived from S6K1 Ϫ/Ϫ mice, S6 phosphorylation was shown to be intact but still rapamycin sensitive (51), leading to the discovery of S6K2 (17,47,51). Although studies with ES cells have been difficult to rationalize, some investigators recently demonstrated that growth factor stimulation of these same cells leads to S6K2 activation and increased S6 phosphorylation in a rapamycinsensitive manner (31,60). ...
Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1(-/-) mice are significantly smaller, whereas S6K2(-/-) mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1(-/-)/S6K2(-/-) mice, cell cycle progression and the translation of 5'-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1(-/-)/S6K2(-/-) cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1(-/-), and S6K2(-/-) cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.
... In mammals, two separate genes encode S6Ks: 2 S6K1 (or S6K␣) and S6K2 (or S6K). The use of alternative ATG start codons makes two protein isoforms for each S6 kinase (for S6K1, p85 and p70; for S6K2, p56 and p54) (3)(4)(5). Among these S6Ks, the p70 S6K1 (S6K␣2), termed S6K1 in this study, is the best characterized isoform for its function and regulation. ...
... Throughout this study, the following designations were used for mutants: KR, acetylation-defective mutation by replacing lysine with arginine; KQ, acetylation-mimetic mutation by replacing lysine with glutamine; KA, acetylation-mimetic mutation by replacing lysine with alanine; KT, acetylation-mimetic mutation by replacing lysine with threonine.4 Throughout this study, the following designations were used for multiple mutants: TKR, K484R/K485R/K493R; TKQ, K484Q/K485Q/K493Q; TKA, K484A/K485A/K493A; TKT, K484T/K485T/K493T. ...
p70 ribosomal S6 kinase (S6K1), a major substrate of the mammalian target of rapamycin (mTOR) kinase, regulates diverse cellular
processes including protein synthesis, cell growth, and survival. Although it is well known that the activity of S6K1 is tightly
coupled to its phosphorylation status, the regulation of S6K1 activity by other post-translational modifications such as acetylation
has not been well understood. Here we show that the acetylation of the C-terminal region (CTR) of S6K1 blocks mTORC1-dependent
Thr-389 phosphorylation, an essential phosphorylation site for S6K1 activity. The acetylation of the CTR of S6K1 is inhibited
by the class III histone deacetylases, SIRT1 and SIRT2. An S6K1 mutant lacking acetylation sites in its CTR shows enhanced
Thr-389 phosphorylation and kinase activity, whereas the acetylation-mimetic S6K1 mutant exhibits decreased Thr-389 phosphorylation
and kinase activity. Interestingly, relative to the acetylation-mimetic S6K1 mutant, the acetylation-defective mutant displays
higher affinity toward Raptor, an essential scaffolding component of mTORC1 that recruits mTORC1 substrates. These observations
indicate that sirtuin-mediated regulation of S6K1 acetylation is an additional important regulatory modification that impinges
on the mechanisms underlying mTORC1-dependent S6K1 activation.
... The predominantly expressed in cells short (70 kDa) isoform of S6K1 is localized mainly but not exclusively within the cytoplasm. A nuclear localization sequence (NLS) found within the N-terminal (23 aa) extension of long S6K1 isoform (85 kDa) may target it to the nucleus [3,4]. Similarly, the long (56 kDa) isoform of S6K2 differs from the short (54 kDa) splicing version by the presence of the NLS [1,4] located within the N-terminal (13 aa) extension. ...
... A nuclear localization sequence (NLS) found within the N-terminal (23 aa) extension of long S6K1 isoform (85 kDa) may target it to the nucleus [3,4]. Similarly, the long (56 kDa) isoform of S6K2 differs from the short (54 kDa) splicing version by the presence of the NLS [1,4] located within the N-terminal (13 aa) extension. At the same time, both S6K2 isoforms contain additional NLS at the C-terminus, which can determine their nuclear localization [5,6]. ...
Our previous studies have shown that S6K1 and S6K2 protein kinases form the complexes with newly identified adaptor protein TDRD7, which is involved in regulation of cytoskeleton dynamics, mRNA transport, protein translation, piRNAs processing and transposons silensing. Aim Determination the subcellular localization of S6K1-TDRD7 and S6K2-TDRD7 protein complexes. Methods. Immunofluorescense microscopy was used to study co-localization of S6K1/S6K2 and TDRD7 in HEK293, HEPG2 cell lines as well as in rat primary hippocampal neurons using primary polyclonal anti-S6K1 antibodies, monoclonal anti-S6K2 and anti- TDRD7 antibodies. Results. It was found that S6K1 is co-localized with TDRD7 in perinuclear region of HEK293 cells. S6K1 and S6K2 were also co-localized with TDRD7 in perinuclear region of HEPG2 cells and in soma of primary rat hippocampal neurons. Conclusions. In this report we provide an additional experimental evidences of possible S6K1-TDRD7 and S6K2-TDRD7 complexes formation in cells of different tissue origins that may reflect their potential physiological importance. However, elucidation of the exact composition of these complexes and their role in cell physiology requires additional studies.
... In mammals, S6K represents a family composed of two distinct genes, S6K1 (RPS6KB1; also known as S6Kα) and S6K2 (RPS6KB2; also known as S6Kβ) [12][13][14][15][16][17][18]. Mice singly null for S6K1 or S6K2 are born at normal Mendelian ratios, whereas mice null for both S6K1 and S6K2 display perinatal lethality [14,19]. ...
... S6K1 and S6K2 belong to the AGC kinase family, named for its three founding members PKA, PKG and PKC [43,44]. Although S6K1 was cloned in 1990 [12,13], S6K2 was not cloned until almost a decade later [14][15][16][17][18]. AGC kinases share several structural features that confer similar modes of regulation. ...
The ribosomal protein S6K (S6 kinase) represents an extensively studied effector of the TORC1 [TOR (target of rapamycin) complex 1], which possesses important yet incompletely defined roles in cellular and organismal physiology. TORC1 functions as an environmental sensor by integrating signals derived from diverse environmental cues to promote anabolic and inhibit catabolic cellular functions. mTORC1 (mammalian TORC1) phosphorylates and activates S6K1 and S6K2, whose first identified substrate was rpS6 (ribosomal protein S6), a component of the 40S ribosome. Studies over the past decade have uncovered a number of additional S6K1 substrates, revealing multiple levels at which the mTORC1-S6K1 axis regulates cell physiology. The results thus far indicate that the mTORC1-S6K1 axis controls fundamental cellular processes, including transcription, translation, protein and lipid synthesis, cell growth/size and cell metabolism. In the present review we summarize the regulation of S6Ks, their cellular substrates and functions, and their integration within rapidly expanding mTOR (mammalian TOR) signalling networks. Although our understanding of the role of mTORC1-S6K1 signalling in physiology remains in its infancy, evidence indicates that this signalling axis controls, at least in part, glucose homoeostasis, insulin sensitivity, adipocyte metabolism, body mass and energy balance, tissue and organ size, learning, memory and aging. As dysregulation of this signalling axis contributes to diverse disease states, improved understanding of S6K regulation and function within mTOR signalling networks may enable the development of novel therapeutics.
... The cloning of a close paralogue of S6K1 by several groups (Gout et al., 1998;Koh et al., 1999;Lee-Fruman et al., 1999;Saitoh et al., 1998;Shima et al., 1998) added further to the S6K family. This gene, RPS6KB2, often referred to as S6Kˇor S6K2 also utilizes alternative translational start sites to produce two isoforms. ...
... The catalytic domains of S6K1 and S6K2 share 83% identity at the amino acid level (Gout et al., 1998). The kinase extension and pseudosubstrate domains immediately C-terminal to the kinase domain are also very similar, and contain several conserved serine and threonine residues critical to the activation of S6K1 and S6K2 Gout et al., 1998;Saitoh et al., 1998). Interesting differences lie in the extreme N-and C-terminal regions, also thought to be important in the regulation of these kinases. ...
The 70kDa ribosomal protein S6 kinases, S6K1 and S6K2 are two highly homologous serine/threonine kinases that are activated in response to growth factors, cytokines and nutrients. The S6 kinases have been linked to diverse cellular processes, including protein synthesis, mRNA processing, glucose homeostasis, cell growth and survival. Studies in model organisms have highlighted the roles that S6K activity plays in a number of pathologies, including obesity, diabetes, ageing and cancer. The importance of S6K function in human diseases has led to the development of S6K-specific inhibitors by a number of companies, offering the promise of improved tools with which to study these enzymes and potentially the effective targeting of deregulated S6K signalling in patients. Here we review the current literature on the role of S6Ks in the regulation of cell growth, survival and proliferation downstream of various signalling pathways and how their dysregulation contributes to the pathogenesis of human diseases.
... The p70b1 and p70b2 isoforms consist of 495 and 482 residues, respectively; the sequence of p70b2 is identical to the part (14±495) of p70b1. Other groups have also cloned the p70b2 isoform but not the p70b1 isoform (Koh et al. 1999;Lee Fruman et al. 1999;Saitoh et al. 1998;Shima et al. 1998). The overall sequence of p70b1 is very close to that of p70a1, with 70% identity and 85% similarity at the protein level. ...
... One such different role may be related to the different cellular distribution of the two isoforms as described below. In addition, this may account for the reason why other groups cloned only the p70b2 isoform (Koh et al. 1999;Lee Fruman et al. 1999;Saitoh et al. 1998;Shima et al. 1998). ...
A novel ribosomal S6 kinase, termed p70 S6 kinase beta (p70beta), has a highly homologous amino acid sequence to that of p70/p85 S6 kinase (p70alpha). This includes the critical phosphorylation sites, Thr252, Ser394 and Thr412 in p70alpha1, which correspond to Thr241, Ser383 and Thr401 in p70beta1, respectively. However, the regulatory mechanism for p70beta remains to be elucidated.
We report here the expression and the mechanism of in vivo regulation of p70beta. Two isoforms, p70beta1 and p70beta2, were expressed in a variety of tissues at a different level. p70beta1 was mainly targeted to the nucleus, whereas p70beta2 dispersed throughout the cytoplasm including nucleoplasm. The kinase activity of p70beta1 was less sensitive to the inhibition induced by rapamycin, wortmannin and amino acid withdrawal than that of p70alpha. The portion of p70beta activity inhibited by rapamycin was rescued by the rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR). Mutational analysis revealed that the phosphorylation of Thr241 and Thr401 in p70beta1 was indispensable for the kinase activity. In contrast, a p70beta1 mutant in which Ser383 was substituted with Gly (S383G) still retained nearly the half maximal activity. Sequential phosphorylation of wild-type and S383G mutant of p70beta1 with mTOR and 3-phosphoinositide-dependent protein kinase 1 (PDK1) in vitro synergistically activated their kinase activities.
These results indicate that p70beta is regulated by the mTOR- and PDK1-signalling pathways through a synergistic interaction between phosphorylated Thr241 and Thr401, while Ser383 plays minor role in their activation mechanism. Activated p70beta may be less sensitive to dephosphorylation mediated by putative phosphatases activated by rapamycin, amino acid withdrawal, and probably wortmannin.
... To better understand the downstream events of the mTOR pathway in this system, we evaluated the role of S6K2, one of the downstream targets of mTOR, in IL-3-mediated cell function. S6K2 was first identified via its sequence homology to S6K1 [8][9][10][11][12]. S6K2 is regulated by proteins in the PI-3K, MAPK kinase (MEK), and mTOR pathways in fibroblasts [8][9][10][11][12][13][14][15][16][17][18], and S6K2 has been shown to phosphorylate S6 in vivo [19,20]. ...
... S6K2 was first identified via its sequence homology to S6K1 [8][9][10][11][12]. S6K2 is regulated by proteins in the PI-3K, MAPK kinase (MEK), and mTOR pathways in fibroblasts [8][9][10][11][12][13][14][15][16][17][18], and S6K2 has been shown to phosphorylate S6 in vivo [19,20]. However, the cellular function of S6K2 is largely unknown at this time. ...
Interleukin-3 (IL-3) mediates hematopoietic cell survival and proliferation via several signaling pathways such as the Janus kinase/signal transducer and activator of transcription pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphoinositide-3 kinase (PI-3K) pathway. Mammalian target of rapamycin (mTOR) is one of the downstream targets of the PI-3K pathway, and it plays an important role in hematopoiesis and immune cell function. To better elucidate how mTOR mediates proliferation signals from IL-3, we assessed the role of S6 kinase 2 (S6K2), one of the downstream targets of mTOR, in IL-3 signaling. We show that S6K2 is activated by IL-3 in the IL-3-dependent Ba/F3 cell line and that this is mediated by mTOR and its upstream activator PI-3K but not by the MAPK kinase/extracellular signal-regulated kinase pathway. S6K2 is also activated in primary mouse bone marrow-derived mast cells upon IL-3 stimulation. Expression of a rapamycin-resistant form of S6K2, T388E, in Ba/F3 cells provides a proliferation advantage in the absence or presence of rapamycin, indicating that S6K2 can potentiate IL-3-mediated mitogenic signals. In cells expressing T388E, rapamycin still reduces proliferation at all doses of rapamycin, showing that mTOR targets other than S6K2 play an important role in IL-3-dependent proliferation. Cell-cycle analysis shows that T388E-expressing Ba/F3 cells enter S phase earlier than the control cells, indicating that the proliferation advantage may be mediated by a shortened G1 phase. This is the first indication that S6K2 plays a role in IL-3-dependent cell proliferation.
... To verify the exploitation of mTORC1 signaling by WSSV for its infection, the expression of another downstream target in the signaling, ribosomal protein S6 kinases (S6K1 and S6K2 in S6A Fig), which are responsible for protein synthesis [30][31], were analyzed. We firstly detected the tissue distribution of S6k1 and S6k2, and found that S6k1 was distributed in all tested tissues, but S6k2 was hardly detected in hemocytes and stomach ( Fig 3A). ...
Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus , which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture.
... RPS6 is the first identified substrate of S6K1 (p70S6Kα) [60], and phosphorylation of RPS6 has been used as a readout of mTORC1 activity [2,[18][19][20]. However, a close homolog of S6K1, S6K2 (p70S6Kβ), is the major kinase of RPS6 [61][62][63][64][65]. The two S6K homologs S6K1 (S6Kα) and S6K2 (S6Kβ) in mammal share extensive but dispersed homology (43%, 84%, and 59% in N-terminus, catalytic domain, and C-terminus, respectively) [63]. ...
Ribosomal protein S6 (RPS6) is a component of the 40S small ribosomal subunit and participates in the control of mRNA translation. Additionally, phospho (p)-RPS6 has been recognized as a surrogate marker for the activated PI3K/AKT/mTORC1 pathway, which occurs in many cancer types. However, downstream mechanisms regulated by RPS6 or p-RPS remains elusive, and the therapeutic implication of RPS6 is underappreciated despite an approximately half a century history of research on this protein. In addition, substantial evidence from RPS6 knockdown experiments suggests the potential role of RPS6 in maintaining cancer cell proliferation. This motivates us to investigate the current knowledge of RPS6 functions in cancer. In this review article, we reviewed the current information about the transcriptional regulation, upstream regulators, and extra-ribosomal roles of RPS6, with a focus on its involvement in cancer. We also discussed the therapeutic potential of RPS6 in cancer.
... However cl.#23 has an internal G/T point mutation which introduces a stop codon at 1093 bp from the start codon (Gout at al., 1998). Other groups have also reported the isolation of S6KpII clones from cDNA libraries of different cell lines (Lee-Fruman et al., 1999;Saitoh, et al., 1998;Shima et al., 1998). cDNA clones described above were used in this study to construct and subclone into expression vectors recombinant S6KpI and S6KpII ...
The ribosomal protein S6 kinase belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of translation and Gl/S transition of the cell cycle. In addition to playing an essential role in regulating cell growth, S6K appears to be a multifunctional protein, involved in the control of other cellular processes such as transcription, RNA processing, and apoptosis. Two human S6K forms have been identified, termed S6Kα and S6Kβ which have cytoplasmic and nuclear splicing variants. Both kinases have very high level of homology in kinase domain, but differ significantly in the N- and C-terminal regulatory regions. These differences and some variations in the pattern of phosphorylation sites may predestine the involvement of both kinases in distinct signaling events and cellular responses. This manuscript presents analysis of regulatory and functional properties of ribosomal S6Kα and its recently identified homologue S6Kβ. Recombinant full-length versions and a panel of deleted and point mutants of S6Kα and S6Kβ were created and used to study the mechanisms of mitogen-induced activation of these kinases. Furthermore, employment of specific inhibitors of mTOR/FRAP, PI-3'-K, PKC and MEK allowed us to study the contribution of these signaling molecules in the activation of S6Kα and S6Kβ by various mitogenic stimuli. Here we also present a detailed analysis of subcellular localisation of S6Kα and S6Kβ isoforms. Using confocal microscopy and a panel of mutants, we have described for the first time mitogen-regulated nucleocytoplasmic shuttling of S6KβII and addressed a critical role of PKC signaling in this process. A novel PKC phosphorylation site, specific for S6Kβ, was identified by mass spectrometry and found to be involved in the regulation of its subcellular distribution. Finally, we have developed tetracycline-inducible stable cell lines, overexpressing activated versions of four different isoforms of S6K. This model system allowed us to initiate studies on the role of individual S6K isoforms in the regulation of gene expression. In conclusion, this study demonstrates the existence of distinct signaling mechanisms, involved in the regulation of S6Kα and S6Kβ.
... It was originally identified as the serine/threonine kinase that mediated the mitogen-inducible phosphorylation of ribosomal protein S6 (rpS6). However, the observation that rpS6 phosphorylation was not affected in S6K1 knockout mice led to the identification of a close homolog of S6K1, S6K2 (p70S6Kβ), which was later shown to be the major kinase mediating rpS6 phosphorylation [15][16][17][18][19]. S6K1 and S6K2 are encoded by RPS6KB1 on chromosome 17 and RPS6KB2 on chromosome 11, respectively (Table 1). ...
The mechanistic target of rapamycin (mTOR) is a master regulator of protein translation, metabolism, cell growth and proliferation. It forms two complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). mTORC1 is frequently deregulated in many cancers, including breast cancer, and is an important target for cancer therapy. The immunosuppressant drug rapamycin and its analogs that inhibit mTOR are currently being evaluated for their potential as anti-cancer agents, albeit with limited efficacy. mTORC1 mediates its function via its downstream targets 40S ribosomal S6 kinases (S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). There are two homologs of S6K: S6K1 and S6K2. Most of the earlier studies focused on S6K1 rather than S6K2. Because of their high degree of structural homology, it was generally believed that they behave similarly. Recent studies suggest that while they may share some functions, they may also exhibit distinct or even opposite functions. Both homologs have been implicated in breast cancer, although how they contribute to breast cancer may differ. The purpose of this review article is to compare and contrast the expression, structure, regulation and function of these two S6K homologs in breast cancer.
... Two distinct genes encode different S6Ks, S6K1 (RPS6KB1, also known as S6Kα) and S6K2 (RPS6KB2, also known as S6Kβ), in mammals (Banerjee et al. 1990;Saitoh et al. 1998). Human S6K1 and S6K2 have conserved kinase domains with 84% identity but have less conserved N-and C-terminal regions with 43% and 59% identity, respectively (Lee-Fruman et al. 1999). ...
Ribosomal protein S6 kinases (S6Ks) are key targets downstream of the mechanistic target of rapamycin (mTOR) signaling pathway that are activated by mTOR complex 1 phosphorylation to promote protein synthesis, and are involved in muscle growth and pathology. In this study, we cloned a 1560-base pair (bp) complementary DNA sequence encoding a unique S6K1, designated EsS6K1, in the Chinese mitten crab Eriocheir sinensis. EsS6K1 contains a 54-bp 5′ untranslated region, a 246-bp 3′ untranslated region and a 1260-bp coding sequence that encodes 419 amino acids. EsS6K1 contains a conserved kinase domain, a TOR signaling motif, and a turn motif; it lacks a C-terminal autoinhibitory domain, and the conventional threonine in the hydrophobic motif is replaced with serine. Real-time quantitative reverse transcription–polymerase chain reaction experiments showed that EsS6K1 expression was highest in the ovaries, followed by the testes and the eyestalks, in mature crabs. In juvenile crabs, the relative messenger RNA levels of EsS6K1 in the claw, walking leg and abdominal muscles were significantly higher in the late premolt (D3-4) stage than in the intermolt (C) stage. After ecdysis, the expression of EsS6K1 in the walking leg and abdominal muscles declined to the level in the C stage, but the expression in claw muscles remained as high as that in the D3-4 stage. The findings showed that EsS6K1 likely plays roles in muscle changes during crustacean molting. These results provide valuable insights into the mechanism underlying crustacean muscle growth induced by molting.
... Cloning of 70 kDa ribosomal S6 kinase was soon accomplished after protein sequencing of a rat and rabbit cDNA [6][7][8]. Subsequent identification and cloning of human orthologs of S6K genes RPS6KB1 and RPS6KB2 encoding S6K1 and S6K2 thus, respectively, led to the emergence of a second family of S6 kinases, a principal kinase of rps6 in somatic cells [2,[9][10][11][12][13][14]. ...
... Cloning of 70 kDa ribosomal S6 kinase was soon accomplished after protein sequencing of a rat and rabbit cDNA [6][7][8]. Subsequent identification and cloning of human orthologs of S6K genes RPS6KB1 and RPS6KB2 encoding S6K1 and S6K2 thus, respectively, led to the emergence of a second family of S6 kinases, a principal kinase of rps6 in somatic cells [2,[9][10][11][12][13][14]. ...
... RPS6KB2 and RPS6KB1 diverge in the C and N terminus; a nuclearlocalizing sequence (NLS) is found in the C terminus of the former protein and in RPS6KB1 a suppressor zona occludens-1 protein (PDZ)-binding domain occurs in the S6K1 C terminus (Burnet et al. 1998). Many studies of RPS6KB2 exist in mammals including humans and rats (Gout et al. 1998;Saitoh et al. 1998;Shima et al. 1998;Lee-Fruman et al. 1999;Koh et al. 1999;Martin et al. 2001;Pende et al. 2004;Cruz et al. 2005;Karlsson et al. 2013;Yoshida et al. 2013;Chauvin et al. 2014;Karlsson et al. 2015;Pai et al. 2016) but are scarce in teleosts, particularly during flatfish metamorphosis. In the present study, we screened a novel metamorphosis-related gene, rps6kb2, obtained the full-length complementary DNA (cDNA) of rps6kb2, and illustrated its expression pattern in flatfish metamorphosis. ...
Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. This unique process involves eye migration, a 90° rotation in posture, and asymmetrical pigmentation for adaptation to a benthic lifestyle. In the present study, we used genetics to map a metamorphosis-related locus (q-10M) in the male linkage group (LG10M), a small interval of 0.9 cM corresponding to a 1.8 M-bp physical area in chromosome 9 in the Chinese tongue sole (Cynoglossus semilaevis). Combined with single-marker analysis, ribosomal protein S6 kinase 2 (rps6kb2) a member of the family of AGC kinases was identified as a novel metamorphosis-related candidate gene. Its expression pattern during metamorphosis was determined by quantitative RT-PCR and whole-mount in situ hybridization analysis. rps6kb2 gene was significantly expressed in metamorphic climax stage larvae and distributed in all the tissues transforming during metamorphosis, including tail, jaw, eye and skin of larvae. The results suggest that rps6kb2 has a general role in tissue transformations during flatfish metamorphosis including tail changes, skull remodeling, eye migration, and asymmetrical pigmentation.
... The catalytic and regulatory domains as well as the regulatory phosphorylation sites are conserved between S6K1 and S6K2 (Shima et al., 1998). However, S6K2 contains a proline rich region ( Fig. 8) in the carboxyl-terminal tail and is less sensitive to inhibition by rapamycin compared to S6K1 (Gout et al., 1998;Saitoh et al., 1998). The presence and upregulation of S6K2 may obscure and compensate some functions of S6K1 and also the characterization of the S6K1 and S6K2 double knockout mouse is anticipated. ...
The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.
... The p90 ribosomal S6 kinases (RSKs), comprising RSK1-4 (1), were first identified followed by the p70 ribosomal S6 kinase, S6K1 (2,3). It took an additional 10 years for the p70 ribosomal S6 kinase homolog, S6K2, to be discovered (4)(5)(6). The high degree of homology between S6K1 and S6K2 has for many years led researchers to assume that these were redundant kinases with essentially overlapping functions. ...
S6 kinase 2 (S6K2) is a member of the AGC kinases super-family. Its closest homolog, S6K1, has been extensively studied along the years. However, due to the belief in the community that the high degree of identity between these two isoforms would translate in essentially identical biological functions, S6K2 has been largely neglected. Nevertheless, recent research has clearly highlighted that these two proteins significantly differ in their roles in vitro as well as in vivo. These findings are significant to our understanding of S6 kinase signaling and the development of therapeutic strategies for several diseases including cancer. Here, we will focus on S6K2 and review the protein-protein interactions and specific substrates that determine the selective functions of this kinase.
... However, two lines of evidence are inconsistent with the involvement of S6K2 in this mode of regulation. (i) Reports from several laboratories have shown that rapamycin, at the concentration used here (20 nM), also completely inhibits S6K2 in 293, mouse ES, and COS cells (30,33,52,56), but has only a minor effect on translation of TOP mRNAs (58; this study). (ii) It has been shown that S6K2 is quite refractory to inhibition by amino acid withdrawal (38), yet this resistance was insufficient to prevent the repression of TOP mRNA translation upon amino acid starvation in 293 or p70 S6K1Ϫ/Ϫ cells (58). ...
... p110δ is not expressed in muscle [115] Akt/PKB 5 Akt1/PKBa, Akt2/PKBb & Akt3/PKBg. Akt3 is not expressed in muscle [4,116] mTOR 6 No isoforms, but mTOR can interact with different partners: Raptor in the rapamycin-sensitive complex mTORC1, or Rictor in the rapamycin-insensitive complex mTORC2 [5] S6K 7 S6K1 and S6K2117118119 4EBP 8 4EBP1, 4EBP2, 4EBP3. 4EBP3 is not expressed in muscle [120] FoxO 4 isoforms: FoxO1, FoxO3, FoxO4 and FoxO6. ...
A highly conserved signaling pathway involving insulin-like growth factor 1 (IGF1), and a cascade of intracellular components that mediate its effects, plays a major role in the regulation of skeletal muscle growth. A central component in this cascade is the kinase Akt, also called protein kinase B (PKB), which controls both protein synthesis, via the kinases mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3β (GSK3β), and protein degradation, via the transcription factors of the FoxO family. In this paper, we review the composition and function of this pathway in skeletal muscle fibers, focusing on evidence obtained in vivo by transgenic and knockout models and by muscle transient transfection experiments. Although this pathway is essential for muscle growth during development and regeneration, its role in adult muscle response to mechanical load is less clear. A full understanding of the operation of this pathway could help to design molecularly targeted therapeutics aimed at preventing muscle wasting, which occurs in a variety of pathologic contexts and in the course of aging.
... Also GAB2 expression is suggested to be oestrogen regulated and amplification and overexpression of GAB2 has been reported in breast cancer cell lines and specimens (Daly et al., 2002;Bentires-Alj et al., 2006;Bocanegra et al., 2010). S6K2 in the proximal amplification core, encodes an mTOR regulated serine/threonine kinase, initially identified as a homologue to p70S6 kinase (S6K1) (Gout et al., 1998;Saitoh et al., 1998). In a recent study, gene copy gain and nuclear protein expression of S6K2 were shown to predict tamoxifen response (Perez-Tenorio et al., 2010). ...
The chromosomal region 11q13 is amplified in 15-20% of breast cancers; an event not only associated with estrogen receptor (ER) expression but also implicated in resistance to endocrine therapy. Coamplifications of the 11q13 and 8p12 regions are common, suggesting synergy between the amplicons. The aim was to identify candidate oncogenes in the 11q13 region based on recurrent amplification patterns and correlations to mRNA expression levels. Furthermore, the 11q13/8p12 coamplification and its prognostic value, was evaluated at the DNA and the mRNA levels. Affymetrix 250K NspI arrays were used for whole-genome screening of DNA copy number changes in 29 breast tumors. To identify amplicon cores at 11q13 and 8p12, genomic identification of significant targets in cancer (GISTIC) was applied. The mRNA expression levels of candidate oncogenes in the amplicons [RAD9A, RPS6KB2 (S6K2), CCND1, FGF19, FGF4, FGF3, PAK1, GAB2 (11q13); EIF4EBP1 (4EBP1), PPAPDC1B, and FGFR1 (8p12)] were evaluated using real-time PCR. Resulting data revealed three main amplification cores at 11q13. ER expression was associated with the central 11q13 amplification core, encompassing CCND1, whereas 8p12 amplification/gene expression correlated to S6K2 in a proximal 11q13 core. Amplification of 8p12 and high expression of 4EBP1 or FGFR1 was associated with a poor outcome in the group. In conclusion, single nucleotide polymorphism arrays have enabled mapping of the 11q13 amplicon in breast tumors with high resolution. A proximal 11q13 core including S6K2 was identified as involved in the coamplification/coexpression with 8p12, suggesting synergy between the mTOR targets S6K2 and 4EBP1 in breast cancer development and progression.
... As shown in Fig. 5A, URI was efficiently We then transfected MAF1-HT into control MEFs or MEFs lacking S6 kinase. Mammalian cells have two S6 kinases that are more than 80% homologous in the kinase and linker domains, S6K1 and S6K2 (8,29,32). To ensure a complete lack of S6K activity, we used cells deleted for both genes (25). ...
mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified.
RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other
processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1
becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations
nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum
starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75,
and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our
results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription,
and add a new branch to the signal transduction cascade immediately downstream of TORC1.
... Phosphorylation of ribosomal protein S6 is a common effect of mitogens and has been proposed to regulate translation of TOP mRNAs (4)(5)(6). S6 phosphorylation requires activation of the S6 kinase, S6K (S6K1 or its isoform S6K2) (7,8), which is dependent upon activation of both phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) (9)(10)(11). This latter kinase has also been demonstrated to phosphorylate S6K1 in vitro at a physiologically relevant site (12), suggesting that it directly regulates S6K activity. ...
Synthesis of new ribosomes is an energy costly and thus highly regulated process. Ribosomal protein synthesis is controlled
by regulating translation of the corresponding ribosomal protein (rp)mRNAs. In mammalian cells a 5′-terminal oligopyrimidine
tract (TOP) is a conserved feature of these mRNAs that has been demonstrated to be essential for their translational regulation.
Translation of TOP mRNAs has been proposed to be regulated by phosphorylation of ribosomal protein S6, which is a common effect
of mitogenic stimulation of cells. However, as demonstrated here, S6 phosphorylation is not detectable in murine erythroleukemia
(MEL) or other hematopoietic cells. The absence of S6 phosphorylation appears to be due to the action of a phosphatase that
acts downstream of S6 kinase, presumably on S6 itself. Despite the absence of changes in S6 phosphorylation, translation of
TOP mRNAs is repressed during differentiation of MEL cells. These data demonstrate the existence of a mechanism for regulating
S6 phosphorylation that is distinct from kinase activation, as well as the existence of mechanisms for regulating translation
of TOP mRNAs that are independent of S6 phosphorylation.
... Surprisingly, the deletion of both P70S6K alleles did not affect S6 phosphorylation induced by mitogens, and this activation was still inhibited by rapamycin (14). These data led to the identification of a gene that encodes a kinase highly homologous to P70S6K (S6K1), which was named S6K2 (15,16). The high degree of protein identity and the up-regulation of S6K2 mRNA in S6K1-deficient mice suggest that the two genes perform overlapping functions and that each gene partially compensates for the lack of the other. ...
We report here that apelin (65-77) activates p70 S6 kinase (p70S6K), not only in CHO cells that have been stably transfected with the apelin receptor, but also in umbilical endothelial cells (HUVEC), which express it endogenously. Apelin (65-77) induces a time-dependent phosphorylation of p70S6K at residues T421/S424 and T389. This dual phosphorylation is associated with two transduction cascades, involving a PI3K pathway and an ERK pathway, respectively. The PI3K pathway, which can be blocked by wortmannin, leads to phosphorylation of Akt at residues T308 or S473, which then promotes the phosphorylation of p70S6K at T421/S424 and T389. The ERK pathway is blocked by PD 098059, a MEK inhibitor, and results in the phosphorylation of p70S6K at T421/S424. Phosphorylation both of Akt and p70S6K is abrogated by pretreatment with pertussis toxin (PTX) and an inhibitor of atypical PKCs. In addition, we demonstrate that apelin (65-77) also increases the enzymatic activity of p70S6K and that the effects of the previously mentioned inhibitors on the level of T389 phosphorylation correlate with their action on enzyme activity. Interestingly, the main findings were reproduced in umbilical endothelial cells and apelin (65-77) promoted thymidine incorporation into DNA of these cells, revealing that apelin is a new mitogenic peptide for the endothelial cell.
... Phosphorylation of rpS6 at multiple sites is one of the early intracellular responses to growth factor stimulation. Two families of kinases, S6K (26)(27)(28)(29) and RSK (30), are known to phosphorylate rpS6 in vitro and in vivo. Until recently, it was generally believed that S6K is the sole kinase responsible for the rpS6 phosphorylation in somatic cells in vivo as the capability of RSK to phosphorylate rpS6 in these cells has been controversial. ...
Although big mitogen-activated protein kinase 1 (BMK1) has been shown to be critical for embryonic angiogenesis, the role of BMK1 in tumor-associated neovascularization is poorly understood. Exogenous tumors were established in BMK1+/+, BMK1flox/+, or BMK1flox/flox mice carrying the Mx1-Cre transgene. Induced deletion of host BMK1 gene significantly reduced the volumes of B16F10 and LL/2 tumor xenografts in BMK1flox/flox mice by 63% and 72%, respectively. Examining the tumors in these induced BMK1-knockout animals showed a significant decrease in vascular density. Localized reexpression of BMK1 in BMK1-knockout mice by administration of adenovirus encoding BMK1 restored tumor growth and angiogenesis to the levels observed in wild-type mice. These observations were further supported by in vivo Matrigel plug assays in which vascular endothelial growth factor- and basic fibroblast growth factor-induced neovacularization was impaired by removing BMK1. Through screening with the Pepchip microarray, we discovered that in BMK1-knockout endothelial cells, phosphorylation of ribosomal protein S6 (rpS6) at Ser235/236 was mostly abrogated, and this BMK1-dependent phosphorylation required the activity of p90 ribosomal S6 kinase (RSK). Immunofluorescent analysis of tumor vasculature from BMK1-knockout and control animals revealed a strong correlation between the presence of BMK1 and the phosphorylation of rpS6 in tumor-associated endothelial cells of blood vessels. As both RSK and rpS6 are known to be important for cell proliferation and survival, which are critical endothelial cell functions during neovascularization, these findings suggest that the BMK1 pathway is crucial for tumor-associated angiogenesis through its role in the regulation of the RSK-rpS6 signaling module.
The mRNA translation machinery is tightly regulated through several, at times overlapping, mechanisms that modulate its efficiency and accuracy. Due to their fast rate of growth and metabolism, cancer cells require an excessive amount of mRNA translation and protein synthesis. However, unfavorable conditions, such as hypoxia, amino acid starvation, and oxidative stress, which are abundant in cancer, as well as many anti-cancer treatments inhibit mRNA translation. Cancer cells adapt to the various internal and environmental stresses by employing specialised transcript-specific translation to survive and gain a proliferative advantage. We will highlight the major signaling pathways and mechanisms of translation that regulate the global or mRNA-specific translation in response to the intra- or extra-cellular signals and stresses that are key components in the process of tumourigenesis.
S6 kinase, a member of the AGC family of kinases, is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth and proliferation. The activation process involves a complex sequential series of ten or more serine and threonine phosphorylations. In this thesis it was investigated if S6K activation would involve translocation to the plasma membrane and if tyrosine phosphorylation would occur as was previously shown for some other AGC kinases. It is demonstrated for the first time that S6 kinase is associated with receptor protein tyrosine kinases (RTKs) upon growth factor stimulation. The binding occurs via the kinase or kinase extension domain of S6K as shown by the use of truncation mutants in co-immunoprecipitation studies. Furthermore, both isoforms of S6 kinase were shown to be phosphorylated on tyrosine in a RTK- and Src-dependent manner. Using mass spectrometry and truncation mutants, two phosphorylation sites were mapped: one is located in the activation loop and the second one in the N-terminus of the kinase. Inhibitor studies reveal that phosphorylation occurs independently of PI3 Kinase and mTor but is inhibited by Src family kinase inhibitors. It has already been established that expression of Src kinase leads to an activated S6K. However, as shown in this study, this effect is not mediated through tyrosine phosphorylation. Subcellular localisation and S6K stability are also not altered in a Src-dependent manner. Taken together, the presented work shows for the first time an additional mechanism of S6 kinase regulation via association with receptor tyrosine kinases and tyrosine phosphorylation. The phosphotyrosine site/s possibly create/s recognition sites for SH2 domain containing proteins and therefore may be important for recruiting S6K into a multienzyme complex with other signalling molecules at the plasma membrane.
Aim: to evaluate p70S6 kinase α (p70S6K α) content in epithelial cells of thyroid gland in normal state and upon pathology. Methods: the study was carried out by quantitative immunohistochemical analysis with the use of anti-p70S6K α-MoAbs, image computer analysis and statistical analysis on autopsy samples of thyroid gland in normal state (3 cases), and on surgical samples of diffuse toxic goiter (3 cases) and papillary thyroid carcinoma (6 cases). Results: normal state of thyroid gland is characterised by low content of p70S6K α. However, upon hyperthyreosis and papillary thyroid cancer, p70S6K α content is 4-folds and 9-folds higher respectively. Conclusion: hyperfunction and carcinogenesis of thyroid gland is accompanied by increase of the content of p70S6K α in thyroid epithelial cells.
Ribosomal protein S6 kinases (S6Ks) are principal regulators of cell size, growth and metabolism. Signaling
via the PI3K/mTOR pathway mediates the activation of S6Ks in response to various mitogenic stimuli, nutrients
and stresses. To date, the regulation and cellular functions of S6Ks are not fully understood. Our aim was
to investigate and characterize the interaction of S6Ks with the novel binding partner of S6Ks, Tudor domain
containing 7 protein (TDRD 7), which is a scaffold protein detected in complexes involved in the regulation of
cytoskeleton dynamics, mRNA transport and translation, non-coding piRNAs processing and transposons silencing.
This interaction was initially detected in the yeast two-hybrid screening of HeLa cDNA library and further
confirmed by pull-down and co-immunoprecipitation assays. In addition we demonstrated that TDRD 7 can
form a complex with other isoform of S6K - S6K2. Notably, both isoforms of S6K were found to phosphorylate
TDRD 7 in vitro at multiple phosphorylation sites. Altogether, these findings demonstrate that TDRD 7 is a novel
substrate of S6Ks, suggesting the involvement of S6K signaling in the regulation of TDRD 7 cellular functions.
This review summarizes experimental data related to the studies of PI3K/mTOR/S6K signaling conducted at the department of cell signaling. Analysis of novel S6Ks protein-protein interactions provided valuable information for understanding molecular mechanisms of regulation of S6Ks functional activity and subcellular localization mediated by PKC, CK2 and ROC1 ubiquitin ligase. We discuss the identification and functional analysis of novel isoform of ribosomal protein S6 kinase – S6K2 and of mTOR kinase – mTOR, as well as their oncogenic properties. Identification of CoA synthase responsible for last two steps in CoA biosynthesis and characterization of its interactions with S6K1 and other signaling molecules uncovere a potential link between mTOR/S6K signaling pathway and energy metabolism through regulation of CoA biosynthesis. The data concerning new molecular mechanisms of CoA synthase regulation are presented.
Alteration of expression levels of p70S6 kinase (p70S6K) in Guerin’s carcinoma cells in vivo upon dynamics of tumor development has been studied. The expression levels of p70S6K were studied by Western blot analysis with the use of polyclonal anti-p70S6K-antibodies in the samples of tumor tissues, uteral tissues of tumor-bearing animals (control) and uteral tissues of healthy animals (negative control). The data have demonstrated that in tumor tissues the level of p70S6K expression is higher than in normal uteral tissue, and that development of Guerin’s carcinoma is accompanied by increase of p70S6K expression on the initial stages and by its decrease on the terminal stages
RPS6KB1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) plays a key role in regulating protein translation. The role of RPS6KB1 in HCC (hepatocellular carcinoma) has not been fully investigated. This study was undertaken to determine the clinicopathological features and prognostic value of RPS6KB1 in HCC. We examined RPS6KB1 expression in 30 paired liver cancer tissues and adjacent noncancerous tissues by reverse transcription real-time PCR and Western blotting. In addition, we analyzed RPS6KB1 expression in 87 HCC samples by immunohistochemistry. The expression of RPS6KB1 was significantly increased in cancer tissues. Clinicopathological analysis showed that the expression of RPS6KB1 was significantly correlated with tumor size, histopathologic classifications, and serum alpha-fetoprotein (AFP). Kaplan-Meier survival curves revealed that increased expression of RPS6KB1 correlated with poor prognosis of HCC patients. RPS6KB1 expression was an independent prognostic marker for overall survival of HCC patients by multivariate analysis. Our data suggest that RPS6KB1 may play an important role in the progression of HCC and could serve as a potential molecular target for HCC therapy.
The 70kDa ribosomal protein S6 kinases (S6K1 and S6K2) play important roles in the regulation of protein synthesis, cell growth and survival. S6Ks are activated in response to mitogen stimulation and nutrient sufficiency by the phosphorylation of conserved serine and threonine residues. Here we show for the first time, that in addition to phosphorylation, S6Ks are also targeted by lysine acetylation. Following mitogen stimulation, S6Ks interact with the p300 and p300/CBP-associated factor (PCAF) acetyltransferases. S6Ks can be acetylated by p300 and PCAF in vitro and S6K acetylation is detected in cells expressing p300. Furthermore, it appears that the acetylation sites targeted by p300 lie within the divergent C-terminal regulatory domains of both S6K1 and S6K2. Acetylation of S6K1 and 2 is increased upon the inhibition of class I/II histone deacetylases (HDACs) by trichostatin-A, while the enhancement of S6K1 acetylation by nicotinamide suggests the additional involvement of sirtuin deacetylases in S6K deacetylation. Both expression of p300 and HDAC inhibition cause increases in S6K protein levels, and we have shown that S6K2 is stabilized in cells treated with HDAC inhibitors. The finding that S6Ks are targeted by histone acetyltransferases uncovers a novel mode of crosstalk between mitogenic signalling pathways and the transcriptional machinery and reveals additional complexity in the regulation of S6K function.
Rapamycin is an immunosuppressant which antagonizes cellular proliferation by inhibiting the function of mTOR. The mTOR:FKBP12: rapamycin complex blocks G1/S transition by inhibiting downstream targets essential for cell cycle progression. One such target is p70S6k1 (S6K1), a serine/threonine kinase which is inactivated by the mTOR : FKBP12 : rapamycin complex, and which has been linked to translational control by virtue of its ability to phosphorylate the ribosomal protein S6. In the current work, we describe cloning and characterization of a novel S6K1 homolog, p54 S6 kinase 2 (p54S6k2/S6K2). Similar to S6K1, S6K2 is activated by mitogens and by constitutively active PI3K, and is inhibited by rapamycin as well as wortmannin. Differences between activation of S6K1 and S6K2 by PDK1 were observed, suggesting potential differences in the regulation of these homologs. Strikingly, S6K2 activity and S6 phosphorylation were both intact in S6K1-/-ES cell, indicating a possible role for S6K2 in in vivo S6 phosphorylation. Interestingly, we found two isoforms of S6K2 which are localized to distinct cellular compartments; the smaller form resides in the detergent-soluble fraction, whereas the larger form is found in the particulate fraction. Our findings demonstrate the existence of a family of rapamycin-sensitive protein kinases potentially involved in S6 phosphorylation, translational control, and transduction of mTOR signals.
Insulin treatment of Drosophila melanogaster Kc 167 cells induces the multiple phosphorylation of a Drosophila ribosomal protein, as judged by its decreased electrophoretic mobility on two-dimensional polyacrylamide gels. The extent to which insulin induces this response is potentiated by cycloheximide and blocked by pretreatment with rapamycin. Isolation and mass spectrometric analysis revealed that the multiply phosphorylated protein was the larger of two Drosophila melanogaster orthologues of mammalian 40S ribosomal protein S6, termed here DS6A. Proteolytic cleavage of DS6A derived from stimulated Kc 167 cells with the endoproteinase Lys-C released a number of peptides, one of which contained all the putative phosphorylation sites. Conversion of phosphoserines to dehydroalanines with Ba(OH)(2) showed that the sites of phosphorylation reside at the carboxy terminus of DS6A. The sites of phosphorylation were identified by Edman degradation after conversion of the phosphoserine residues to S-ethylcysteine as Ser(233), Ser(235), Ser(239), Ser(242), and Ser(245). Finally, phosphopeptide mapping of individual phosphoderivatives, isolated from two-dimensional polyacrylamide gels, indicated that DS6A phosphorylation, in analogy to mammalian S6 phosphorylation, appears to proceed in an ordered fashion. The importance of these observations in cell growth and development is discussed.
This article reviews our current knowledge of the role of ribosomal protein S6 phosphorylation and the S6 kinase (S6K) signaling pathway in the regulation of cell growth and proliferation. Although 40S ribosomal protein S6 phosphorylation was first described 25 years ago, it only recently has been implicated in the translational up-regulation of mRNAs coding for the components of protein synthetic apparatus. These mRNAs contain an oligopyrimidine tract at their 5' transcriptional start site, termed a 5'TOP, which has been shown to be essential for their regulation at the translational level. In parallel, a great deal of information has accumulated concerning the identification of the signaling pathway and the regulatory phosphorylation sites involved in controlling S6K activation. Despite this knowledge we are only beginning to identify the direct upstream elements involved in growth factor-induced kinase activation. Use of the immunosupressant rapamycin, a bacterial macrolide, in conjunction with dominant interfering and activated forms of S6K1 has helped to establish the role of this signaling cascade in the regulation of growth and proliferation. In addition, current studies employing the mouse as well as Drosophila melanogaster have provided new insights into physiological function of S6K in the animal. Deletion of the S6K1 gene in mouse cells led to an animal of reduced size and the identification of the S6K1 homolog, S6K2, whereas loss of dS6K function in Drosophila demonstrated its paramount importance in development and growth control.
PS6K, a putative oncogene mapped to chromosome 17q23, encodes a serine/threonine kinase, which phosphorylates ribosomal subunit 6 and is part of the insulin receptor signal transduction pathway involved in the regulation of messenger RNA translation, protein synthesis, cell cycle progression, and cell size. Comparative genomic hybridization studies have detected 17q23 amplifications in a subset of meningiomas, particularly those with aggressive histologic features. PS6K amplifications have been reported in breast cancer, another hormonally driven neoplasm. We assessed PS6K dosage in 94 archival paraffin-embedded meningiomas using dual-color fluorescence in situ hybridization. We found high-level PS6K amplifications in 3 of 22 anaplastic grade III meningiomas. Amplification was confirmed by differential polymerase chain reaction in 1 of these cases. In contrast, no amplifications were identified in 37 benign (grade I) and 35 atypical (grade II) meningiomas. To our knowledge, this represents the first report of gene amplification in primary human meningiomas. Given its exclusive association with anaplastic meningiomas, PS6K amplification likely occurs during the malignant progression of a small subset of anaplastic tumors. Further studies are needed to map the 17q23 amplicon to determine whether additional genes in this region are amplified in high-grade meningiomas.
The phosphorylation of ribosomal protein S6 is thought to be required for biosynthesis of the cell's translational apparatus, a critical component of cell growth and proliferation. We have studied the signal transduction pathways involved in hepatic S6 phosphorylation during late gestation in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of mitogenic signaling pathways that are operative in mature hepatocytes. Our initial studies demonstrated that there was low basal activity of two S6 kinases in liver, S6K1 and S6K2, on embryonic day 19 (2 days preterm). In addition, insulin- and growth factor-mediated S6K1 and S6K2 activation was markedly attenuated compared with that in adult liver. Nonetheless, two-dimensional gel electrophoresis demonstrated that fetal liver S6 itself was highly phosphorylated. To characterize the fetal hepatocyte pathway for S6 phosphorylation, we went on to study the sensitivity of hepatocyte proliferation to the S6 kinase inhibitor rapamycin. Unexpectedly, administration of rapamycin to embryonic day 19 fetuses in situ did not affect hepatocyte DNA synthesis. This resistance to the growth inhibitory effect of rapamycin occurred even though S6K1 and S6K2 were inhibited. Furthermore, fetal hepatocyte proliferation was sustained even though rapamycin administration resulted in the dephosphorylation of ribosomal protein S6. In contrast, rapamycin blocked hepatic DNA synthesis in adult rats following partial hepatectomy coincident with S6 dephosphorylation. We conclude that hepatocyte proliferation in the late gestation fetus is supported by a rapamycin-resistant mechanism that can function independently of ribosomal protein S6 phosphorylation.
Phosphorylation of the ribosomal S6 subunit is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery, which is an important early event that controls mammalian cell growth and proliferation. The recently identified S6 kinase 2 (S6K2), together with its homologue S6K1, is likely responsible for the mitogen-stimulated phosphorylation of S6. Like S6K1, the activation of S6K2 requires signaling from both the phosphatidylinositol 3-kinase and the mammalian target of rapamycin (mTOR). Here we report the investigation of the mechanisms of S6K2 regulation by mTOR. We demonstrate that similar to S6K1 the serum activation of S6K2 in cells is dependent on mTOR kinase activity, amino acid sufficiency, and phosphatidic acid. Previously we have shown that mTOR is a cytoplasmic-nuclear shuttling protein. As a predominantly nuclear protein, S6K2 activation was facilitated by enhanced mTOR nuclear import with the tagging of an exogenous nuclear localization signal and diminished by enhanced mTOR nuclear export with the tagging of a nuclear export sequence. However, further increase of mTOR nuclear import by the tagging of four copies of nuclear localization signal resulted in its decreased ability to activate S6K2, suggesting that mTOR nuclear export may also be an integral part of the activation process. Consistently, the nuclear export inhibitor leptomycin B inhibited S6K2 activation. Taken together, our observations suggest a novel regulatory mechanism in which an optimal cytoplasmic-nuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.
mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo.
Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.
The discussion will focus on the role of the ribosomal protein S6 kinase (S6K) signaling pathway in the regulation of cell growth and proliferation. Although 40S ribosomal protein S6 phosphorylation was first described 25 years ago (Gressner and Wool, 1974), it only recently has been implicated in the translational up-regulation of mRNAs coding for the components of protein synthetic apparatus (Fumagalli and Thomas, 2000). These mRNAs contain an oligopyrimidine tract at their 5' transcriptional start site, termed a 5'TOP, which has been shown to be essential for their regulation at the translational level (Meyuhas et al., 1996). In parallel, a great deal of information has accumulated concerning the identification of the signaling pathway and the regulatory phosphorylation sites involved in controlling S6K activation (Dufner and Thomas, 1999). Despite this knowledge we are only beginning to identify the direct upstream elements involved in growth factor-induced kinase activation (Dennis et al., 2001; Pullen et al., 1998). Use of the immunosuppressant rapamycin, a bacterial macrolide, in conjunction with dominant interfering and activated forms of S6K1 has helped to establish the role of this signaling cascade in the regulation of growth and proliferation (Dennis and Thomas, 2002). In addition, current studies employing the mouse as well as Drosophila melanogaster have provided new insights into physiological function of S6K in the animal (Montagne et al., 1999; Pende et al., 2000). Loss of dS6K function in Drosophila melanogaster demonstrated its paramount importance in development and growth control (Montagne et al., 1999), whereas deletion of the S6K1 gene in the mouse led to an animal of reduced size and the identification of the S6K1 homologue, S6K2 (Shima et al., 1998). Such mice are significantly smaller during fetal development (Shima et al., 1998) and hypoinsulinemic in the adult, conditions known to lead to type 2 diabetes (Pende et al., 2000).
Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. We now show that the ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase Balpha (PKBalpha). Likewise, overexpression of constitutively active PI3-kinase or PKBalpha can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway.
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Modulation of the abundance of the translational apparatus appears to enable eukaryotic cells to cope withchanging requirements for protein synthesis during transitions between extreme growth and nutritional states.Thus, rRNA synthesis is down-regulated when cellscease to proliferate or are deprived of amino acids and isup-regulated upon reversal of such conditions (Grummt1999 and references therein). Likewise, the translationalefficiency of mRNAs encoding many protein components of the translational machinery is similarly regulated. These include ribosomal proteins (Meyuhas et al.1996a; Meyuhas and Hornstein 2000 and referencestherein); elongation factor 1A (Rao and Slobin 1987;Avni et al. 1994; Jefferies et al. 1994a) and elongationfactor 2 (Terada et al. 1994; Avni et al. 1997); poly(A)-binding protein (Hornstein et al. 1999), which has beenimplicated in both translation initiation and ribosome assembly (Sachs 2000); and a few other proteins that havebeen implicated in ribosome assembly or nuclear-cytoplasmic transport of RNA (for review, see Meyuhas2000). The corresponding mRNAs are characterized bythe presence of a 5′ terminal oligopyrimidine tract(5′TOP) and therefore are referred to as TOP mRNAs.This structural motif comprises the core of the translational cis-regulatory element of these mRNAs, and itsfeatures are summarized elsewhere (Meyuhas and Hornstein 2000)...
The mammalian target of rapamycin, mTOR, is a protein Ser-Thr kinase that functions as a central element in a signaling pathway involved in the control of cell growth and proliferation. The activity of mTOR is controlled not only by amino acids, but also by hormones and growth factors that activate the protein kinase Akt. The signaling pathway downstream of Akt leading to mTOR involves the protein products of the genes mutated in tuberous sclerosis, TSC1 and TSC2, and the small guanosine triphosphatase, Rheb. In cells, mTOR is found in a complex with two other proteins, raptor and mLST8. In this review, we describe recent progress in understanding the control of the mTOR signaling pathway and the role of mTOR-interacting proteins.
Various mitogenic or growth inhibitory stimuli induce a rapid change in the association of terminal oligopyrimidine (TOP) mRNAs with polysomes. It is generally believed that such translational control hinges on the mammalian target of rapamycin (mTOR)-S6 kinase pathway. Amino acid availability affects the translation of TOP mRNAs, although the signaling pathway involved in this regulation is less well characterized. To investigate both serum- and amino acid-dependent control of TOP mRNA translation and the signaling pathways involved, HeLa cells were subjected to serum and/or amino acid deprivation and stimulation. Our results indicate the following. 1). Serum and amino acid deprivation had additive effects on TOP mRNA translation. 2). The serum content of the medium specifically affected TOP mRNA translation, whereas amino acid availability affected both TOP and non-TOP mRNAs. 3). Serum signaling to TOP mRNAs involved only a rapamycin-sensitive pathway, whereas amino acid signaling depended on both rapamycin-sensitive and rapamycin-insensitive but wortmannin-sensitive events. 4). Eukaryotic initiation factor-2alpha phosphorylation increased during amino acid deprivation, but not following serum deprivation. Interestingly, rapamycin treatment suggests a novel connection between the mTOR pathway and eukaryotic initiation factor-2alpha phosphorylation in mammalian cells, which may not, however, be involved in TOP mRNA translational regulation.
To perform an immunohistochemical analysis of human breast adenomas and adenocarcinomas as well as normal breast tissues in respect of S6 ribosomal protein kinase (S6K) expression and localization in normal and transformed cells.
The expression level and localization of S6K have been detected in formalin fixed, paraffin embedded sections of normal human breast tissues, adenomas and adenocarcinomas with different grade of differentiation. Immunohistochemical detection of S6K1 and S6K2 in normal human breast tissues and breast tumors were performed using specific monoclonal and polyclonal antibodies against S6K1 and S6K2 with following semiquantitative analysis.
The increase of S6K content in the cytoplasm of epithelial cells in benign and malignant tumors has been detected. Nuclear accumulation of S6K1 and to a greater extend S6K2 have been found in breast adenocarcinomas. About 80% of breast adenocarcinomas cases revealed S6K2 nuclear staining comparing to normal tissues. In 31% of cases more then 50% of cancer cells had strong nuclear staining. Accumulation of S6K1 in the nucleus of neoplastic cells has been demonstrated in 25% of cases. Nuclear localization of S6K in the epithelial cells in normal breast tissues has not been detected.
Immunohistochemical analysis of S6K1 and S6K2 expression in normal human breast tissues, benign and malignant breast tumors clearly indicates that both kinases are overexpressed in breast tumors. Semiquantitative analysis of peculiarities of S6K localization in normal tissues and tumors revealed that nucleoplasmic accumulation of S6K (especially S6K2) is a distinguishing feature of cancer cells.
The immunosuppressants rapamycin and FK506 bind to the same intracellular protein, the immunophilin FKBP12. The FKB12-FK506 complex interacts with and inhibits the Ca(2+)-activated protein phosphatase calcineurin. The target of the FKBP12-rapamycin complex has not yet been identified. We report that a protein complex containing 245 kDa and 35 kDa components, designated rapamycin and FKBP12 targets 1 and 2 (RAFT1 and RAFT2), interacts with FKBP12 in a rapamycin-dependent manner. Sequences (330 amino acids total) of tryptic peptides derived from the 245 kDa RAFT1 reveal striking homologies to the yeast TOR gene products, which were originally identified by mutations that confer rapamycin resistance in yeast. A RAFT1 cDNA was obtained and found to encode a 289 kDa protein (2549 amino acids) that is 43% and 39% identical to TOR2 and TOR1, respectively. We propose that RAFT1 is the direct target of FKBP12-rapamycin and a mammalian homolog of the TOR proteins.
Here, we disrupted the p70 S6 kinase (p70s6k) gene in murine embryonic stem cells to determine the role of this kinase in cell growth, protein synthesis, and rapamycin
sensitivity. p70s6k−/− cells proliferated at a slower rate than parental cells, suggesting that p70s6k has a positive influence on cell proliferation but is not essential. In addition, rapamycin inhibited proliferation of p70s6k−/− cells, indicating that other events inhibited by the drug, independent of p70s6k, also are important for both cell proliferation and the action of rapamycin. In p70s6k−/− cells, which exhibited no ribosomal S6 phosphorylation, translation of mRNA encoding ribosomal proteins was not increased
by serum nor specifically inhibited by rapamycin. In contrast, rapamycin inhibited phosphorylation of initiation factor 4E-binding
protein 1 (4E-BP1), general mRNA translation, and overall protein synthesis in p70s6k−/− cells, indicating that these events proceed independently of p70s6k activity. This study localizes the function of p70s6k to ribosomal biogenesis by regulating ribosomal protein synthesis at the level of mRNA translation.
Employing oligonucleotide primers derived from the DNA sequence of rat p70s6k a homologous sequence was shown by polymerase chain reaction (PCR) to be present as a maternal transcript in stage IV-VI Xenopus laevis oocytes. The sequence covered 665 bp of p70s6k and was 97% identical at the amino acid level. When used to probe a Northern blot of the poly(A)+ mRNA from stage VI oocytes, this sequence recognized four transcripts of 1.9, 2.5, 3.2 and 5.2 kb. Specific rat p70s6k antibodies immunoprecipitated active S6 kinase from stage VI oocytes but not unfertilized eggs. The basal level of activity was 3- to 5-fold higher in primed versus non-primed oocytes indicating that p70s6k activation was an early event in maturation. Consistent with this observation, progesterone induced a 10-fold activation of the kinase in non-primed oocytes within 1 h post-induction at a time critical for early activation of protein synthesis. A much smaller, variable peak of activation was observed at 85% germinal vesicle breakdown (GVBD), which was dependent on the rate of maturation. Two members of the pp90rsk family, thought to be the sole S6 kinases present in X.laevis oocytes, exhibited distinct kinetics of activation. Finally, the S6 kinase activity present 1 h post-progesterone stimulation was purified and shown to have a Mr of 70K.
Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.
Treatment of quiescent 3T3 cells with sodium orthovanadate induces a 10-fold stimulation of a kinase that phosphorylates ribosomal protein S6. The kinase in crude extracts is extremely labile and rapidly loses activity when incubated at 37 degrees C. This reaction is blocked by phosphatase inhibitors such as p-nitrophenyl phosphate and beta-glycerophosphate, suggesting that dephosphorylation of the kinase leads to its inactivation (Novak-Hofer, I., and Thomas, G. (1985) J. Biol. Chem. 260, 10314-10319). After three steps of purification the kinase can be separated from greater than 99% of the cellular phosphorylase a phosphatases. At this stage the kinase preparation is almost completely stable but can be inactivated by readdition of specific column fractions that contain both phosphorylase phosphatase and protease activity. However, employing a number of specific inhibitors it is shown that the inactivating agent in these fractions is a protein phosphatase. Furthermore, the physical and enzymatic properties of the kinase inactivator argue that it can be classified as a type 2A phosphatase. These results are consistent with the finding that the purified catalytic subunits of phosphatase type 1 and type 2A also inactivate the kinase. At equivalent phosphorylase a phosphatase activities, the type 2A catalytic subunit is 3 times more potent than the type 1 enzyme in carrying out this reaction. These data indicate that the major S6 kinase inactivator in 3T3 cell extracts is a type 2A phosphatase, supporting the hypothesis that the orthovanadate-stimulated S6 kinase is regulated in vivo by a phosphorylation-dephosphorylation mechanism.
The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen-activated kinases. Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404. T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen-induced kinases. However, the principal target of rapamycin-induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain. Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance. The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.
Activation of p70s6k in cells stimulated with serum correlates with the phosphorylation of seven sites. Pretreatment of Swiss 3T3 cells with the
immunosuppressant rapamycin blocks phosphorylation of four of these sites (Thr229, Thr389, Ser404, and Ser411), whereas phosphorylation proceeds in the remaining three sites (Ser418, Thr421, and Ser424). If rapamycin is added post-serum stimulation, the pattern of phosphorylation is qualitatively similar except that Ser411 is still highly phosphorylated. The inhibitory effect of rapamycin on serum-induced p70s6k activation and the phosphorylation of Thr229, Thr389, Ser404, and Ser411 is rescued by FK506, providing further evidence that the inhibitory effect is exerted through a complex of rapamycin-FKBP12.
Wortmannin treatment pre- or post-serum stimulation inhibits phosphorylation of the same set of sites as rapamycin, supporting
the argument that both agents act on the same pathway. Likewise, methylxanthine phosphodiesterase inhibitors block p70s6k activation and phosphorylation of the same set of sites as wortmannin and rapamycin. However, other agents that raise intracellular
cAMP levels have no inhibitory effect, leading to the hypothesis that the inhibitory actions of methylxanthines on p70s6k activity are not through activating protein kinase A but through inhibition of an upstream kinase. Together the results indicate
that there are two kinase signaling pathways that must converge to activate p70s6k and that only one of these pathways is sensitive to rapamycin, wortmannin, and methylxanthine inhibition.
When complexed with the intracellular protein FKBP12, rapamycin is a potent immunosuppressant and an inhibitor of a mitogen-stimulated signalling pathway that leads to activation of p70 S6 kinase (p70S6k) and cyclin-dependent kinases (CDKs). A recently cloned FKBP12-rapamycin-associated protein (FRAP/RAFT) is the likely mediator of these effects. Using FRAP variants that do not bind FKBP12-rapamycin, we demonstrate here that FRAP is a rapamycin-sensitive regulator of p70S6k in vivo and that the kinase activity of FRAP is required for this regulation. In addition, we show that FRAP autophosphorylates in vitro. Consistent with an essential role for FRAP kinase activity in vivo, autophosphorylation of FRAP is inhibited by FKBP12-rapamycin. Deletion studies indicate that the kinase activity of FRAP alone is not sufficient for control of p70S6k and that an amino-terminal domain in FRAP is also required.
Elevation of intracellular cAMP by forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and prostaglandin E1, in synergy with insulin, stimulated DNA synthesis in quiescent Swiss 3T3 cells to the same level achieved by platelet-derived growth factor (PDGF) or bombesin. Both forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate stimulated a significant increase in cell number which, in the presence of insulin, reached the same levels achieved with PDGF. Treatment with either PDGF or bombesin caused a marked and persistent stimulation of p42MAPK and p44MAPK. In striking contrast, no activation was seen with mitogenic combinations of cAMP as shown by three different assays. Swiss 3T3 cells stably transfected with a constitutively activated Gs alpha subunit were 100-fold more sensitive to the mitogenic effects of forskolin but in this distinct cellular model forskolin did not activate p42MAPK. Swiss 3T3 cells stably transfected with interfering mutants of MEK-1 showed a 60% decrease in PDGF-stimulated p42 MAPK activation, but there was no inhibition of the mitogenic effect of forskolin in these cells. Furthermore, the upstream kinases MEK-1/MEK-2 and p74raf-1 were not activated by mitogenic combinations of cAMP while PDGF caused marked stimulation of their activity. Treatment of 3T3 cells with forskolin attenuated PDGF-stimulated p74raf-1 and p42MAPK activation but enhanced the mitogenic effects of this agent. Mitogenic combinations of cAMP strongly stimulated the phosphorylation and activation of p70s6k an effect that was inhibited by rapamycin. This agent markedly inhibited cAMP-stimulated DNA synthesis suggesting a critical role for p70s6k in cAMP mitogenic signaling. These results demonstrate that cAMP-induced mitogenesis can be dissociated from activation of the mitogen-activated protein kinase cascade and that this is not an obligatory point of convergence in mitogenic signaling in Swiss 3T3 cells.
Transforming growth factor-beta (TGF-beta) and activin exert their effects by binding to heteromeric complexes of type I and type II receptors. The type II receptors for TGF-beta and activin are transmembrane serine-threonine kinases; a series of related receptors, denoted activin receptor-like kinase (ALK) 1 to 5, have recently been identified, and ALK-6 is described here. ALK-5 has been shown to be a functional TGF-beta type I receptor. A systematic analysis revealed that most ALKs formed heteromeric complexes with the type II receptors for TGF-beta and activin after overexpression in COS cells; however, among the six ALKs, only ALK-5 was a functional TGF-beta type I receptor for activation of plasminogen activator inhibitor-1, and only ALK-2 and ALK-4 bound activin with high affinity.
Immunolocalization of a newly described isoform of p70s6k, termed p85s6k, demonstrated a predominantly nuclear location in rat embryo fibroblasts (REF-52), a compartment in which growth factor-mediated phosphorylation of S6 has recently been reported. Microinjection of expression vectors encoding either p85s6k or a fusion protein containing only the putative nuclear localization motifs led to the exclusive accumulation of both products in the nucleus. Consistent with such a localization, microinjection of affinity-purified anti-p85s6k IgG into the nucleus, but not the cytoplasm, blocked serum-induced initiation of DNA synthesis. Co-injection into the nucleus of the anti-p85s6k IgG with activated p70s6k, which lacks the antigenic epitope, rescued the S phase block, arguing that the antibody exerts its effects through inhibiting p85s6k function. The results indicate a novel role for S6 phosphorylation in the nucleus distinct from that in the cytoplasm, a role essential for mitogenesis.
The immunosuppressant rapamycin blocks p70s6k/p85s6k activation and phosphorylation of 40S ribosomal protein S6 in Swiss 3T3 cells. The same net result is obtained when the macrolide is added 3 hr after serum stimulation. In stimulated cells p70s6k/p85s6k inactivation is achieved within minutes, whereas S6 dephosphorylation requires 1-2 hr, supporting the concept that S6 dephosphorylation results from kinase inactivation. In parallel, rapamycin treatment causes a small, but significant, reduction in the initiation rate of protein synthesis, as measured both by [35S]methionine incorporation into protein and by recruitment of 80S ribosomes into polysomes. More striking, analysis of individual mRNA transcripts revealed that rapamycin selectively suppresses the translation of a family of mRNAs that is characterized by a polypyrimidine tract immediately after their N7-methylguanosine cap, a motif that can act as a translational modulator. This family includes transcripts for ribosomal proteins, elongation factors of protein synthesis, and proteins of as-yet-unknown function. The results imply that (i) 40S ribosomes containing phosphorylated S6 may selectively recognize this motif or proteins which bind to it and (ii) rapamycin may inhibit cell growth by blocking S6 phosphorylation and, thus, translation of these mRNAs.
Employing specific inhibitors and docking-site mutants of growth factor receptors, recent studies have indicated that the insulin-induced increase in 40S ribosomal protein S6 and initiation factor 4E binding protein 1 (4E-BP1) phosphorylation is mediated by the mTOR/FRAP-p70s6k signal transduction pathway. However, it has not been resolved whether the phosphorylation of both proteins is mediated by p70s6k or whether they reside on parallel pathways which bifurcate upstream of p70s6k. Here we have used either rapamycin-resistant, kinase-dead, or wild-type p70s6k variants to distinguish between these possibilities. The rapamycin-resistant p70s6k, which has high constitutive activity, was able to signal to S6 in the absence of insulin and to prevent the rapamycin-induced block of S6 phosphorylation. This same construct did not increase the basal state of 4E-BP1 phosphorylation or protect it from the rapamycin-induced block in phosphorylation. Unexpectedly, the rapamycin-resistant p70s6k inhibited insulin-induced 4E-BP1 phosphorylation in a dose-dependent manner. This effect was mimicked by the kinase-dead and wild-type p70s6k constructs, which also blocked insulin-induced dissociation of 4E-BP1 from initiation factor 4E. Both the kinase-dead and wild-type constructs also blocked reporter p70s6k activation, although only the kinase-dead p70s6k had a dominant-interfering effect on S6 phosphorylation. Analysis of phosphopeptides from reporter 4E-BP1 and p70s6k revealed that the kinase-dead p70s6k affected the same subset of sites as rapamycin in both proteins. The results demonstrate, for the first time, that activated p70s6k mediates increased S6 phosphorylation in vivo. Furthermore, they show that increased 4E-BP1 phosphorylation is controlled by a parallel signalling pathway that bifurcates immediately upstream of p70s6k, with the two pathways sharing a common rapamycin-sensitive activator.
Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein
synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the
phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.
The macrolide rapamycin blocks cell cycle progression in yeast and various animal cells by an unknown mechanism. We demonstrate that rapamycin blocks the phosphorylation and activation of the 70 kd S6 protein kinases (pp70S6K) in a variety of animal cells. The structurally related drug FK506 had no effect on pp70S6K activation but at high concentrations reversed the rapamycin-induced block, confirming the requirement for the rapamycin and FK506 receptor, FKBP. Rapamycin also interfered with signaling by these S6 kinases, blocking serum-stimulated S6 phosphorylation and delaying entry of Swiss 3T3 cells into S phase. Neither rapamycin nor FK506 blocked activation of a distinct family of S6 kinases (RSKs) or the MAP kinases. These studies identify a rapamycin-sensitive signaling pathway, argue for a ubiquitous role for FKBPs in signal transduction, indicate that FK506-FKBP-calcineurin complexes do not interfere with pp70S6K signaling, and show that in fibroblasts pp70S6K, not RSK, is the physiological S6 kinase.
Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.
Recently we reported the purification of a mitogen-activated S6 kinase from Swiss mouse 3T3 fibroblasts and rat liver. The rat liver protein was cleaved with cyanogen bromide or trypsin and 17 of the resulting peptides were sequenced. DNA primers were generated from 3 peptides that had homology to sequences of the conserved catalytic domain of protein kinases. These primers were used in the polymerase chain reaction to obtain a 0.4-kilobase DNA fragment. This fragment was either radioactively labeled and hybridized to Northern blots of poly(A)+ mRNA or used to screen a rat liver cDNA library. Northern blot analysis revealed four transcripts of 2.5, 3.2, 4.0, and 6.0 kilobases, and five S6 kinase clones were obtained by screening the library. Only two of the clones, which were identical, encoded a full-length protein. This protein had a molecular weight of 56,160, which correlated closely to that of the dephosphorylated kinase determined by SDS/PAGE. The catalytic domain of the kinase resembles that of other serine/threonine kinases belonging to the second messenger subfamily of protein kinases.
Publisher Summary To help cope with the rapidly expanding protein kinase family, a database of the catalytic domain amino acid sequences has been established. This database would be a useful resource for the initial classification of novel protein kinases and for other studies that require extensive sequence comparisons. The catalytic domain database is updated frequently and often includes new sequences before they can be found in the Genbank/EMBL/PIR resources. More importantly, the availability of this large group of sequences in a single file saves investigators from the tedious task of collecting them themselves. This chapter describes the current makeup of the catalytic domain database and present two examples of its use: analysis and graphic display of conserved catalytic domain residues using conservation plots and classification of protein kinases by phylogenetic mapping. A prerequisite for both of these tasks is a multiple sequence alignment. The protein kinase catalytic domain database file can be obtained electronically over Internet using the standard network file transfer program (FTP). The database file PKINASES.IG contains brief descriptions of the kinases and references for the sequences. One hundred and seventeen distinct sequences had been entered by mid-February, 1990. Seventy-five of these are taken from protein-serine/threonine kinases and 42 from protein-tyrosine kinases. Sixty-eight of the sequences are from vertebrate species, 24 from yeasts (both budding and fission), 18 from Drosophila, two from nematode, and one each from Aplysia , Aspergillus , Hydra , bean plant ( Phaseolus ), and avian erythroblastosis virus S13. 43 of the 68 current vertebrate entries are taken from human sources and twenty two of the remaining vertebrate sequences derive from four other mammals: bovine, rabbit, rat, and mouse.
The molecular structure of a rat hepatoma 70-kDa insulin/mitogen-stimulated S6 protein kinase, obtained by molecular cloning, is compared to that of a rat homolog of the 85-kDa Xenopus S6 protein kinase alpha; both kinases were cloned from H4 hepatoma cDNA libraries. The 70-kDa S6 kinase (calculated molecular mass of 59,186 Da) exhibits a single catalytic domain that is most closely related in amino acid sequence (56% identity) to the amino-terminal, kinase C-like domain of the rat p85 S6 kinase (calculated molecular mass of 82,695 Da); strong similarity extends through a further 67 residues carboxyl-terminal to the catalytic domain (40% identity), corresponding to a region also conserved among the kinase C family. Outside of this segment of approximately 330 amino acids, the structures of the p70 and p85 S6 kinases diverge substantially. The p70 S6 kinase is known to be activated through serine/threonine phosphorylation by unidentified insulin/mitogen-activated protein kinases. A model for the regulation of p70 S6 protein kinase activity is proposed wherein the low activity of the unphosphorylated enzyme results from the binding of a basic, inhibitory pseudosubstrate site (located carboxyl-terminal to the extended catalytic domain) to an acidic substrate binding region (located amino-terminal to the catalytic domain); substrate binding is thereby prevented. S6 kinase activation requires displacement of this inhibitory segment, which is proposed to occur consequent to its multiple phosphorylation. The putative autoinhibitory segment contains several serine and threonine residues, each followed directly by a proline residue. This motif may prevent autophosphorylation but permit transphosphorylation; two of these serine residues reside in a maturation promoting factor (MPF)/cdc-2 consensus motif. Thus, hormonal regulation of S6 kinase may involve the action of MPF/cdc-2 or protein kinases with related substrate specificity.
Treatment of Swiss mouse 3T3 cells with epidermal growth factor, orthovanadate, or serum results in the activation of a kinase that phosphorylates protein S6 of the 40S ribosomal subunit in vitro. This kinase is eluted as a single peak of activity from either a Mono Q anion-exchange column at 0.34 M NaCl or a Mono S cation-exchange column at 0.20 M NaCl. Treatment of the peak fraction from the Mono S column with phosphatase 2A completely abolishes the activity of the enzyme. The kinase appears to be distinct from protein kinase C, cAMP-dependent protein kinase, and two protease-activated kinases, PAK II and H4P. The kinase has been purified to apparent homogeneity and migrates as a single band at Mr 70,000 in NaDodSO4/polyacrylamide gels. The kinase exhibits the ability to autophosphorylate, and this activity directly parallels S6 phosphorylation activity on the final step of purification. In vitro, the kinase incorporates up to 5 mol of phosphate into S6, and the tryptic phosphopeptide maps obtained are equivalent to those from S6 phosphorylated in vivo. Most important, treatment of the purified kinase with phosphatase 2A results in complete inactivation of the enzyme, arguing that the activity of the kinase is directly controlled by phosphorylation.
In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.
A serine/threonine kinase, named protein kinase B (PKB) for its sequence homology to both protein kinase A and C, has previously been isolated. PKB, which is identical to the kinase Rac, was later found to be the cellular homologue of the transforming v-Akt. Here we show that PKB is activated by stimuli such as insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Activation of PKB was inhibited by the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin and by coexpression of a dominant-negative mutant of PI(3)K. PDGF receptor mutants that lack detectable associated PI(3)K activity also fail to induce PKB activation, PKB kinase activity is correlated with phosphorylation of PKB on serine. Finally, we show that a constructed Gag-PKB fusion protein, homologous to the v-akt oncogene, displays significantly increased ligand-independent kinase activity. Furthermore, this activity is sufficient to activate the p70 S6-kinase (p70S6K). These results suggest a role for PKB in PI(3)K-mediated signal transduction.
The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.
Activation of the adenylyl cyclase signaling pathway elicits the induction of genes via activators binding to cAMP-responsive elements (CREs). Nuclear factor CRE modulator (CREM) is activated by PKA-mediated phosphorylation on a serine at position 117. We show that Ser-117 is also phosphorylated by the mitogen-activated p70 S6 kinase (p70S6K) in vitro. Activation of cellular p70S6K by serum factors enhances Ser-117 phosphorylation and CREM transactivation. Coexpression of p70S6K significantly increases transactivation by a GAL4-CREM fusion. The macrolide rapamycin, a potent and specific inhibitor of p70S6K in vivo, completely blocks CREM activation induced by serum and by p70S6K. Thus, CREM constitutes a target for mitogenic signaling through p70S6K and may acts as a nuclear effector in which transduction pathways may converge and cross-talk.
A cDNA clone encoding a 53 kd serine/threonine kinase receptor with an overall structure similar to that of the type II receptor for transforming growth factor beta (TGF beta) was obtained. 125I-TGF beta 1 bound to porcine endothelial cells transfected with the cDNA and formed a cross-linked complex of 70 kd, characteristic of a TGF beta type I receptor. Immunoprecipitation of the cross-linked complexes by antibodies against the cloned receptor revealed the 70 kd complex as well as a 94 kd TGF beta type II receptor complex. The immunoprecipitated novel serine/threonine kinase receptor had biochemical properties of the TGF beta type I receptor and was observed in different cell types. Transfection of the cloned cDNA into TGF beta type I receptor-deficient cells restored TGF beta-induced plasminogen activator inhibitor 1 production. These results suggest that signal transduction by TGF beta involves the formation of a heteromeric complex of two different serine/threonine kinase receptors.
Transforming growth factor beta (TGF beta) and activin each bind to pairs of membrane proteins, known as receptor types I and II, that associate to form a signaling complex. We report that TSR-I and ActR-I, two human transmembrane serine/threonine kinases distantly related to TGF beta and activin type II receptors, act as type I receptors for these factors. TSR-I is a type I receptor shared by TGF beta and activin, whereas ActR-I is an activin type I receptor. ActR-I, but not TSR-I, signals a particular transcriptional response in concert with activin type II receptors. The results indicate that type I receptors are transmembrane protein kinases that associate with type II receptors to generate diverse heteromeric serine/threonine kinase complexes of different signaling capacities.
An essential step in the pathway by which growth factors trigger cellular proliferation is the induction of high levels of protein synthesis. This appears in part to be controlled by multiple phosphorylation of the ribosomal protein S6 (refs 4, 5). The main kinase responsible, p70s6k (refs 6-8), is activated through the phosphorylation of four sites clustered in a putative autoinhibitory domain, which is mediated by a signalling pathway distinct from those used by other well characterized mitogen-activated serine/threonine kinases (such as p42/p44mapk or p90rsk; refs 10, 11). Here we investigate the role of p70s6k in the mitogenic response. Microinjection of quiescent rat embryo fibroblasts with any of three distinct polyclonal antibodies to p70s6k abolishes serum-induced entry into S phase of the cell cycle. This effect is preceded by almost complete abrogation of the activation of protein synthesis and the expression of an essential immediate early gene product, c-fos. The inhibitory effect on DNA synthesis is also elicited by microinjection of the antibodies late in G1 phase, consistent with the finding that p70s6k activity remains high throughout G1.
The activation of the 70kDa S6 kinase, pp70S6k, is a well documented mitogenic response, yet until recently little was known of how pp70S6k is activated, or of the identities of its crucial targets. The past year has revealed the complexity of pp70S6k regulation, with the overriding theme being that enzymes which have proven or putative roles in phospholipid metabolism mediate its activation. Studies also indicate that pp70S6k may regulate many more pathways than previously recognized.
The S6 ribosomal protein is phosphorylated by p70 S6 kinase (p70S6k). Although the cellular role of S6 phosphorylation is still not fully clear, studies on p70S6k and its activation have revealed the existence of a novel signalling pathway, clues to the mechanism of action of certain immunosuppressants and insights into the control of gene expression at the levels of transcription and translation.
Mitogen-activated protein (MAP) kinase cascades are activated in response to various extracellular stimuli, including growth factors and environmental stresses. A MAP kinase kinase kinase (MAPKKK), termed ASK1, was identified that activated two different subgroups of MAP kinase kinases (MAPKK), SEK1 (or MKK4) and MKK3/MAPKK6 (or MKK6), which in turn activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and p38 subgroups of MAP kinases, respectively. Overexpression of ASK1 induced apoptotic cell death, and ASK1 was activated in cells treated with tumor necrosis factor-alpha (TNF-alpha). Moreover, TNF-alpha-induced apoptosis was inhibited by a catalytically inactive form of ASK1. ASK1 may be a key element in the mechanism of stress- and cytokine-induced apoptosis.
The activation of p70s6k is accompanied by a complex series of phosphorylation events. In this review we propose a model of activation which divides p70s6k into four functional modules that cooperate in leading to full enzyme activity. In the light of the model, we suggest how candidate effectors of p70s6k activation might function by directing the phosphorylation of specific sites.
The mouse cDNA for apoptosis signal-regulating kinase 1 (ASK)1 was isolated. The overall amino acid sequence identity between the mouse and the human ASK1 was 91.9%. A database search revealed that the kinase domain of ASK1 is evolutionally well-conserved over species among nematode, fly, mouse and human. Northern blot analysis identified a 6-kb transcript of ASK1 which is expressed in the various mouse adult tissues including heart, brain, lung, liver and kidney. Immunohistochemical analysis of mouse embryos (17 days post coitum) revealed a localized expression of ASK1 in developing skin, cartilage and bone, suggesting a possible role for ASK1 in tissue development during embryogenesis as well as cytokine-induced apoptosis.
TOR, phosphatidylinositol 3-kinase, p70s6k, and 4E-BP1 have recently emerged as components of a major signalling pathway that is dedicated to protein translation and thus to cell growth. This pathway appears to be conserved, at least in part, in yeast, slime molds, plants, flies, and mammals. TOR and phosphatidylinositol 3-kinase control p70s6k and 4E-BP1, which, in turn, directly control the translation initiation machinery.
The p70 S6 kinase, an enzyme critical for cell-cycle progression through the G1 phase, is activated in vivo by insulin and mitogens through coordinate phosphorylation at multiple sites, regulated by signaling pathways, some of which depend on and some of which are independent of phosphoinositide 3-kinase (Pl 3-kinase). It is not known which protein kinases phosphorylate and activate p70.
Co-expression of p70 with 3-phosphoinositide-dependent protein kinase 1 (PDK1), a protein kinase that has previously been shown to phosphorylate and activate protein kinase B (PKB, also known as c-Akt), resulted in strong activation of the S6 kinase in vivo. In vitro, PDK1 directly phosphorylated Thr252 in the activation loop of the p70 catalytic domain, the phosphorylation of which is stimulated by PI 3-kinase in vivo and is indispensable for p70 activity. Whereas PDK1-catalyzed phosphorylation and activation of PKB in vitro was highly dependent on the presence of phosphatidylinositol 3,4,5-trisphosphate (Ptdlns (3,4,5)P3), PDK1 catalyzed rapid phosphorylation and activation of p70 in vitro, independent of the presence of Ptdlns(3,4,5)P3. The ability of PDK1 to phosphorylate p70 Thr252 was strongly dependent on the phosphorylation of the p70 noncatalytic carboxy-terminal tail (amino acids 422-525) and of amino acid Thr412. Moreover, once Thr252 was phosphorylated, its ability to cause activation of the p70 S6 kinase was also controlled by the p70 carboxy-terminal tail and by phosphorylation of p70 Ser394, and most importantly, Thr412. The overriding determinant of the absolute p70 activity was the strong positive cooperativity between Thr252 and Thr412 phosphorylation; both sites must be phosphorylated to achieve substantial p70 activation.
PDK1 is one of the components of the signaling pathway recruited by Pl 3-kinase for the activation of p70 S6 kinase as well as of PKB, and serves as a multifunctional effector downstream of the Pl 3-kinase.
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