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Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families

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Abstract

Highly degenerate oligonucleotide primers designed from regions conserved between protein-serine kinases have been used specifically to amplify human epithelial (HeLa) cDNA by the polymerase chain reaction (PCR). Of several novel cDNA fragments encoding putative kinases thus isolated, one was further characterised. Screening of human fibroblast and bovine brain cDNA libraries with the PCR fragment yielded several clones with an open reading frame of 479 amino acids containing all of the conserved sequence motifs of protein-serine kinases. The predicted protein was most similar to the protein kinase C (PKC)/cAMP-dependent protein kinase (PKA) families and its gene has thus been termed pkb. Expression of the pkb gene is general but highest in brain, heart and lung. Translation of pkb RNA in vitro generated a 57-kDa protein (PKB) recognised by antisera raised to a bacterially expressed PKB/TrpE fusion protein. Transfection of COS cells with the kinase cDNA resulted in the synthesis of a 60-kDa protein which was partially purified by Mono Q anion-exchange chromatography. Column fractions containing PKB-immunoreactive protein exhibited elevated histone H1 kinase activity compared with similar fractions from control cells, demonstrating the enzymatic activity of this protein kinase.

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... The former isolated them by cDNA library screening from porcine kidney LLC-PK 1 cells using a porcine cAMP-PK probe, and then from human epithelial MCF-7 or lung fibroblast WI38 cell line (9,11). The latter did so from human fibroblast using an amplified cDNA probe with degenerate oligonucleotide primers designed from regions conserved in the serine/threonine protein kinase catalytic domains (10). They were termed rac kinases (related to the A and C kinases) (9) or PKB (for Protein Kinase B most similar to the PKC/PKA families) (10). ...
... The latter did so from human fibroblast using an amplified cDNA probe with degenerate oligonucleotide primers designed from regions conserved in the serine/threonine protein kinase catalytic domains (10). They were termed rac kinases (related to the A and C kinases) (9) or PKB (for Protein Kinase B most similar to the PKC/PKA families) (10). ...
... Knowledge on kinase expression in all life stages relevant for drug targeting is desirable, as well as a reliable quantification of expression during in vitro culture, for instance as part of knockdown experiments. Therefore, we identified orthologs of above mentioned kinases by BLASTp search ( Table 2): Fhabl1 and Fhabl2 as orthologs of the protein tyrosine kinases abl1 and abl2, which play roles in a variety of cellular processes including cell differentiation and cytoskeletal rearrangements 31 ; Fhakt1 and Fhpkc as orthologs of the serine/threonine-protein kinases B and C, which are known to regulate many processes including cell metabolism, proliferation, and survival 32,33 ; Fhplk1 as an ortholog of polo-like kinase 1 with important roles during cell cycle progression 34 . The presence of conserved protein domains was confirmed by SMART analysis (see Supplementary Fig. S4). ...
... Opposite to Fhplk1, orthologs of the two Abl kinases and two serine/threonine-protein kinases showed a peak of expression in NEJ. In other organisms, these kinases are amongst others involved in cytoskeleton remodeling in response to extracellular stimuli such as growth factors, and in the regulation of cell metabolism [31][32][33][34] . Thus, these kinases might play important roles during early growth and development of flukes, which still has to be substantiated in functional studies in the future. ...
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The liver fluke Fasciola hepatica causes fasciolosis, a foodborne zoonosis affecting humans and livestock worldwide. A reliable quantification of gene expression in all parasite life stages relevant for targeting by anthelmintics in the mammalian host is fundamental. The aim of this study was to define a set of stably expressed reference genes for qRT-PCR in Fasciola studies. We determined the expression stabilities of eight candidate reference genes by the algorithms NormFinder, geNorm, BestKeeper, and comparative ΔCT method. The most stably expressed reference genes for the comparison of intra-mammalian life stages were glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). The two best reference genes for analysis of in vitro-cultured juveniles were Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7). These genes should replace the housekeeping gene gapdh which is used in most Fasciola studies to date, but in fact was differentially expressed in our analysis. Based on the new reference genes, we quantified expression of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) discussed as targets in other parasitic flatworms. Distinct expression patterns throughout development were revealed and point to interesting biological functions. We like to motivate using this set of validated reference genes for future F. hepatica research, such as studies on drug targets or parasite development.
... At a similar time, the high frequency of adenomatous polyposis coli and ␤-catenin mutations in colorectal cancer cemented the fields of cancer and Wnt signaling. In another twist, Paul Coffer, my first graduate student, and Brian Hemmings independently cloned a novel protein kinase we termed protein kinase B because it was similarly related by sequence to protein kinases A and C (Coffer et al. 1991;Jones et al. 1991). Brian Hemmings and Philip Cohen later showed that PKB phosphorylated GSK-3 at an inhibitory residue, and since PKB has been shown to be activated by the product of phosphatidylinositol 3= kinase that is induced by stimuli in-cluding insulin, a compelling mechanism by which insulin could act to reduce glycogen synthase phosphorylation was assembled (Fig. 2) (Cross et al. 1995). ...
Article
The strength of the scientific process is its immunity from human frailties. The built-in error correction and robustness of principles protect and nurture truth, despite both intended and unintended errors and naivety. What it doesn't secure is understanding of how the scientific sausage is made. Here, a scientific journey revolving around a single protein that spans nearly 35 years is used to illustrate the twists and turns that can accompany any scientific path. Lessons learned from such exploration speak to the need for story-telling in communicating scientific meaning - and the effectiveness of this will influence future investment and understanding of the scientific endeavor.
... Several studies have reported on the role of PI3K as a pro-survival signaling molecule that can be activated by growth factors [81]. Upon activation, PI3K phosphorylates AKT in a serine/threonine residue, thereby activating the PI3K/AKT pathway [82][83][84][85]. The phosphorylated AKT subsequently inactivates Bad, a pro-apoptotic element of the Bcl-2 group [86]. ...
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Embelin is a naturally-occurring benzoquinone compound that has been shown to possess many biological properties relevant to human cancer prevention and treatment, and increasing evidence indicates that embelin may modulate various characteristic hallmarks of tumor cells. This review summarizes the information related to the various oncogenic pathways that mediate embelin-induced cell death in multiple cancer cells. The mechanisms of the action of embelin are numerous, and most of them induce apoptotic cell death that may be intrinsic or extrinsic, and modulate the NF-κB, p53, PI3K/AKT, and STAT3 signaling pathways. Embelin also induces autophagy in cancer cells; however, these autophagic cell-death mechanisms of embelin have been less reported than the apoptotic ones. Recently, several autophagy-inducing agents have been used in the treatment of different human cancers, although they require further exploration before being transferred from the bench to the clinic. Therefore, embelin could be used as a potential agent for cancer therapy.
... The signaling downstream of PI3Ks and the generation of PI(3,4,5)P 3 is mediated through the recruitment of Pleckstrin homology (PH)-domain containing proteins which can bind to PI(3,4,5)P 3 [10,11]. Among PH-containing proteins, Akt, which was first discovered as the cellular homolog of the transforming retroviral oncogene v-Akt [12,13], represents the canonical signaling pathway downstream of PI3Ks [14]. Akt is recruited to the membrane by its PH domain following PI3K activation where it is activated by phosphorylation on two residues: T308 in the kinase domain is phosphorylated by another PH-containing protein, phosphoinositide-dependent kinase 1 (PDK1) [15], and S473 in the hydrophobic motif which is phosphorylated by the mammalian target of Rapamycin complex 2 (mTORC2) [16]. ...
Article
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Phosphoinositide 3-kinases (PI3Ks) are central regulators of cellular responses to extracellular stimuli, and are involved in growth, proliferation, migration, and metabolism. The Class I PI3Ks are activated by Receptor Tyrosine Kinases (RTKs) or G Protein-Coupled Receptors (GPCRs), and their signaling is commonly deregulated in disease conditions. Among the class I PI3Ks, the p110β isoform is unique in being activated by both RTKs and GPCRs, and its ability to bind Rho-GTPases and Rab5. Recent studies have characterized these p110β interacting partners, defining the binding mechanisms and regulation, and thus provide insight into the function of this kinase in physiology and disease. This review summarizes the developments in p110β research, focusing on the interacting partners and their role in p110β-mediated signaling.
... Akt was originally found from v-Akt oncogene in the Akt8 transforming retrovirus [8], and the cellular homolog of v-Akt was cloned [9]. It was named protein kinase B (PKB), as it had a similarity to PKA and PKC [10]. As a serine/threonine kinase, Akt is activated by external signals through membrane receptor activation [11]. ...
Article
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Keratin 8 and keratin 18 (K8/K18) are intermediate filament proteins that form the obligate heteropolymers in hepatocytes and protect the liver against toxins. The mechanisms of protection include the regulation of signaling pathway associated with cell survival. Previous studies show K8/K18 binding with Akt, which is a well-known protein kinase involved in the cell survival signaling pathway. However, the role of K8/K18 in the Akt signaling pathway is unclear. In this study, we found that K8/K18-Akt binding is downregulated by K8/K18 phosphorylation, specifically phosphorylation of K18 ser7/34/53 residues, whereas the binding is upregulated by K8 gly-62-cys mutation. K8/K18 expression in cultured cell system tends to enhance the stability of the Akt protein. A comparison of the Akt signaling pathway in a mouse system with liver damage shows that the pathway is downregulated in K18-null mice compared with nontransgenic mice. K18-null mice with Fas-induced liver damage show enhanced apoptosis combined with the downregulation of the Akt signaling pathway, i.e., lower phosphorylation levels of GSK3β and NFκB, which are the downstream signaling factors in the Akt signaling pathway, in K18-null mice compared with the control mice. Our study indicates that K8/K18 expression protects mice from liver damage by participating in enhancing the Akt signaling pathway.
... To date, the protein is most commonly referred to as Akt or PKB (protein kinase B). Akt/PKB is the prototype of a family of kinases that includes three known members, Akt1/PKBα [2,4,5], Akt2/PKBβ [3,6] and Akt3/PKBγ [7]. These have ∼80% amino acid sequence homology and share a common domain structure, with an N-terminal PH domain, a central kinase domain and a C-terminal regulatory domain [8] ( Figure 1). ...
Article
The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase.
... In response to glucagon, for example, PKA reprograms the entire metabolic profile of a cell such as the liver turning off glycolysis and turning on gluconeogenesis. Later the insulin receptor was found to also be a protein kinase (8), and this was followed by Akt (PKB) (30)(31)(32) and AMPK (33). All reinforce the importance of dynamic protein phosphorylation as one of the fundamental challenges for physiology and metabolism and one of the most important questions for diabetes, which has become an epidemic in this country. ...
Article
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In recognition of the first protein kinase structure that was solved 25 years ago, we review the history of the Structural Kinome. What did we learn prior to that first structure of the PKA catalytic subunit, what have we learned since the structure was solved, and what are our remaining challenges for the future?
... This 58 kDa serine/threonine kinase was identified through molecular cloning by three independent groups and was named PKB (due to homology to PKA and PKC kinase domains), c-Akt and RAC (for Related to A and C kinase) (Bellacosa, etal., 1991, Coffer and Woodgett, 1991, Jones, etal., 1991. It is the cellular homologue v-Akt, the product of the AKT-8 transforming retrovirus. ...
Thesis
Protein phosphorylation and its control by the action of protein kinases and phosphatases has been shown to play an essential role in signal transduction pathways and hence cell functions. More recent discoveries have implicated GTP hydrolysing enzymes and lipids; adding to the depth of complexity required for the balance and amplification of signals. The Protein Kinase C (PKC) family of kinases can be subdivided based on their requirement for Ca2+, Diacylglycerol (DAG) and phosphor-lipids. Previously, a PCR-based screen for novel PKC isoforms produced two new gene products, which were subsequently fully cloned: Protein Kinase C-Related Kinases (PRK 1 and 2). These were seen to be highly homologous to the PKCs in their kinase domains but had novel N-terminal regulatory regions. Their serine/threonine kinase activity was insensitive to both Ca2+ and DAG but was activated in vitro by limited proteolysis and polyphosphoinositides Ptdlns(4,5)P2 and Ptdlns(3,4,5)P3. The aim of this thesis was to investigate the control of the PRKs. Initial work focused on the potential interaction between the PRKs and Small GTPases of the Rho family, and assessed what role such an interaction would have on the kinase's activity. A variety of in vitro techniques were used to characterise the site of contact on both the PRKs and the Rho proteins. The nucleotide-specificity of binding was also analysed. The definition of the Rho binding region within the PRKs novel regulatory region (Homology Region 1) suggested the GTPase disrupted an intramolecular, pseudo-substrate site interaction with the kinase domain. However, our data showed only a two-fold increase in the PRK's kinase activity when co-expressed in cultured cells with an active Rho. Nevertheless we were able to show translocation of PRK to early endosomes with RhoB overexpression and further that a hyperphosphorylated form of the kinase existed within this sub-cellular compartment. This suggested that other components were involved in the control of the kinase. On consideration of the similarities between members of the AGO kinase subfamilies within the activation-loop, the involvement of the upstream kinase PDK1 in PRK activation was assessed. Co-expression studies in cultured cells allowed the definition of a Rho-dependent interaction between the two kinases and a three-way co-localisation of PRK, RhoB and PDK1 on early endosomes. An activation loop phosphorylation event was analysed using phospho-specific polyclonal antisera. In vitro and in vivo studies using both wild type and mutant PRKs demonstrated that this phosphorylation event was necessary for kinase activity. It has thus been shown that both Rho family GTPases and PDK kinases, which depend on the products of PI3-Kinase activity, are required for the cellular localisation and activation of the PRK sub-family of kinases.
... Akt is a serine/threonine protein kinase originally identified as a cellular homolog of the viral oncogene Akt8 (2). The three isoforms of Akt (Akt1/PKB␣, Akt2/PKB␤, Akt3/PKB␥) share a high degree of structural similarity and sequence homology (3)(4)(5)(6)(7)(8). It also appears that each isoform may play unique as well as common roles in cells (9 -12); Akt1 knock-out mice are growth-retarded (9), Akt2 knock-out mice develop diabeteslike symptoms because of the impaired insulin response (10,12), and Akt3 knock-out mice show reduced brain size (11). ...
Article
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The serine/threonine kinases Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ have been implicated in preventing cells from undergoing apoptosis. Although several small molecule inhibitors of Akt have been reported to induce apoptosis in cancer cells, these inhibitors may have additional targets. In the current study, we used an Akt3 small interfering RNA (Akt3 siRNA) to analyze apoptosis induction in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells (MEF-Akt1,2-DKO). Our data indicated that Akt3 siRNA inhibited Akt3 protein expression in a dose-dependent manner. As a result, phosphorylation of Akt and its downstream targets, including FKHRL1 and GSK3α/β, were reduced accordingly. The treatment also induced apoptosis in MEF-Akt1,2-DKO cells. However, apoptosis induction is significant only when more than 80% of Akt3 protein was depleted. Reintroducing Akt3 totally rescued Akt3-siRNA-induced apoptosis in MEF-Akt1,2-DKO cells. In addition, reintroducing Akt1 also inhibited apoptosis induced by Akt3 siRNA. Moreover, Akt3 siRNA potentiated different stress-induced apoptosis in MEF-Akt1,2-DKO cells at a lower dose when compared with what is required for apoptosis induction by itself. Our study suggests that only a small portion of Akt is active in wild-type MEF cells and a threshold of Akt inhibition is required to induce apoptosis by pure Akt inhibitors. In addition, our data indicate that cells under stress require more Akt for its survival.
... Protein Kinase B PKB a (protein kinase Ba) (Coffer & Woodgett, 1991), also known as c-Akt (BeUacosa et a l, 1991) and RACa (Jones et a l, 1991) is the cellular homologue of the transforming v-Akt (Staal et a l, 1977). It is a serine/threonine kinase which is activated within one minute of stimulating cells with insulin (Cross et a l, 1995;Kohn et a l, 1995;Moule et a l, 1997). ...
Thesis
Malarial extracts (MEs) derived from the erythrocytic stages of Plasmodium infection in mice have been shown to synergise with insulin in both stimulating lipogenesis and inhibiting lipolysis in rat adipocytes in vitro. In this study, rat adipocytes were used as a test system in an attempt to determine where in the insulin signalling network these malarial extracts were exerting their effects. Initial studies demonstrated that the MEs had no effect on insulin receptor binding or internalisation, nor did they enhance insulin stimulated 3-O-methylglucose uptake. Investigation of their effects on the activity of the downstream enzymes involved in lipogenesis showed that they had no effect on the stimulation of pyruvate dehydrogenase by insulin. They did however potentiate insulin's stimulation of the committal enzyme in fatty acid synthesis, acetyl-CoA carboxylase (ACC), without changing its total activity. The MEs were also shown to synergise with insulin in the activation of glycogen synthesis. They appeared to exert this effect by enhancing the insulin stimulation of glycogen synthase, having no effect on the inactivation of glycogen phosphorylase activity by insulin. Neither epidermal growth factor nor isoproterenol had any effect on the ability of the MEs to synergise with insulin and enhance glycogen synthesis and the MEs did not induce EGF to stimulate glycogen synthesis. Isoproterenol did however block any insulin-mimetic effect of the MEs. The MEs synergistically enhanced the effect of insulin to increase anti- phosphotyrosine immunoprecipitatable phosphatidylinositol 3-kinase (PI3-kinase) activity. Since both ACC and glycogen synthase have been shown to be stimulated by insulin through a PI3-kinase dependent pathway, these results suggest that the ME's effects on PI3-kinase may be a key step in their synergistic enhancement of insulin signalling. As well as synergising with insulin, some of the ME preparations were also able to stimulate the production of tumour necrosis factor (TNF) by isolated macrophages. However there was no correlation between these two properties of the MEs. There was also no correlation between the synergistic effects of the MEs and their ability to mimic insulin. While these results were very interesting it should be noted that the malarial extracts used in this project were specifically chosen for their ability to synergise with insulin. Of the total number of malarial extracts prepared only approximately 40% actually demonstrated this ability.
... The reason being it has a catalytic domain similar to PKA and PKC (Coffer & Woodgett, 1991). ...
Thesis
Three skeletal muscle fibre types (type I, type IIa and type IIb) were assayed for the presence of the following enzymes ATP-citrate lyase, citrate-dependent acetyl-CoA carboxylase, fatty acid synthase and malonyl-CoA decarboxylase. All activities were present in each of the muscle fibre types studied. A cytosolic activity of malonyl-CoA decarboxylase would appear to offer a feasible route for the disposal of malonyl-CoA in skeletal muscle. Rat soleus muscle strips were incubated with 5mM glucose followed by measurements of tissue contents of malonyl-CoA, long chain fatty acyl-CoA and carnitine esters. Alternatively muscle strips were incubated with 5mM glucose and 0.2mM palmitate followed by measurements of [14C] palmitate CO2 formation from exogenous palmitate or from fatty acids released from prelabelled glycerolipids. Etomoxir at high concentrations (150µM) significantly decreased the malonyl-CoA content by 50% and at low concentrations (50 & 100µM) had no effect on malonyl-CoA. Etomoxir had no effect on the total long chain ester pool but significantly increased long chain acyl-CoA and decreased the ratio of acyl-carnitine/acyl-CoA suggesting that such changes could be diagnostic for inhibition of CPT 1. Insulin and DC A (3mM) increased both malonyl-CoA and long chain fatty acyl-CoA content and decreased the ratio of fatty acylcarnitine/acyl-CoA and β-oxidation. Isoprenaline and palmitate (0.5mM) opposed the effects of insulin, by decreasing the contents of malonyl-CoA and long chain fatty acyl-CoA, increasing the ratio of fatty acylcarnitine/ acyl-CoA and increasing β-oxidation. These findings are consistent with the notion that all these agents can cause acute regulation of CPT 1 in type I skeletal muscle. In the presence of 5-amino-4-imidazolecarboxamide ribonucloeside (AICAR) (1mM) the malonyl-CoA content decreased by 65% and decreased the content of both long chain esters significantly, in particular long chain acyl-CoA. Oxidation of 14C-labelled exogenous and endogenous fatty acid was measured in soleus muscle strips incubated with insulin as mentioned above. Isoprenaline (10-7M) increased both processes (28% and 103% respectively). Indicating that isoprenaline has a lipolytic effect. AICAR (1mM) increased oxidation of exogenous fatty acid by 102% but had no effect on endogenous oxidation. It is therefore concluded that AICAR causes a decrease in lipolysis in muscle. Similar experiments led to the suggestion that dichloroacetate (3mM) had a lipolytic effect in muscle.
... Une autre dénomination de la protéine AKT est celle de protéine kinase B (PKB) puisque dans les mêmes années, deux autres groupes reconnurent des séquences apparentées aux protéines kinases A et C dans les ADNc codant AKT (Coffer and Woodgett, 1991;Jones et al., 1991). ...
Thesis
La protéine AKT1, appelée aussi protéine kinase B ou PKB, est un composant central de la voie de signalisation PI3K/AKT/mTOR qui représente une des voies majoritaires des cellules régulant un nombre varié de processus biologiques tels que la prolifération cellulaire, l’apoptose, la survie cellulaire ainsi que l’angiogenèse parmi d’autres. Sa nature de serine-thréonine kinase lui confère la capacité de phosphoryler une multitude de substrats, aussi impliqués dans d’autres voies de signalisation et créant ainsi un réseau de connexion très vaste avec comme finalité le bon fonctionnement cellulaire. Une faille dans le système de régulation d’un ou de plusieurs composants de la voie de signalisation PI3K/AKT/mTOR provoque l’apparition de cancers. C’est le cas de la protéine AKT1 qui est fréquemment dérégulée dans les cellules cancéreuses, pouvant être mutée, délétée ou surexprimée selon les cas.Les voies de signalisation ne sont pas les seuls événements assurant l’homéostasie cellulaire. De nombreux systèmes de surveillance sont présents, de la transcription de l’ADN jusqu’à la synthèse en protéine. Un des mécanismes de contrôle-qualité est le Nonsense-mediated mRNA decay (NMD), qui cible spécifiquement les ARNm porteurs d’un codon stop prématuré (PTC) afin d ’empêcher la synthèse de protéines tronquées qui pourraient avoir des effets néfastes pour la cellule, garder une partie ou la totalité de leur activité ou tout simplement être non fonctionnelles. Les PTC sont retrouvés dans les séquences géniques responsables d’un tiers des maladies génétiques ainsi que de nombreuses formes de cancer, mais le NMD est aussi capable de réguler l’expression physiologique de différents ARNm.Le NMD fait intervenir une vingtaine de facteurs protéiques dont les protéines up-frameshift, UPF1, UPF2 et UPF3X. Les facteurs UPF1 et UPF2 sont des phosphoprotéines qui jouent un rôle central dans l’activation du NMD, et en particulier la protéine UPF1 qui doit être phosphorylée et déphosphorylée afin d'induire la dégradation d'un ARNm par le NMD. La seule kinase identifiée aujourd’hui dans le NMD est la protéine SMG1, une PI3-kinase related kinase qui phosphoryle le facteur UPF1. La probabilité que d’autres protéines kinases soient impliquées dans le NMD est par conséquent élevée.Afin d’identifier de nouvelles protéines jouant un rôle dans le NMD, le criblage d’une banque d'inhibiteurs de kinases sur une construction luciférase sujette à une dégradation par le NMD nous a permis de découvrir trois inhibiteurs montrant une très forte capacité à inhiber le NMD. De manière intéressante, ces derniers ont tous les trois pour cible la protéine AKT1.Nous avons ainsi entrepris d’étudier l’implication et l’éventuelle activité de la protéine AKT1 dans le mécanisme de NMD. La réalisation d’expériences capables de déterminer la possible intervention d’AKT1 dans le NMD a permis la validation des résultats obtenus lors du crible. Les interactions protéiques entre AKT1 et les différents facteurs du NMD ont été examinées et les données obtenues montrent que la kinase AKT1 est capable d’interagir avec deux facteurs essentiels du NMD, les protéines UPF1 et UPF3X.L'implication de la protéine AKT1 dans le NMD suggère une régulation de ce dernier par une nouvelle kinase et une efficacité modulée dans les cancers ou dans des situations spécifiques en fonction du niveau d'expression de la protéine AKT1 mais aussi des facteurs du NMD. Cette nouvelle kinase identifiée dans le NMD pourrait ouvrir de nouvelles possibilités de stratégies thérapeutiques dans des pathologies telles que le cancer.
Chapter
The PI3K/PTEN/AKT/mTOR signaling axis has been intensively studied in many cancer systems. Current evidence suggests deregulation of this pathway plays a unique role in the initiation, development, and recurrence of head and neck squamous cell carcinoma (HNSCC). A heterogeneous disease by nature, HNSCC encompasses a disparate collection of anatomical sites with complex tumor biology. Yet, PI3K/PTEN/AKT/mTOR signaling has an intimate role in nearly every facet of this disease. In this chapter, we will provide a brief introduction to the mechanisms involved in PI3K/PTEN/AKT/mTOR signaling and how specific alterations in these signaling nodes enable HNSCC development. We will also discuss differences in PI3K/PTEN/AKT/mTOR signaling with respect to HPV status. A number of inhibitors targeting multiple nodes in this pathway have been developed as agents have broad application across many cancer types. We will briefly review how these therapeutic agents are being evaluated and what predictive biomarkers have been established in HNSCC for these drugs. Finally, PI3K/PTEN/AKT/mTOR signaling represents an important source of resistance to radiation, chemotherapy, and other targeted agents. We will also speculate on how PI3K/PTEN/AKT/mTOR inhibitors may increase the efficacy of these established therapies. Although PI3K/PTEN/AKT/mTOR investigations are relatively new to HNSCC research, early evidence suggests further evaluation of this essential signal transduction pathway is warranted.
Chapter
Akt is the main constituent of the PI3K/mTOR pathway that plays a crucial role in cell survival, proliferation, growth, and metabolism. The Akt signaling pathway is the most commonly altered pathway network in several human cancers. The deregulation of the Akt pathway contributes an essential direction for therapeutic cancer targets. Targeting the Akt pathway represents a pretty treatment choice for many cancer types, particularly those resistant to chemotherapy treatments. Targeted tumor therapies are utilized to block the progression, growth, metastasis, and survival of the tumor through interfering with unique targets that are presently the center of antitumor drug development. The finding process of inhibitors of Akt by multiple strategies has guided the detection of inhibitors with huge selectivity. Several drugs and agents targeting Akt/PI3K pathway are in various phases of clinical studies. Many Akt inhibitors have been presently developed and broadly tested in phase I and phase II clinical trial studies either alone or with chemotherapy. In this chapter, we discuss the role of the Akt pathway in several cancers and target Akt for cancer therapy. Furthermore, summarize the details concerning synthetic and natural Akt targeting inhibitors and their links with clinical studies.
Chapter
The phosphorylation of signal transduction molecules is of central importance for growth factor-induced transduction of mitogenic signals, cell growth and differentiation. Stimulation of growth factor-receptor uses by their ligands can activate several signalling modules depending on the cell type. Activation of receptor tyrosine tease cascades can be either independent of a second messenger system or dependent on a second messenger.
Chapter
The chapter discusses the RAC (Related to PKA and PKC) PK subfamily that was initially identified by homology cloning. Further work has established that an oncogenic form (v-akt) exists, resulting from the addition of a truncated gag protein 6 bp upstream of the initiator methionine. Sequence analysis of the RAC kinase domain reveals that it is related to the PKA and PKC subfamilies, and to Sgk kinase. Three isoforms of RAC PK have been identified in vertebrates (a, b1, and b2), and a single form in Drosophila melanogaster (DmRAC). The b2 isoform has a different C-terminus to b1, which may arise from alternative splicing. No regulatory subunits have been identified. However, three domains have been identified: an N-terminal pleckstrin homology (PH) domain; a kinase domain closely related to that of PKA and PKC; a C-terminal domain similar to the C-terminal region of the PKC subfamily. Pleckstrin, the major PKC substrate in platelets, contains N- and C-terminal regions of ≈100 amino acids with very similar sequences. PH domains have been identified in a number of other proteins and are proposed to function by promoting protein–protein interactions.
Article
The phosphatidylinositol 3'-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is a highly conserved signaling network, tightly controlled via multistep processes. It plays a pivotal role in the regulation of numerous cellular processes, such as transcription, translation, proliferation, growth, apoptosis, metabolism, motility, and intracellular vesicular transport. This pathway is used by cell-surface receptors to control intracellular events, principally by activating downstream kinases. Indeed, the PI3K/Akt/mTOR pathway is regulated through phosphorylation and dephosphorylation by specific kinases and phosphatases, as well as GTP/GDP exchange proteins, adaptor proteins, and scaffolding proteins.
Chapter
Glucose metabolism plays an important role in hydroperoxide detoxification and the inhibition of glucose metabolism has been shown to increase prooxidant production and cytotoxicity in cancer cells. Increased Akt pathway signaling has been shown to be directly correlated with increased rates of glucose metabolism observed in cancer cells versus normal cells. These observations have led to the proposal that inhibition of Akt signaling would inhibit glycolysis and increase hydroperoxide production which would preferentially kill tumor cells versus normal cells via oxidative stress. The current study shows that inhibition of the Akt pathway inhibits glucose consumption and induces parameters indicative of oxidative stress such as glutathione disulfide (%GSSG) and thioredoxin reductase (TR) activity in human head and neck cancer (HNSCC) cells. A theoretical model to explain the results is presented and implications for the use of Akt pathway inhibitors in combination with glycolytic inhibitors and/or manipulations that increase prooxidant production are discussed.
Article
The protein kinase B (PKB/Akt) is found over-expression in many kinds of cancer. Here Lipofectamin was used to transfect a consistent active form of PKB (gagPKB) into SMMC 7721 cells to study the ability of the influence of this protein on proliferation and apoptosis of human hepatocellular carcinoma SMMC 7721 cells. The stably over expressing PKB/Akt cell line was identified by Northern blot, Western blot and the assay of PKB activity. Over expressing PKB/Akt promoted cell growth in serum culture and anchorage-indpendent growth in agarose with high efficiency. Alternatively, over-expressing PKB/Akt was sufficient to promote the cells into the S phase of the cell cycle and decreased the expression of the cyclin-dependent kinase inhibitor p27 Kip1. Furthermore, over-expression of PKB/Akt suppressed the apoptosis of cells induced by the detachment of the cells from extracelluar matrix. These results suggest the ability of PKB/Akt to promote proliferation and suppress apoptosis in cancer cells.
Article
Hepatitis C virus (HCV) activates PI3K-Akt signaling to enhance entry and replication. Here, we found that this pathway also increased HCV translation. Knocking down the three Akt isoforms significantly decreased, whereas ectopic expression increased HCV translation. HCV translation upregulation by Akt required their kinase activities because Akt kinase-dead mutants downregulated HCV translation; and was dependent on PI3K activity since it was sensitive to PI3K inhibitor wortmannin. The viral 3'UTR was not involved in translation upregulation by Akt. HCV NS5A increased Akt phosphorylation/activity and HCV translation in the absence of the viral 3'UTR. Sterol regulatory element-binding proteins (SREBPs) were the downstream effectors of the PI3K-Akt pathway in regulating HCV translation because Akt1 and Akt2 activated both SREBP-1 and SREBP-2, whereas Akt3 upregulated SREBP-1. Knocking down SREBPs significantly decreased, while ectopic expression of SREBPs increased HCV translation. Taken together, we showed that the PI3K-Akt signaling pathway positively regulates HCV translation through SREBPs.
Article
The serine/threonine kinases, Akti/PKBα, Akt2/PKBβ, and Akt3/PKBγ, play a critical role in preventing cancer cells from undergoing apoptosis. However, the function of individual Akt isoforms in the tumorigenicity of cancer cells is still not well defined. In the current study, we used an AM antisense oligonucleotide (AS) to specifically downregulate Akti protein in both cancer and normal cells. Our data indicate that AM AS treatment inhibits the ability of MiaPaCa-2, H460, HCT-15, and HT1080 cells to grow in soft agar. The treatment also induces apoptosis in these cancer cells as demonstrated by FRCS analysis and a caspase activity assay. Conversely, Akti AS treatment has little effect on the cell growth and survival of normal human cells including normal human fibroblast (NHF), fibroblast from muscle (FBM), and mammary gland epithelial 184135 cells. In addition, AM AS specifically sensitizes cancer cells to typical chemotherapeutic agents. Thus, Akti is indispensable for maintaining the tumorigenicity of cancer cells. Inhibition of AM may provide a powerful sensitization agent for chemotherapy specifically in cancer cells.
Article
This chapter reviews the past and current information on the biosynthesis of mammalian and bacterial glycogen, and of starch in plants. It first discusses the role of these storage polysaccharides and later discusses the biosynthetic reactions involved in the synthesis of the polysaccharides in the above three systems. In plants and in bacteria, the synthesis of starch or glycogen occurs by utilizing adenosine disphosphate (ADP)-glucose as the glucosyl donor for the elongation of α-1,4-glucosidic chains. The first committed step in these pathways is the synthesis of ADP-glucose in a reaction catalyzed by ADP-glucose pyrophosphorylase and the reaction is the regulatory step in plants and in bacteria. The enzyme is allosterically activated by glycolytic intermediates and inhibited by either 5′-adenylate or adenosine diphosphate. In mammals uridine diphosphate glucose is the glucosyl donor and the regulatory step is glycogen synthase. Glycogen synthase activity is regulated allosterically and most important by covalent modification by protein kinases and protein phosphatases that are under hormonal control. A detailed description of the chemical, structural, kinetic, and regulatory properties of the biosynthetic enzymes involved in the three systems, bacterial, plant, and mammalian, is provided.
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Aims: The pleiotropic effects of HMG-CoA reductase inhibitors (statins) independent of cholesterol-lowering effects have attracted much attention. We have recently demonstrated that the pleiotropic effects of statins are partly mediated through up-regulation of small GTP-binding protein dissociation stimulator (SmgGDS) with a resultant Rac1 degradation and reduced oxidative stress. However, it remains to be elucidated what molecular mechanisms are involved. Methods and results: To first determine in what tissue statins up-regulate SmgGDS expression, we examined the effects of 2 statins (atorvastatin 10 mg/kg/day or pravastatin 50 mg/kg/day for 1 week) on SmgGDS expression in mice in vivo. The 2 statins increased SmgGDS expression especially in the aorta. Atorvastatin also increased SmgGDS expression in cultured human umbilical venous endothelial cells (HUVEC) and human aortic endothelial cells (HAoEC), but not in human aortic vascular smooth muscle cells (HAoVSMC). Furthermore, Akt phosphorylation was transiently enhanced only in HUVEC in response to atorvastatin. Then, in order to examine whether Akt is involved for up-regulation of SmgGDS by statins, we knocked out Akt1 by its siRNA in HUVEC, which abolished the effects by atorvastatin to up-regulate SmgGDS. Furthermore, when we knocked down β1-integrin in order to elucidate the upstream molecule of Akt1, the effect of atorvastatin to up-regulate SmgGDS was abolished. Finally, we confirmed that Akt activator, SC79, significantly up-regulate SmgGDS in HUVEC. Conclusions: These results indicate that statins selectively up-regulate SmgGDS in endothelial cells, for which the β1-integrin/Akt1 pathway may be involved, demonstrating the novel aspects of the pleiotropic effects of statins.
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Aim: To investigate the effect of intrathecal injection of PI3K inhibitor LY294002 on pain behaviour and expression of p-Akt in spinal dorsal horns in bone cancer pain(BCP) rats. Methods: Forty female SD rats weighing 180-200g were randomly divided into five groups (n = 8 each): (I) sham group; (H) sham + LY294002 group; (III) BCP group; (IV) BCP + DMSO group; (V) BCP + LY294002 group. BCP rat model was induced by inoculating Walker 256 mammary gland carcinoma cells into the medullary cavity of the left tibia. Rats received i. t. injections of either PI3K inhibitor LY294002 10μX(2.5g · L-1) or 5% DMSO 10 μL at the time of d 7-9 after the operation. Mechanical withdrawal threshold (MWT) test was performed before and after i. t. injections on d7(till 8h). The rats were sacrificed after inoculation and the L4 ∼ 6 segments of the spinal cords were removed for immu-nohistochemistry to determinate the expression changes of spinal p-Akt. Results: Compared with I group, the rats in III, IV, V group showed obvious mechanical hyperalgesia. The MWT of V group increased apparently from 2nd hour to 4th hour(P < 0.05), and reached the peak in 3rd hour(P < 0.01). Compared with I group, the expression of p-Akt in the spinal cord in III, IV group increased obviously (P < 0.01). Compared with III, IV group, i. t. injections of LY294002 obviously cut down the expression of p-Akt in the spinal cord (P < 0.05). Conclusion: PI3K/Akt singaling pathway may take part in the development of bone cancer pain.
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Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.
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Since its discovery more than 25 years ago, the kinase AKT has become a central figure in cell signaling. We highlight some of the landmark findings in those 25 years that contributed to our understanding of the regulation and function of AKT in directing cellular processes and behavior. Future progress toward fully understanding the roles of AKT in cell, tissue, and organismal biology will depend on technological innovations and the combination of in-depth reductionist analyses with systems-based strategies.
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Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of cold hardiness to survive the winter. Here we report that protein kinase-dependent signal transduction featuring mitogen-activated protein kinase (MAPK) signalling cascades (extracellular signal regulated kinase, c-jun N-terminal kinase and p38 MAPK pathways) and the Akt (also known as protein kinase B, or PKB) pathway could be integral parts of the development of cold hardiness by E. scudderiana. We used Luminex technology to assess the protein levels and phosphorylation status of key components and downstream targets of those pathways in larvae in response to low temperature acclimation. The data showed that MAPK pathways (both total protein and phosphorylated MAPK targets) were inhibited after 5°C acclimation, but not −15°C exposure, as compared with the 15°C control group. However, total heat shock protein 27 (HSP27) levels increased dramatically by ∼12-fold in the −15°C acclimated insects. Elevated HSP27 may facilitate anti-apoptotic mechanisms in an Akt-dependent fashion. By contrast, both 5 and −15°C acclimation produced signs of Akt pathway activation. In particular, the inhibitor phosphorylated Glycogen Synthase Kinase 3a (p-GSK3) levels remained high in cold-exposed larvae. Additionally, activation of the Akt pathway might also facilitate inhibition of apoptosis independently of GSK3. Overall, the current study indicates that both MAPK and Akt signal transduction may play essential roles in freeze avoidance by E. scudderiana.
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At present, diseases such as obesity, type Ⅱ diabetes and cancer have brought serious health problems, which are closely related to mTOR pathway. 70 kDa ribosomal protein S6 kinase (p70S6K), as a significant downstream effector of mTOR, mediates protein synthesis, RNA processing, glucose homeostasis, cell growth and apoptosis. Inhibiting the function of p70S6K can reduce the risk of obesity which helps to treat dyslipidemia, enhance insulin sensitivity, and extend the life span of mammals. Therefore, p70S6K has become a potential target for the treatment of these diseases. So far, except for the first p70S6K specific inhibitor PF-4708671 developed by Pfizer and LY2584702 developed by Lilai, all of them are in preclinical research. This paper briefly introduces the general situation of p70S6K and reviews their inhibitors in recent years, which are mainly classified into two categories: natural compounds and synthetic compounds. In particular, their inhibitory activities, structure-activity relationships (SARs) and mechanisms are highlighted.
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Interferon regulatory factor 3 (IRF3)-induced type I interferon (I-IFN) production plays key roles in both antiviral and autoimmune responses. IRF3 phosphorylation, dimerization, and nuclear localization are needed for its activation and function, but the precise regulatory mechanisms remain to be explored. Here, we show that the serine/threonine kinase AKT2 interacts with IRF3 and phosphorylates it on Thr207, thereby attenuating IRF3 nuclear translocation in a 14-3-3ε-dependent manner and reducing I-IFN production. We further find that AKT2 expression is downregulated in viral-infected macrophages or in monocytes and tissue samples from systemic lupus erythematosus (SLE) patients and mouse models. Akt2-deficient mice exhibit increased I-IFN induction and reduced mortality in response to viral infection, but aggravated severity of SLE. Overexpression of AKT2 kinase-inactive or IRF3-T207A mutants in zebrafish supports that AKT2 negatively regulates I-IFN production and antiviral response in a kinase-dependent manner. This negative role of AKT2 in IRF3-induced I-IFN production suggests that AKT2 may be therapeutically targeted to differentially regulate antiviral infection and SLE.
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Akt, also known as protein kinase B (PKB), belongs to the AGC family of protein kinases. It acts downstream of the phosphatidylinositol 3-kinase (PI3K) and regulates diverse cellular processes, including cell proliferation, cell survival, metabolism, tumor growth and metastasis. The PI3K/Akt signaling pathway is frequently deregulated in breast cancer and plays an important role in the development and progression of breast cancer. There are three closely related members in the Akt family, namely Akt1(PKBα), Akt2(PKBβ) and Akt3(PKBγ). Although Akt isoforms share similar structures, they exhibit redundant, distinct as well as opposite functions. While the Akt signaling pathway is an important target for cancer therapy, an understanding of the isoform-specific function of Akt is critical to effectively target this pathway. However, our perception regarding how Akt isoforms contribute to the genesis and progression of breast cancer changes as we gain new knowledge. The purpose of this review article is to analyze current literatures on distinct functions of Akt isoforms in breast cancer.
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PI3K-AKT/mTOR signaling pathway represents an essential signaling mechanism for mammalian enzyme-related receptors in transducing signals or biological processes such as cell development, differentiation, cell survival, protein synthesis, and metabolism. Upregulation of the PI3K-AKT/mTOR signaling pathway involves many human brain abnormalities, including autism and other neurological dysfunctions. Autism is a neurodevelopmental disorder associated with behavior and psychiatric illness. This research-based review discusses the functional relationship between the neuropathogenic factors associated with PI3K-AKT/mTOR signaling pathway. Ultimately causes autism-like conditions associated with genetic alterations, neuronal apoptosis, mitochondrial dysfunction, and neuroinflammation. Therefore, inhibition of the PI3K-AKT/mTOR signaling pathway may have an effective therapeutic value for autism treatment. The current review also summarizes the involvement of PI3K-AKT/mTOR signaling pathway inhibitors in the treatment of autism and other neurodegenerative disorders.
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Skin cancer is more prevalent than any other cancer in the United States. Non-melanoma skin cancers are the more common forms of skin cancer that affect individuals. The development of squamous cell carcinoma, the second most common type of skin cancer, can be stimulated by exposure of environmental carcinogens, such as chemical toxicants or UVB. It is developed by three distinct stages: initiation, promotion, and progression. During the initiation, the fate of DNA-damaged skin cells is determined by the homeostatic regulation of pro-apoptotic and anti-apoptotic signaling pathways. The imbalance or disruption of either signaling will lead to the survival of initiated cells, resulting in the development of skin cancer. In this chapter, we will discuss signaling pathways that regulate apoptosis and the impact of their dysfunction during skin tumor initiation.
Chapter
The chapter focuses on five major groups of targets that have been the object of particularly vigorous drug-discovery research over the past decade, which include the erbB family of growth factor receptors, the ras/MAPK pathway components, src family kinases, the AKT family, and the nuclear hormone receptor family. The erbB receptor family comprises four distinct membrane glycoproteins consisting of epidermal growth factor (EGF) receptors erbB1, erbB2, erbB3, and erbB4. They are widely expressed in epithelial, mesenchymal, and neuronal tissues, and are structurally similar. They are composed of an extracellular ligand-binding domain, a single transmembrane region, an intracellular domain possessing protein tyrosine kinase activity, and a C-terminal tail that contains specific tyrosine containing sequences, which, upon phosphorylation, become binding sites for src homology region 2 (SH2)–containing signaling proteins. The receptors also contain other phosphorylation sites that are believed to have a role in the regulation and function of the protein. Another family of tyrosine kinases that may contain appropriate targets for therapeutic intervention in cancer is the nonreceptor, cytoplasmic c-Src kinase, and other members of that kinase family. The amino acid sequence of this kinase family is composed of three major structural domains numbered from the C terminus. The first domain is the Src Homology1, or SH1, which encompasses the catalytic domain of the kinase. The SH2 domain, which contains tyrosine phosphorylation sites, and the SH3 domain containing proline-rich regions are likely to be involved in regulatory functions via intra and intermolecular interactions. Akt is a critical mediator of cell survival, and activated Akt protects cells from apoptosis induced by ultraviolet radiation, ionizing radiation, GF withdrawal, detachment from ECM, and cell cycle irregularities.
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Cancer is a devastating disease that has plagued humans from ancient times to this day. After decades of slow research progress, promising drug development, and the identification of new targets, the war on cancer was launched in 1972. The P13K/Akt pathway is a growth-regulating cellular signaling pathway, which in many human cancers is over-activated. Studies have demonstrated that a decrease in Akt phosphorylation by Akt inhibitors is associated with a reduction in tumor cell proliferation. There have been several promising drug candidates that have been studied, including but not limited to ipatasertib (RG7440), 1; afuresertib (GSK2110183), 2; uprosertib (GSK2141795), 3; capivasertib (AZD5363), 4; which reportedly bind to the ATP active site and inhibit Akt activity, thus exerting cytotoxic and antiproliferative activities against human cancer cells. For most of the Akt inhibitors listed, preclinical data reveals robust mechanistic rationales evaluating their effects in various cancer types with hyperactivated Akt signaling. Allosteric inhibitors are also known to alter the activity of kinases. Perifosine (KRX-0401), 5, an alkylphospholipid, is known as the first allosteric Akt inhibitor to enter clinical development and is mechanistically characterized as a PH-domain dependent inhibitor, non-competitive with ATP. This results in a reduction in Akt enzymatic and cellular activities. Other small molecule (MK-2206, 6, PHT‐427, Akti‐1/2) inhibitors with a similar mechanism of action, alter Akt activity through the suppression of cell growth mediated by inhibition of Akt membrane localization and subsequent activation. The natural product solenopsin has been identified as a downstream inhibitor of Akt. A few promising solenopsin derivatives have emerged through pharmacophore modeling, energy-based calculations, and property predictions.
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Insulin signaling is initiated by the activation of the insulin receptor (IR) through autophosphorylation of the tyrosine residue in the IR, and then many signaling molecules including IR substrate 1 and 2, phosphoinositide‐3‐kinase (PI3K) and AKT/protein kinase B (PKB) are involved to modulate downstream signaling pathways. This chapter focuses on the role of PI3K and AKT/PKB in insulin signaling related to pathophysiology in type 2 diabetes mellitus, non‐alcoholic fatty liver disease, and liver cancer. To understand the physiologic role of the signaling molecules in insulin action and glucose homeostasis, the chapter reviews the biochemical characteristics of the PI3K and AKT kinases. The secretion of insulin and glucagon are tightly regulated to modulate gluconeogenesis and lipogenesis in hepatocytes, and dysregulation of the proximal signaling pathways of the ligands results in the hyperglycemia and hyperlipidemia seen in metabolic diseases.
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Background: AKT, also known as protein kinase B, is a key element of the PI3K/AKT signaling pathway. Moreover, AKT regulates the hallmarks of cancer, e.g. tumor growth, survival and invasiveness of tumor cells. After AKT was discovered in the early 1990s, further studies revealed that there are three different AKT isoforms, namely AKT1, AKT2 and AKT3. Despite their high similarity of 80%, the distinct AKT isoforms exert non-redundant, partly even opposing effects under physiological and pathological conditions. Breast cancer as the most common cancer entity in women, frequently shows alterations of the PI3K/AKT signaling. Main content: A plethora of studies addressed the impact of AKT isoforms on tumor growth, metastasis and angiogenesis of breast cancer as well as on therapy response and overall survival in patients. Therefore, this review aimed to give a comprehensive overview about the isoform-specific effects of AKT in breast cancer and to summarize known downstream and upstream mechanisms. Taking account of conflicting findings among the studies, the majority of the studies reported a tumor initiating role of AKT1, whereas AKT2 is mainly responsible for tumor progression and metastasis. In detail, AKT1 increases cell proliferation through cell cycle proteins like p21, p27 and cyclin D1 and impairs apoptosis e.g. via p53. On the downside AKT1 decreases migration of breast cancer cells, for instance by regulating TSC2, palladin and EMT-proteins. However, AKT2 promotes migration and invasion most notably through regulation of β-integrins, EMT-proteins and F-actin. Whilst AKT3 is associated with a negative ER-status, findings about the role of AKT3 in regulation of the key properties of breast cancer are sparse. Accordingly, AKT1 is mutated and AKT2 is amplified in some cases of breast cancer and AKT isoforms are associated with overall survival and therapy response in an isoform-specific manner. Conclusions: Although there are several discussed hypotheses how isoform specificity is achieved, the mechanisms behind the isoform-specific effects remain mostly unrevealed. As a consequence, further effort is necessary to achieve deeper insights into an isoform-specific AKT signaling in breast cancer and the mechanism behind it.
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In the present study, four AKT isoforms termed AKT1, AKT2, AKT3a and AKT3b were isolated and characterized from yellow catfish. Their molecular characterizations, tissue expressions and transcriptional responses to insulin and/or wortmannin were determined. The validated complementary DNA (cDNA) of yellow catfish AKT1, AKT2, AKT3a and AKT3b were 1422, 1431, 1389 and 1440 bp in length, encoding the peptide of 472, 475, 462 and 479 amino acid residues, respectively. The amino acid sequences of yellow catfish AKTs possessed all the characteristics of AKTs in other species. AKT1, AKT2 and AKT3b contained a conserved domain structure including a specific PH domain, a central catalytic domain and a C-terminal regulatory domain, while AKT3a lacked the C-terminal regulatory domain. All mRNAs of AKTs were expressed at the highest levels in the ovary. Among other tissues, the messenger RNA (mRNA) of AKT1 was widely distributed in all tested tissues, and AKT2 mRNA was more abundant in the muscle, liver and fat and lowest in other tested tissues, while AKT3a mRNA was predominant in the brain and showed no significant difference among other tested tissues, and AKT3b mRNA was highly expressed in the ovary, followed by the brain, muscle and fat and was relatively low in other tissues. Intraperitoneal insulin injection and incubation increased the mRNA expression of AKT1 and AKT2, but not that of AKT3a and AKT3b in the liver and hepatocytes of yellow catfish. Wortmannin reduced the mRNA level of all AKT isoforms and also alleviated the insulin-induced changes of AKT2 expression. The present study cloned full-length cDNA sequences of four AKTs in fish and determined their tissue expression profiles and studied their transcriptional responses to insulin and/or wortmannin, which serves to increase our understanding of their physiological function in lipid metabolism in fish.
Article
Activation of the serine/threonine kinase Akt and the regulation of its activation are recognized as critical in controlling proliferative/survival signals via many hematopoietic receptors. In B lymphocytes, the B-cell receptor (BCR)-mediated activation of Akt is attenuated by co–cross-linking of BCR with the inhibitory receptor FcγRIIB1, and the binding of the SH2 domain-containing inositol phosphatase, SHIP, to FcγRIIB1. Because SHIP dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PIP3) and activation of Akt requires PIP3, the destruction of this phospholipid has been proposed as the mechanism for Akt inhibition. However, upstream kinases that activate Akt, such as PDK1, also require PIP3 for activation. In this report, we addressed whether SHIP inhibits Akt directly at the level of Akt recruitment to the membrane, indirectly through PDK recruitment/phosphorylation of Akt, or both. We generated stable B-cell lines expressing a regulatable, but constitutively membrane-bound Akt that still required PDK-dependent phosphorylation for activation. Several lines of evidence suggested that activation of this membrane-targeted Akt is not inhibited by FcγRIIB1/SHIP and that PDK is not a target for SHIP-mediated inhibition. These data demonstrate that SHIP inhibits Akt primarily through regulation of Akt membrane localization. We also observed during these studies that FcγRIIB1/SHIP does not inhibit p70S6k activation, even though several other PIP3-dependent events were down-regulated. Because the enhanced activation of Akt in the absence of SHIP correlates with hyperproliferation in the myeloid lineage, our data have implications for SHIP and Akt-dependent regulation of proliferation in the hematopoietic lineage.
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Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction–based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin–dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34+ stem cells. CKLiK kinase activity was dependent on Ca++ and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinaseα enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca++/calmodulin-dependent PMN- specific kinase that may play a role in Ca++-mediated regulation of human granulocyte functions.
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YAP (also known as YAP1 or YAP65) is a transcriptional coactivator that interacts with a number of transcription factors including RUNX and TEAD and plays a pivotal role in controlling cell growth. YAP is classified as a proto-oncogene. However, the mechanism by which activated YAP induces cancerous changes is not well known. Here we demonstrate that overexpression of YAP in NIH3T3 cells was sufficient for inducing tumorigenic transformation of cells. Mechanistically, YAP exerts its function in cooperation with the TEAD transcription factor. Our data also show that cMYC is a critical factor that acts downstream of the YAP/TEAD complex. Furthermore, we also found that aberrant activation of YAP is sufficient to drive tumorigenic transformation of non-immortalized mouse embryonic fibroblasts. Together our data indicate that YAP can be categorized as a new type of proto-oncogene distinct from typical oncogenes, such as H-RAS, whose expression in non-immortalized cells is tightly linked to senescence.
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Protein kinase B (Akt), similar to many other protein kinases, is at the crossroads of cell death and survival, playing a pivotal role in multiple interconnected cell signaling mechanisms implicated in cell metabolism, growth and division, apoptosis suppression and angiogenesis. Akt protein kinase displays important metabolic effects, among which are glucose uptake in muscle and fat cells or the suppression of neuronal cell death. Disruptions in the Akt‑regulated pathways are associated with cancer, diabetes, cardiovascular and neurological diseases. The regulation of the Akt signaling pathway renders Akt a valuable therapeutic target. The discovery process of Akt inhibitors using various strategies has led to the identification of inhibitors with great selectivity, low side‑effects and toxicity. The usefulness of Akt emerges beyond cancer therapy and extends to other major diseases, such as diabetes, heart diseases, or neurodegeneration. This review presents key features of Akt structure and functions, and presents the progress of Akt inhibitors in regards to drug development, and their preclinical and clinical activity in regards to therapeutic efficacy and safety for patients.
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The PI3K/PTEN/AKT/mTOR signaling axis has been intensively studied in many cancer systems. Extensive evidence suggests deregulation of this pathway plays an important role in the initiation, development, and recurrence of head and neck squamous cell carcinoma (HNSCC). A heterogeneous disease by nature, HNSCC encompasses a disparate collection of anatomical sites with complex tumor biology. Nevertheless, PI3K/PTEN/AKT/mTOR signaling has a critical role in nearly every facet of this disease. In this chapter we will provide a brief introduction to the mechanisms involved in PI3K/PTEN/AKT/mTOR signaling and how specific alterations in these signaling nodes enable HNSCC development and progression. We will also discuss differences in PI3K/PTEN/AKT/mTOR signaling with respect to human papillomavirus (HPV) status. A number of inhibitors targeting multiple nodes in this pathway have been developed, with these agents having potential application and in some cases demonstrated clinical activity in HNSCC. We will briefly review how these therapeutic agents are being evaluated and what predictive biomarkers have been established for them in HNSCC. Finally, PI3K/PTEN/AKT/mTOR signaling represents an important source of resistance to radiation and chemotherapy as well as other targeted agents. We will speculate on how PI3K/PTEN/AKT/mTOR inhibitors may increase the efficacy of these established therapies.
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The Ser and Thr kinase AKT, also known as protein kinase B (PKB), was discovered 25 years ago and has been the focus of tens of thousands of studies in diverse fields of biology and medicine. There have been many advances in our knowledge of the upstream regulatory inputs into AKT, key multifunctional downstream signaling nodes (GSK3, FoxO, mTORC1), which greatly expand the functional repertoire of AKT, and the complex circuitry of this dynamically branching and looping signaling network that is ubiquitous to nearly every cell in our body. Mouse and human genetic studies have also revealed physiological roles for the AKT network in nearly every organ system. Our comprehension of AKT regulation and functions is particularly important given the consequences of AKT dysfunction in diverse pathological settings, including developmental and overgrowth syndromes, cancer, cardiovascular disease, insulin resistance and type 2 diabetes, inflammatory and autoimmune disorders, and neurological disorders. There has also been much progress in developing AKT-selective small molecule inhibitors. Improved understanding of the molecular wiring of the AKT signaling network continues to make an impact that cuts across most disciplines of the biomedical sciences.
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Having healthy adipose tissue is essential for metabolic fitness. This is clear from the obesity epidemic, which is unveiling a myriad of comorbidities associated with excess adipose tissue including type 2 diabetes, cardiovascular disease, and cancer. Lipodystrophy also causes insulin resistance, emphasizing the importance of having a balanced amount of fat. In cells, the mechanistic target of rapamycin (mTOR) complexes 1 and 2 (mTORC1 and mTORC2, respectively) link nutrient and hormonal signaling with metabolism, and recent studies are shedding new light on their in vivo roles in adipocytes. In this review, we discuss how recent advances in adipose tissue and mTOR biology are converging to reveal new mechanisms that maintain healthy adipose tissue, and discuss ongoing mysteries of mTOR signaling, particularly for the less understood complex mTORC2.
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A natural tolerance of various environmental stresses is typically supported by various cytoprotective mechanisms that protect macromolecules and promote extended viability. Among these are antioxidant defenses that help to limit damage from reactive oxygen species and chaperones that help to minimize protein misfolding or unfolding under stress conditions. To understand the molecular mechanisms that act to protect cells during primate torpor, the present study characterizes antioxidant and heat shock protein (HSP) responses in various organs of control (aroused) and torpid grey mouse lemurs, Microcebus murinus. Protein expression of HSP70 and HSP90α was elevated to 1.26 and 1.49 fold, respectively, in brown adipose tissue during torpor as compared with control animals, whereas HSP60 in liver of torpid animals was 1.15 fold of that in control (P < 0.05). Among antioxidant enzymes, protein levels of thioredoxin 1 were elevated to 2.19-fold in white adipose tissue during torpor, whereas Cu-Zn superoxide dismutase 1 levels rose to 1.1-fold in skeletal muscle (P < 0.05). Additionally, total antioxidant capacity was increased to 1.6-fold in liver during torpor (P < 0.05), while remaining unchanged in the five other tissues. Overall, our data suggest that antioxidant and HSP responses are modified in a tissue-specific manner during daily torpor in grey mouse lemurs. Furthermore, our data also show that cytoprotective strategies employed during primate torpor are distinct from the strategies in rodent hibernation as reported in previous studies. Copyright © 2015. Production and hosting by Elsevier Ltd.
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A lambda Insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coli a phagemid, pBluescript SK(−), contained within the vector, can be excised by 11 or M13 helper phage. The excision process elimlnates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the 11 bacteriophage origin of replication (1). Six of 21 restriction sites In the excised pBluescript SK polylinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA Hbrary and the isolatlon of actin clones by screening with oligonucleotide probes. Putative actin clones were excised from the lambda vector and Identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.
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We have identified protein kinase genes of Dictyostelium by using highly conserved amino acid sequence motifs to design the synthesis and amplification of DNA fragments by polymerase chain reactions (PCRs). Cloning and sequencing the PCR products have revealed five different members of the protein kinase multigene family. These five putative kinases showed varying degrees of amino acid sequence similarity (40-70%) to protein kinases in data bases and contained invariant amino acid residues characteristic of protein kinases. DNA from PCR was labeled and used to isolate several lambda gt11 cDNA clones, including one full-length one (Dd kinase-2). The nucleotide sequence of Dd kinase-2 contained a region identical to one of the cloned kinase fragments amplified by PCR, and based on the deduced amino acid sequence Dd kinase-2 encodes a protein of 479 amino acids. A 350-amino acid kinase domain at the C-terminal end shows high homology to the catalytic domains of protein kinase A, protein kinase C, S-6 kinase of Xenopus, and the suppressor of cdc25 of yeast. The N-terminal domain is highly basic and also contains alternating threonine/proline residues. The cDNA hybridized to a single copy gene but to two differentially regulated mRNAs--a 2.0-kilobase mRNA that is expressed in vegetative cells and a 2.2-kilobase mRNA that is expressed during development. The larger mRNA is induced by cAMP by using a cell-surface receptor-mediated signal transduction pathway.
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Glycogen synthase kinase-3 (GSK-3) is a protein-serine kinase implicated in the hormonal control of several regulatory proteins including glycogen synthase and the transcription factor c-jun. Two classes of rat brain cDNA for this enzyme have been isolated termed GSK-3 alpha and GSK-3 beta. The alpha-type encodes a 51 kd polypeptide, the sequence of which includes all of the tryptic peptides determined by protein sequence analysis of purified skeletal muscle GSK-3. The novel beta-type cDNA has the potential to encode a 47 kd protein with 85% amino acid identity to GSK-3 alpha. The two types of cDNA are the products of distinct genes as determined by genomic organization and nucleic acid sequence analysis. Both alpha and beta clones exhibit kinase activity when expressed in COS-1 cells and type-specific antibodies to GSK-3 alpha and beta detect proteins of 51 and 47 kd, respectively, in a variety of rat tissue extracts, with highest levels of both in brain. Partial purification of GSK-3 activity from bovine brain results in the isolation of active alpha and beta proteins. The physiological importance of these two proteins in cellular signal transduction is discussed.
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Cycloheximide injection of rats results in the activation of a protein kinase that phosphorylates 40 S ribosomal protein S6. This Ca2+/cyclic nucleotide-independent kinase exhibits chromatographic properties that are indistinguishable from the S6 kinase in H4 hepatoma cells whose activity is stimulated by insulin and growth factors and the S6 kinase that is activated during liver regeneration. The enzyme has been purified 50,000-fold to near homogeneity: a critical step in purification employs a peptide affinity column using a synthetic peptide corresponding to the carboxyl-terminal 32-amino acid residues of mouse liver S6, which encompasses all S6 phosphorylation sites. The purified enzyme is a 70,000-dalton polypeptide that is reactive with azido-ATP. In addition to 40 S ribosomal S6 and the synthetic peptide, the S6 kinase catalyzes rapid phosphorylation of a number of other protein substrates including histone H2b, glycogen synthase, and ATP citrate lyase; this last protein is phosphorylated by S6 kinase in vitro on the same serine residue that is phosphorylated in response to insulin and epidermal growth factor in intact hepatocytes. Moreover, the S6 kinase catalyzes the phosphorylation of a number of hepatic nonhistone nuclear proteins. This S6 kinase probably underlies the increased hepatic S6 phosphorylation observed after cycloheximide treatment, which in turn corresponds to the mitogen-activated S6 kinase.
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A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.
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A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coli a phagemid, pBluescript SK(-), contained within the vector, can be excised by f1 or M13 helper phage. The excision process eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the f1 bacteriophage origin of replication (1). Six of 21 restriction sites in the excised pBluescript SK polylinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA library and the isolation of actin clones by screening with oligonucleotide probes. Putative actin clones were excised from the lambda vector and identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.
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In recent years, members of the protein kinase family have been discovered at an accelerated pace. Most were first described, not through the traditional biochemical approach of protein purification and enzyme assay, but as putative protein kinase amino acid sequences deduced from the nucleotide sequences of molecularly cloned genes or complementary DNAs. Phylogenetic mapping of the conserved protein kinase catalytic domains can serve as a useful first step in the functional characterization of these newly identified family members.
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5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs. (GCC)GCCAGCCATGG emerges as the consensus sequence for initiation of translation in vertebrates. The most highly conserved position in that motif is the purine in position -3 (three nucleotides upstream from the ATG codon); 97% of vertebrate mRNAs have a purine, most often A, in that position- The periodical occurrence of G (in positions −3, −6, −9) is discussed. Upstream ATG codons occur in fewer than 10% of vertebrate mRNAs-at-large; a notable exception are oncogene transcripts, two-thirds of which have ATG codons preceding the start of the major open reading frame. The leader sequences of most vertebrate mRNAs fall in the size range of 20 to 100 nucleotides. The significance of shorter and longer 5' -noncoding sequences is discussed.
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Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.
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In Xenopus oocytes ribosomal protein S6 becomes phosphorylated on serine residues in response to hormones or growth factors and following microinjection of the tyrosine-specific protein kinases associated with Rous sarcoma virus or Abelson murine leukemia virus. To begin characterization of the enzymes responsible for S6 phosphorylation in this system, we have undertaken the purification of S6 protein kinases from unfertilized Xenopus eggs. DEAE-Sephacel chromatography of crude extracts revealed two peaks of S6 kinase activity, and the peak eluting at 160 mM NaCl was chosen for further purification. Successive chromatography on Mono S, Sephacryl S-200, Mono Q, and heparin-Sepharose resulted in purification of the enzyme to a single protein migrating at Mr = 92,000 on polyacrylamide gels. The final preparation was purified about 500-fold from the DEAE-Sephacel peak with a recovery of 10%. Apparent Km values of the enzyme for ATP and 40 S subunits were 28 and 5 microM, respectively, and the specific activity with 330 microM ATP and 5.6 microM 40 S subunits was 300 nmol/min/mg. The enzyme was inhibited by beta-glycerophosphate, sodium fluoride, potassium phosphate, ADP, heparin, quercetin, and spermine. The availability of a purified S6 protein kinase should facilitate elucidation of the molecular mechanism of S6 phosphorylation during growth stimulation.
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Soluble extracts from serum- or epidermal growth factor-stimulated Swiss 3T3 cells show up to a 25-fold increase in their ability to phosphorylate 40 S ribosomal protein S6. The increased S6 phosphorylation is due to increased protein kinase activity in extracts of stimulated cells and not due to the inactivation of an S6 phosphatase. However, the presence of phosphatase inhibitors as well as EGTA is required during the preparation of cell extracts to obtain fully active S6 kinase(s). Epidermal growth factor has little effect at concentrations below 10(-10) M: activity increases sharply at 10(-9) M epidermal growth factor and reaches saturation at 10(-8) M (50-60% of the activity obtained by stimulating with 10% serum). Activation of the kinase activity in cell extracts is observed as early as 2 min after serum stimulation, reaches 50% between 10 and 15 min, and is maximal by 60 min of serum stimulation. Phosphorylation in vitro of ribosomal protein S6 with extracts from serum-stimulated cells followed by analysis of the tryptic phosphopeptides shows the presence of 9 of the 11 phosphopeptides induced by serum in vivo.
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Three types of cDNA clone of the protein kinase C family termed δ, ϵ and ξ were newly identified by molecular cloning and sequence analysis. These members have a common structure that is closely related to, but clearly different from the other four known members of the family which have α-, βI-, βII- and γ-sequences, although the ξ-cDNA available at present does not contain a complete reading frame for a protein kinase C molecule. The diverse heterogeneity of the enzyme seems to be an important factor in determining the mode of response of many tissues and cell types to a variety of external stimuli.
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Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
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Degenerate oligodeoxyribonucleotide (oligo) primers derived from amino acid (aa) sequence motifs held in common between all members of the protein tyrosine kinase (PTK) family were used to prime the amplification of PTK-related sequences from a variety of murine cDNA sources, including the haemopoietic cell lines, FDC-P1 and WEHI-3B D+, peritoneal macrophages and whole brain. Several parameters, such as the length (short, i.e., less than 20 nucleotides (nt) vs. long, i.e., greater than 30 nt) and degeneracy (i.e., moderately degenerate vs. highly degenerate) of the oligo primers and the temperature of the extension phase of the reaction, were examined. The data from these analyses suggest that the most effective type of primer in this application of the polymerase chain reaction is a short, moderately degenerate oligo such as that which might be derived from the small patches of aa sequence homology that are frequently found to be held in common among members of protein families. In addition to a number of previously described PTK sequences, a novel mammalian PTK-related sequence was uncovered.
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The role of cyclic AMP (cAMP) in the regulation of mammalian cell proliferation has been the subject of controversy. Negative control was demonstrated in the 1970s, but evidence of positive control in other cell types has been neglected. Recent evidence which demonstrates such a control in the yeast Saccharomyces cerevisiae has now made this concept acceptable.
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Mixed oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identity with rabbit skeletal muscle phosphorylase kinase gamma-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.
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Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
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Protein kinase C is now known to be a large family of proteins, with multiple subspecies that have subtle individual enzymological characteristics. Some members of the family exhibit distinct patterns of tissue expression and intracellular localization; different kinases probably have distinct functions in the processing and modulation of a variety of physiological and pathological responses to external signals.
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Proto-oncogenes encode proteins with three main sites of action: the cell-surface membrane, the cytoplasm and the nucleus. Although the exact biochemical function of most proto-oncogene products is not understood, several of them are known to be involved in signal transduction. A role in gene regulation through DNA binding has been suggested for a recently isolated member of the group of oncogenes acting at the nucleus, v-jun. The C-terminus of the putative v-jun-encoded protein is similar in sequence to the C-terminus of the yeast transcriptional activator GCN4 (refs 8, 9), which forms its minimal DNA-binding domain. GCN4 binds to specific sites whose consensus sequence is highly similar to the recognition sequence of the mammalian transcriptional activator AP-1 (refs 12, 13). Like GCN4, AP-1 binds to promoter elements of specific genes and activates their transcription. Because of the similarity between the recognition sites for GCN4 and AP-1, we examined the possibility that AP-1 could be the product of the c-jun proto-oncogene. The experimental results reported here indicate that the JUN oncoprotein is a sequence-specific transcriptional activator similar to AP-1.
Article
Excerpt The discovery of specific restriction endonucleases (Smith and Wilcox 1970) made possible the isolation of discrete molecular fragments of naturally occurring DNA for the first time. This capability was crucial to the development of molecular cloning (Cohen et al. 1973); and the combination of molecular cloning and endonuclease restriction allowed the synthesis and isolation of any naturally occurring DNA sequence that could be cloned into a useful vector and, on the basis of flanking restriction sites, excised from it. The availability of a large variety of restriction enzymes (Roberts 1985) has significantly extended the utility of these methods. The de novo organic synthesis of oligonucleotides and the development of methods for their assembly into long double-stranded DNA molecules (Davies and Gassen 1983) have removed, at least theoretically, the minor limitations imposed by the availability of natural sequences with fortuitously unique flanking restriction sites. However, de novo synthesis, even with automated...
Article
The cdc2+ gene function has an important role in controlling the commitment of the fission yeast cell to the mitotic cycle and the timing of mitosis. We have raised antibodies against the cdc2+ protein using synthetic peptides and have demonstrated that it is a 34 kd phosphoprotein with protein kinase activity. The protein level and phosphorylation state remain unchanged during the mitotic cycle of rapidly growing cells. When cells cease to proliferate and arrest in G1 the protein becomes dephosphorylated and loses protein kinase activity. Exit from the mitotic cycle and entry into stationary phase may be controlled in part by modulation of the cdc2 protein kinase activity by changes in its phosphorylation state.
Article
Three types of cDNA clone of the protein kinase C family termed delta, epsilon and zeta were newly identified by molecular cloning and sequence analysis. These members have a common structure that is closely related to, but clearly different from the other four known members of the family which have alpha-, beta I-, beta II- and gamma-sequences, although the zeta-cDNA available at present does not contain a complete reading frame for a protein kinase C molecule. The diverse heterogeneity of the enzyme seems to be an important factor in determining the mode of response of many tissues and cell types to a variety of external stimuli.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
It is now feasible, through use of recombinant DNA technology, to perform direct genomic comparisons between MSV isolates. We have cloned the integrated proviral DNA sequences of two MSV isolates, m1 and HT1 in bacteriophage λ. Both cloned fragments isolated from transformed mink cells possess full copies of the sarcoma viruses along with flanking host sequences. These cloned fragments have provided the means for direct comparison of the MSV isolates, information on the integration of MSV, and a method of identifying the regions of the viral genome possessing full transforming potential in tissue culture.
Article
Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate co-precipitation methods. Exposure to chloroquine increased the proportion of transfected mouse cells to approximately 40′. From a culture of one million such cells, microgram quantities of newly synthesized viral DNA could be isolated. Similarly, the transformation frequency of rat cells following polyoma DNA transfection was approximately 6-fold increased by chloroquine treatment. The effect of the compound was even more pronounced in transfections with linear forms of polyoma DNA, suggesting that chloroquine inhibits degradation of DNA absorbed by the cells.
Article
We have molecularly cloned a unique acutely transforming replication-defective mouse type C virus (3611-MSV) and characterized its acquired oncogene. The viral genome closely resembles Moloney (M) murine leukemia virus (MuLV), except for a substitution in M-MuLV in the middle of p30 and the middle of the polymerase gene (pol). Heteroduplex analysis revealed that 2.4 kilobases of M-MuLV DNA were replaced by 1.2 kilobases of cellular DNA. The junctions between viral and cellular sequences were determined by DNA sequence analysis to be 517 nucleotides into the p30 sequence and 1,920 nucleotides into the polymerase sequence. Comparison of the transforming gene from 3611-MSV, designated v-raf, with previously isolated retrovirus oncogenes either by direct hybridization or by comparison of restriction fragments of their cellular homologs shows it to be unique. Transfection of NIH 3T3 cells with cloned 3611-MSV proviral DNA leads to highly efficient transformation and the recovered virus elicits tumors in mice typical of the 3611-MSV virus. Transfected NIH 3T3 cells express two 3611-MSV-specific polyproteins (P75 and P90), both of which contain NH2-terminal gag gene-encoded components linked to the acquired sequence (v-raf) translational product. The cellular homolog, c-raf, is present in one or two copies per haploid genome in mouse and human DNA.
Article
The 350-residue amino acid sequence of the catalytic subunit of bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase is described. The protein has a molecular weight of 40 862, which includes an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in the S-carboxymethylated protein were cleaved with cyanogen bromide to yield eight primary peptides. These fragments, and subpeptides generated by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and Myxobacter AL-1 protease II, were purified and analyzed to yield the majority of the sequence. The primary peptides were aligned by analyses of overlapping peptides, particularly of methione-containing tryptic peptides generated after in vitro [14C]methyl exchange labeling of methionyl residues in the intact protein.
Article
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
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