[Show abstract][Hide abstract] ABSTRACT: Leukotrienes (LTs) are potent proinflammatory mediators, and many important aspects of innate and adaptive immune responses are regulated by LTs. Key members of the LT synthesis pathway are overexpressed in adipose tissue (AT) during obesity, resulting in increased LT levels in this tissue. We observed that several mouse adipocyte cell lines and primary adipocytes from mice and humans both can secrete large amounts of LTs. Furthermore, this production increases with a high-fat diet (HFD) and positively correlates with adipocyte size. LTs produced by adipocytes play an important role in attracting macrophages and T cells in in vitro chemotaxis assays. Mice that are deficient for the enzyme 5-lipoxygenase (5-LO), and therefore lack LTs, exhibit a decrease in HFD-induced AT macrophage and T-cell infiltration and are partially protected from HFD-induced insulin resistance. Similarly, treatment of HFD-fed wild-type mice with the 5-LO inhibitor Zileuton also results in a reduction of AT macrophages and T cells, accompanied by a decrease in insulin resistance. Together, these findings suggest that LTs represent a novel target in the prevention or treatment of obesity-associated inflammation and insulin resistance.
[Show abstract][Hide abstract] ABSTRACT: Suppressor of cytokine signalling (SOCS) proteins are powerful inhibitors of pathways involved in survival and function of pancreatic beta cells. Whereas SOCS1 and SOCS3 have been involved in immune and inflammatory processes, respectively, in beta cells, nothing is known about SOCS2 implication in the pancreas.
Transgenic (tg) mice were generated that constitutively produced SOCS2 in beta cells (betaSOCS2) to define whether this protein is implicated in beta cell functioning and/or survival.
Constitutive production of SOCS2 in beta cells leads to hyperglycaemia and glucose intolerance. This phenotype is not a consequence of decreased beta cell mass or inhibition of insulin synthesis. However, insulin secretion to various secretagogues is profoundly altered in intact animals and isolated islets. Interestingly, constitutive SOCS2 production dampens the rise in cytosolic free calcium concentration induced by glucose, while glucose metabolism is unchanged. Moreover, tg islets have a depletion in endoplasmic reticulum Ca(2+) stores, suggesting that SOCS2 interferes with calcium fluxes. Finally, in betaSOCS2 mice proinsulin maturation is impaired, leading to an altered structure of insulin secretory granules and augmented levels of proinsulin. The latter is likely to be due to decreased production of prohormone convertase 1 (PC1/3), which plays a key role in proinsulin cleavage.
SOCS2 was shown to be a potent regulator of proinsulin processing and insulin secretion in beta cells. While its constitutive production is insufficient to induce overt diabetes in this mouse model, it causes glucose intolerance. Thus, increased SOCS2 production could be an important event predisposing to beta cell failure.
[Show abstract][Hide abstract] ABSTRACT: Due to their ability to regulate various signalling pathways (cytokines, hormones, growth factors), the suppressor of cytokine signalling (SOCS) proteins are thought to be promising therapeutic targets for metabolic and inflammatory disorders. Hence, their role in vivo has to be precisely determined.
We generated transgenic mice constitutively producing SOCS-3 in skeletal muscle to define whether the sole abundance of SOCS-3 is sufficient to induce metabolic disorders and whether SOCS-3 is implicated in physiological roles distinct from metabolism.
We demonstrate here that chronic expression of SOCS-3 in skeletal muscle leads to overweight in mice and worsening of high-fat diet-induced systemic insulin resistance. Counter-intuitively, insulin sensitivity in muscle of transgenic mice appears to be unaltered. However, following constitutive SOCS-3 production, several genes had deregulated expression, among them other members of the SOCS family. This could maintain the insulin signal into skeletal muscle. Interestingly, we found that SOCS-3 interacts with calcineurin, which has been implicated in muscle contractility. In Socs-3 transgenic muscle, this leads to delocalisation of calcineurin to the fibre periphery. Relevant to this finding, Socs-3 transgenic animals had dilatation of the sarcoplasmic reticulum associated with swollen mitochondria and decreased voluntary activity.
Our results show that constitutive SOCS-3 production in skeletal muscle is not in itself sufficient to induce the establishment of metabolic disorders such as diabetes. In contrast, we reveal a novel role of SOCS-3, which appears to be important for muscle integrity and locomotor activity.
[Show abstract][Hide abstract] ABSTRACT: Defects in insulin secretion, resulting from loss of function or destruction of pancreatic beta-cells, trigger diabetes. Interleukin (IL)-1beta is a proinflammatory cytokine that is involved in type 1 and type 2 diabetes development and impairs beta-cell survival and function. Because effective insulin signaling is required for the optimal beta-cell function, we assessed the effect of IL-1beta on the insulin pathway in a rat pancreatic beta-cell line. We show that IL-1beta decreases insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS) proteins as well as phosphatidylinositol 3-kinase (PI3K) activation, and that this action is not due to the IL-1beta-dependent nitric oxide (NO) production in RINm5F cells. We next analyzed if suppressor of cytokine signaling (SOCS)-3, which can be induced by multiple cytokines and which we identified as an insulin action inhibitor, was implicated in the IL-1beta inhibitory effect on insulin signaling in these cells. We show that IL-1beta increases SOCS-3 expression and induces SOCS-3/IR complex formation in RINm5F cells. Moreover, we find that ectopically expressed SOCS-3 associates with the IR and reduces insulin-dependent IR autophosphorylation and IRS/PI3K pathway in a way comparable to IL-1beta treatment in RINm5F cells. We propose that IL-1beta decreases insulin action in beta-cells through the induction of SOCS-3 expression, and that this effect potentially alters insulin-induced beta-cell survival.
[Show abstract][Hide abstract] ABSTRACT: SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr(960) of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-alpha (TNF-alpha), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-alpha induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-alpha target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.
[Show abstract][Hide abstract] ABSTRACT: Suppressor of cytokine signaling (SOCS) proteins were originally described as cytokine-induced molecules involved in negative feedback loops. We have shown that SOCS-3 is also a component of the insulin signaling network (). Indeed, insulin leads to SOCS-3 expression in 3T3-L1 adipocytes. Once produced, SOCS-3 binds to phosphorylated tyrosine 960 of the insulin receptor and inhibits insulin signaling. Now we show that in 3T3-L1 adipocytes and in transfected COS-7 cells insulin leads to SOCS-3 tyrosine phosphorylation. This phosphorylation takes place on Tyr(204) and is dependent upon a functional SOCS-3 SH2 domain. Purified insulin receptor directly phosphorylates SOCS-3. However, in intact cells, a mutant of the insulin receptor, IRY960F, unable to bind SOCS-3, was as efficient as the wild type insulin receptor to phosphorylate SOCS-3. Importantly, IRY960F is as potent as the wild type insulin receptor to activate janus-activated kinase (Jak) 1 and Jak2. Furthermore, expression of a dominant negative form of Jak2 inhibits insulin-induced SOCS-3 tyrosine phosphorylation. As transfected Jaks have been shown to cause SOCS-3 phosphorylation, we propose that insulin induces SOCS-3 phosphorylation through Jak activation. Our data indicate that SOCS-3 belongs to a class of tyrosine-phosphorylated insulin signaling molecules, the phosphorylation of which is not dependent upon a direct coupling with the insulin receptor but relies on the Jaks.
[Show abstract][Hide abstract] ABSTRACT: The SOCS proteins are induced by several cytokines and are involved in negative feedback loops. Here we demonstrate that in 3T3-L1 adipocytes, insulin, a hormone whose receptor does not belong to the cytokine receptor family, induces SOCS-3 expression but not CIS or SOCS-2. Using transfection of COS-7 cells, we show that insulin induction of SOCS-3 is enhanced upon Stat5B expression. Moreover, Stat5B from insulin-stimulated cells binds directly to a Stat element present in the SOCS-3 promoter. Once induced, SOCS-3 inhibits insulin activation of Stat5B without modifying the insulin receptor tyrosine kinase activity. Two pieces of evidence suggest that this negative regulation likely results from competition between SOCS-3 and Stat5B binding to the same insulin receptor motif. First, using a yeast two-hybrid system, we show that SOCS-3 binds to the insulin receptor at phosphotyrosine 960, which is precisely where Stat5B binds. Second, using confocal microscopy, we show that insulin induces translocation of SOCS-3 from an intracellular compartment to the cell membrane, leading to colocalization of SOCS-3 with the insulin receptor. This colocalization is dependent upon phosphorylation of insulin receptor tyrosine 960. Indeed, in cells expressing an insulin receptor mutant in which tyrosine 960 has been mutated to phenylalanine, insulin does not modify the cellular localization of SOCS-3. We have thus revealed an insulin target gene of which the expression is potentiated upon Stat5B activation. By inhibiting insulin-stimulated Stat5B, SOCS-3 appears to function as a negative regulator of insulin signaling.
[Show abstract][Hide abstract] ABSTRACT: Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.
[Show abstract][Hide abstract] ABSTRACT: Involvement of the pleckstrin homology (PH) domain in the insulin-stimulated activation of protein kinase B (PKB) was investigated in human embryonic kidney 293 cells. Different PKB constructs that contain mutations or deletions in the PH domain were transfected into cells, and the results on the basal and insulin-induced kinase activities were analyzed. Deletion of the entire PH domain (DeltaPH-PKB) did not impair the kinase activity; in contrast, the basal activity was elevated with respect to wild-type PKB. In addition, DeltaPH-PKB was responsive to insulin, and as for wild-type PKB, this was dependent on phosphoinositide 3-kinase. By contrast, a point mutation within the PH domain that impairs phospholipid binding (R25C) resulted in a construct that was not responsive to insulin. However, this defect was overcome by mutations that mimic the phosphorylation state of the active kinase. The increase in the basal activity of DeltaPH-PKB was shown to be due to an elevation in the level of phosphorylation of this construct. In addition, the subcellular localization of DeltaPH-PKB, as determined by both immunofluorescence and fractionation, was predominately cytosolic, and DeltaPH-PKB was present in the plasma membrane at much lower levels compared with wild-type PKB. These data show that phosphorylation is the major factor regulating the activity of PKB and that either removal of the PH domain or binding of phospholipids is required to permit this phosphorylation. In addition, membrane localization does not appear to be required for the activation process, but instead, binding of PKB to membrane phospholipids permits a conformational change in the molecule that allows for phosphorylation.
[Show abstract][Hide abstract] ABSTRACT: Bombesin has been reported to stimulate cholecystokinin (CCK) secretion from rat duodeno-jejunal I-cells. Bombesin was shown to activate mitogen-activated protein kinases (MAPKs) in cell types such as Swiss 3T3 fibroblasts and rat pancreatic acinar cells. No information is available on whether MAPK is activated in intestinal endocrine cells upon bombesin stimulation. This was studied by using the CCK-producing enteroendocrine cell line STC-1. Bombesin stimulated markedly and transiently both p42(MAPK) and p44(MAPK), with a maximum at 2 min, and a decrease to basal levels within 10 min. As expected, bombesin stimulated MAPK kinase 1 (MEK-1) activity. Activation of protein kinase C (PKC) with PMA also stimulated p42(MAPK), p44(MAPK) and MEK-1. Treatment of cells with PD 098059 (at 10 microM or 30 microM), which selectively inhibits MEK phosphorylation, blocked bombesin-induced p42(MAPK) and p44(MAPK) activation for at least 90 min. However, PD 098059 inhibited bombesin- and PMA-stimulated CCK secretion during the first 15 min, but failed to significantly reduce CCK release at later times. Inhibition of PKC with staurosporine, or PKC down-regulation by prolonged treatment with PMA, both drastically decreased MEK-1, p42(MAPK) and p44(MAPK) activation upon bombesin stimulation. Additionally, PKC activation appeared to be required for both MAPK-dependent (early) and -independent (late) CCK responses to bombesin. It is concluded that the early CCK secretory response of STC-1 cells to bombesin involves MAPK pathway activation through a PKC-dependent mechanism, whereas the late phase of bombesin-induced CCK secretion, that also requires PKC, appears to result from a MAPK-independent process.
[Show abstract][Hide abstract] ABSTRACT: We have screened a human placenta library using the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Stat 5B and the receptor requires a functional insulin-receptor Kinase, Tyr960 of insulin receptor is implicated in the interaction with Stat 5B, whereas asparagine and proline forming the NPEY960-motif are not, and Stat 5B mutated at Thr684, a potential phosphorylation site of mitogen-activated protein Kinase, loses its ability to interact with the insulin receptor. Further, we found that insulin promotes rapid tyrosine phosphorylation of endogenous Stat 5B in 293 EBNA cells overexpressing insulin receptor and in NHIR cells.
Taken together, our finding suggest that Stat 5B corresponds to a substrate for the insulin-receptor Kinase, and this widens the repertoire of insulin-signaling pathways.
European Journal of Biochemistry 01/1998; 250(2):411-7. DOI:10.1111/j.1432-1033.1997.0411a.x · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In a first series of experiments done in the yeast two-hybrid system, we investigated the nature of protein-protein interaction between the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), p55PIK, and several of its potential signaling partners. The region between the Src homology 2 (SH2) domains of p55PIK bound to the NH2 terminus region of p110alpha, as previously shown for p85alpha. Moreover, we found that the insulin-like growth factor-1 receptor (IGF-IR) bound to p55PIK; the interaction occurred at the receptor tyrosine 1316 and involved both p55PIK SH2 domains. Interaction between p55PIK and IGF-IR was seen not only in the yeast two-hybrid system, but also using in vitro binding and coimmunoprecipitation of lysates from IGF-1 stimulated 293 cells overexpressing p55PIK. Further, IGF-I stimulation of these cells led to tyrosine phosphorylation of p55PIK. In 293 cells association of p55PIK with insulin receptor substrate-1 and with IGF-IR was dependent on PI 3-kinase, since it was increased by wortmannin, an inhibitor of PI 3-kinase. Further, by deleting amino acids 203-217 of p55PIK inter-SH2 domain, we engineered a p55PIK mutant unable to bind to the p110alpha catalytic subunit of PI 3-kinase. This mutant had a dominant-negative action on insulin-stimulated glucose transport, since insulin's effect on Glut 4 myc translocation was inhibited in adipocytes expressing mutant p55PIK. Importantly, this dominant-negative mutant was more efficient than wild type p55PIK in associating to IGF-IR and insulin receptor substrate-1 in 293 cells. Taken together, our results show that p55PIK interacts with key elements in the IGF-I signaling pathway, and that these interactions are negatively modulated by PI 3-kinase itself, providing circuitry for regulatory feedback control.
[Show abstract][Hide abstract] ABSTRACT: In a first series of experiments done in the yeast two-hybrid system, we investigated the nature of protein-protein interaction between the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), p55(PIK) and several of its potential signaling partners. The region between the Src homology 2 (SH2) domains of p55(PIK) bound to the NH2 terminus region of p110 alpha, as previously shown for p85 alpha. Moreover, we found that the insulin-like growth factor-1 receptor (IGF-IR) bound to p55(PIK); the interaction occurred at the receptor tyrosine 1316 and involved both p55(PIK) SH2 domains. Interaction between p55(PIK) and IGF-IR was seen not only in the yeast two-hybrid system, but also using in vitro binding and coimmunoprecipitation of lysates from IGF-1 stimulated 293 cells overexpressing p55(PIK). Further, IGF-I stimulation of these cells led to tyrosine phosphorylation of p55(PIK). In 293 cells association of p55(PIK) with insulin receptor substrate-1 and with IGF-IR was dependent on PI 3-kinase, since it was increased by wortmannin, an inhibitor of PI 3-kinase. Further, by deleting amino acids 203-217 of p55(PIK) inter-SH2 domain, we engineered a p55(PIK) mutant unable to bind to the p110 alpha catalytic subunit of PI 3-kinase. This mutant had a dominant-negative action on insulin-stimulated glucose transport, since insulin's effect on Glut 4 myc translocation was inhibited in adipocytes expressing mutant p55(PIK). Importantly, this dominant-negative mutant was more efficient than wild type p55(PIK) in associating to IGF-IR and insulin receptor substrate-1 in 293 cells. Taken together, results show that p55(PIK) interacts with key elements in the IGF-I signaling pathway, and that these interactions are negatively modulated by PI 3-kinase itself, providing circuitry for regulatory feedback control.
[Show abstract][Hide abstract] ABSTRACT: In addition to the pleckstrin homology domain and the phosphotyrosine binding domain in insulin receptor substrate (IRS)-1 and IRS-2, a region between amino acids 591 and 786 in IRS-2 (IRS-2-(591-786)) binds to the insulin receptor. Based on peptide competition studies, this region interacts with the phosphorylated regulatory loop of the insulin receptor; we designate this region the kinase regulatory loop binding (KRLB) domain. Two tyrosine residues in the KRLB domain at positions 624 and 628 are crucial for this interaction. Phosphorylation of tyrosine residues in the KRLB domain by the insulin receptor inhibits the binding to the receptor. These results reveal a novel mechanism regulating the interaction of the insulin receptor and IRS-2 that may distinguish the signal of IRS-2 from IRS-1.
[Show abstract][Hide abstract] ABSTRACT: The mitogen-activated protein kinase (MAP kinase) is a key participant in growth factor-stimulated intracellular events such as proliferation and differentiation. We and others have previously described a cross-talk between the MAP kinase pathway and the cAMP pathway. Indeed, in several cell lines and, in particular in fibroblasts, an increase in the level of cAMP produced an inhibition of MAP kinase together with decreased cell proliferation. In contrast, in PC12 cells, cAMP induced an increase in the NGF-induced activation of MAP kinase concomitantly with augmented NGF-induced differentiation. Therefore, it has been proposed that the cellular context is important for the nature of the cAMP effects on growth factor-stimulated MAP kinase activity. Here we show that the type of tyrosine kinase receptor stimulated also participates in the nature of the cAMP effect. Thus, in NIH3T3 fibroblasts expressing NGF receptors (NIH3T3/trk cells) we found that cAMP potentiates NGF-stimulated ERK1 and MEK1 activities, whereas in NIH3T3 fibroblasts expressing insulin receptors (NIH3T3/IR cells) we saw no effect of cAMP on the activation of insulin-stimulated ERK1 and MEK1. In PC12 cells and in Rat1 fibroblasts expressing insulin receptors (PC12/IR and Rat1/IR cells) we observed, respectively, a potentiation and an inhibition of insulin-stimulated ERK1 activity. In addition, cAMP does not seem to modify the basal nor growth factor-stimulated She or IRS-1 tyrosine phosphorylation in the different cell lines studied. Finally, we observed that cAMP inhibited serum- and insulin-induced, but not NGF-induced, cell proliferation in NIH3T3 cells. However, cAMP potentiated insulin-stimulated cell differentiation in PC12/IR cells. These results led us to conclude that the cAMP effect on cell proliferation in NIH3T3 fibroblasts and PC12/IR cells appears to be correlated, in part, with the effect of cAMP on the MAP kinase pathway, but by itself this pathway cannot fully account for these observations.
[Show abstract][Hide abstract] ABSTRACT: The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-cell line INS-1, major secretagogue-stimulated signaling pathways converge to activate 44-kDa mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin secretion was found to be associated with a small stimulatory effect on 44-kDa MAP kinase, which was synergistically enhanced by increased levels of intracellular cAMP and by the hormonal secretagogues glucagon-like peptide-1 and pituitary adenylate cyclase-activating polypeptide. Activation of 44-kDa MAP kinase by glucose was dependent on Ca2+ influx and may in part be mediated by MEK-1, a MAP kinase kinase. Stimulation of Ca2+ influx by KCl was in itself sufficient to activate 44-kDa MAP kinase and MEK-1. Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP kinase by both Ca(2+)-dependent and -independent pathways. Nerve growth factor, independently of changes in cytosolic Ca2+, efficiently stimulated 44-kDa MAP kinase without causing insulin release, indicating that activation of this kinase is not sufficient for secretion. In the presence of glucose, however, nerve growth factor potentiated insulin secretion. In INS-1 cells, activation of 44-kDa MAP kinase was partially correlated with the induction of early response genes junB, nur77, and zif268 but not with stimulation of DNA synthesis. Our findings suggest a role of 44-kDa MAP kinase in mediating some of the pleiotropic actions of secretagogues on the pancreatic beta-cell.
[Show abstract][Hide abstract] ABSTRACT: In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.
[Show abstract][Hide abstract] ABSTRACT: Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP.
Proceedings of the National Academy of Sciences 06/1994; 91(11):5002-6. DOI:10.1073/pnas.91.11.5002 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In PC12 cells, extracellular signal-regulated kinase-1 (ERK1 or pp44/mitogen-activated protein kinase) is stimulated in response to epidermal growth factor (EGF) and nerve growth factor (NGF). This stimulation is rapid and short-lived after EGF activation. In contrast, NGF promotes a swift, but persistent, ERK1 stimulation. We took advantage of this difference in activation pattern to study the negative regulation of ERK1. Using antibodies to the C-terminus of ERK1, we performed in vitro reconstitution experiments with immunoprecipitated ERK1 from stimulated cells and extracts from PC12 cells incubated with EGF or NGF for various periods of times. Using this approach, we showed that extracts from unstimulated cells reduce ERK1 activity. Upon exposure of cells to NGF or EGF, we found that the inhibitory activity had a pattern opposite that of ERK1 phosphorylation and activity. Indeed, the highest ERK1 activation was associated with the lowest ERK1-repressing activity and vice versa. This ERK1 inhibitory activity was found to be sensitive mainly to sodium orthovanadate and to a lesser extent to zinc acetate. Interestingly, okadaic acid decreased ERK1-repressing activity from unstimulated cells when tested with ERK1 from 5-min NGF-treated cells, but not with ERK1 from 5-min EGF-treated cells. Hence, ERK1 appears to be regulated differently after stimulation of cells with EGF compared to NGF. We show that cell extracts promote ERK1 dephosphorylation. Indeed, we were able to detect a phosphatase activity toward in vivo phosphorylated ERK1 that was regulated differently after NGF and EGF treatments of the cells, and that has a profile of regulation similar to that of the ERK1 inhibitory activity. This regulatable phosphatase activity was also observed using in vitro phosphorylated ERK1. Taken together, our data provide evidence that ERK1 is negatively controlled by a phosphatase(s) that can undergo differential modulation depending on the stimuli used.
[Show abstract][Hide abstract] ABSTRACT: We recently characterized the association of the 44-kDa mitogen-activated protein kinase, also known as extracellular-regulated kinase 1 (ERK1), with the 90-kDa ribosomal S6 kinase (pp90rsk), one of its putative substrates in intact PC12 cells. Using antibodies to ERK1 that precipitate a functional ERK1.pp90rsk phosphoprotein complex, we demonstrate here the regulation of both kinases by various stimuli. In mouse fibroblasts expressing human insulin receptors, insulin and vanadate swiftly stimulated ERK1 activity within 5 min. While the hormonal effect was short-lived, vanadate led to a first peak followed by a progressively increasing second phase. In PC12 cells, epidermal growth factor, which is a growth promoting factor, provokes a rapid but evanescent activation of ERK1. In contrast, nerve growth factor (NGF), which acts as a neuronal differentiation factor for PC12 cells, induced a swift monophasic response followed by a sustained second phase. This strikingly different pattern of ERK1 stimulation by NGF and epidermal growth factor was associated to a contrasting effect on ERK1 cellular translocation. Thus, NGF induced a nuclear translocation of ERK1, while epidermal growth factor was without noticeable effect on ERK1 localization. In both cell systems all effectors tested stimulated ERK1 phosphorylation on both threonine and tyrosine residues in an 1:1 ratio. During ERK1 inactivation, phosphothreonine and phosphotyrosine were dephosphorylated in a similar fashion. Concurrent with ERK1 activation was the de novo appearance of phosphothreonine and an increase in phosphoserine on pp90rsk. The pp90rsk phosphothreonine content paralleled the ERK1 activity more closely than the phosphoserine level. These results provide compelling evidence that in fibroblasts and PC12 cells ERK1 plays a direct role in the phosphorylation of pp90rsk and that pp90rsk represents a physiologically relevant substrate of extracellular-regulated kinases. Finally, we would like to suggest that the differentiating action of NGF in PC12 cells might be due, at least in part, to the conjunction of its sustained and robust stimulation of ERK1 and pp90rsk, and of its induction of ERK1 nuclear translocation.
Journal of Biological Chemistry 06/1993; 268(13):9803-10. · 4.57 Impact Factor