Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.     

Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cdelta (PKCdelta), whereas ERK activation in response to the mitogenic EGF is independent of PKCdelta. Antisense PKCdelta oligonucleotides or the PKCdelta-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCdelta functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCdelta also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCdelta in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCdelta requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCdelta in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCdelta contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling.     

Role of group A p21-activated kinases in activation of extracellular-regulated kinase by growth factors

The canonical extracellular-regulated kinase (ERK) signaling cascade, consisting of the Ras-Raf-Mek-ERK module, is critically important to many cellular functions. Although the general mechanism of activation of the ERK cascade is well established, additional noncanonical components greatly influence the activity of this pathway. Here, we focus on the group A p21-activated kinases (Paks), which have previously been implicated in regulating both c-Raf and Mek1 activity, by phosphorylating these proteins at Ser(338) and Ser(298), respectively. In NIH-3T3 cells, expression of an inhibitor of all three group A Paks reduced activation of ERK in response to platelet-derived growth factor (PDGF) but not to epidermal growth factor (EGF). Similar results were obtained in HeLa cells using small interference RNA-mediated simultaneous knockdown of both Pak1 and Pak2 to reduce group A Pak function. Inhibition of Pak kinase activity dramatically decreased phosphorylation of Mek1 at Ser(298) in response to either PDGF or EGF, but this inhibition did not prevent Mek1 activation by EGF, suggesting that although Pak can phosphorylate Mek1 at Ser(298), this event is not required for Mek1 activation by growth factors. Inhibition of Pak reduced the Ser(338) phosphorylation of c-Raf in response to both PDGF and EGF; however, in the case of EGF, the reduction in Ser(338) phosphorylation was not accompanied by a significant decrease in c-Raf activity. These findings suggest that Paks are required for the phosphorylation of c-Raf at Ser(338) in response to either growth factor, but that the mechanisms by which EGF and PDGF activate c-Raf are fundamentally different.     

Contribution of PKC-dependent and -independent processes in temporal ERK regulation by ET-1, PDGF, and EGF in rat myometrial cells

Endothelin-1 (ET-1), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) stimulated thymidine incorporation with different efficiency (PDGF >> EGF = ET-1) in rat myometrial cells. They also stimulated ERK activation, which culminated at 5 min and then declined to reach a plateau (at 45 min: EGF > 90%, PDGF = 50%, and ET-1 < 10% of maximum). Inhibition and downregulation of PKC demonstrated that ERK activation at 5 min involved PKC and - for ET-1 and PKC plus another PKC isoform for PDGF. By contrast, the EGF response did not involve PKC. Stimulation of Ras was more important with EGF than with PDGF, with ET-1 being the weakest activator. The simultaneous incubation of the cells with EGF and ET-1 potentiated the ERK activation at 5 min and mimicked the plateau phase obtained with PDGF. Under these conditions thymidine incorporation was comparable to that induced by PDGF. Taken together, our results indicated that the kinetic profile of ERK activation and its impact on cell proliferation can be modulated by the differential involvement of PKC isoforms and the amplitude of Ras activation. uterine smooth muscle; phospholipase C; ET_A receptor; thymidine incorporation; Ras     

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.     

Differentiation factors, including nerve growth factor, fibroblast growth factor, and interleukin-6, induce an accumulation of an active Ras.GTP complex in rat pheochromocytoma PC12 cells.

Ras has been thought to be involved in neuronal differentiation of rat pheochromocytoma PC12 cells. PC12 cells are immature adrenal chromaffin-like cells which undergo differentiation to sympathetic neuron-like cells in response to nerve growth factor (NGF). Fibroblast growth factor (FGF) and interleukin (IL)-6 can also induce differentiation of PC12 cells. In this paper, we report that NGF, FGF, and IL-6 induce an accumulation of an active Ras.GTP complex. In the serum-starved culture of PC12 cells, 6% of the Ras protein was complexed with GTP. Upon stimulation with NGF, the percentage of Ras.GTP increased to 24% after 2 min, and the high level of Ras.GTP was maintained for at least 16 h. On the other hand, the activation of Ras by FGF and IL-6 showed distinct kinetics; about 3-fold increase of Ras.GTP was detected at 10 min, and afterward, the level returned to the basal level within 60 min. These observations provide direct evidence that activation of Ras is involved in signal transduction from these differentiation factors. In addition, it was found that growth factors, including epidermal growth factor, insulin, and insulin-like growth factor-I, and a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), can also activate Ras under the same conditions. A tyrosine kinase-specific inhibitor, genistein, inhibited the increase of Ras.GTP induced by NGF and other factors. On the other hand, down-regulation of protein kinase C (PKC) by prolonged treatment with TPA, which sufficiently blocked TPA-induced Ras activation, did not abolish the formation of Ras.GTP by NGF. These results suggest that tyrosine kinases rather than PKC play a major role in the NGF-induced activation of Ras in PC12 cells.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.     

Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.     

Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.     

IGF-2R-mediated signaling results in hypertrophy of cultured cardiomyocytes from fetal sheep

Activation of the insulin-like growth factor-1 receptor (IGF-1R) is known to play a role in cardiomyocyte hypertrophy. While IGF-2R is understood to be a clearance receptor for IGF-2, there is also evidence that it may play a role in the induction of pathological cardiomyocyte hypertrophy. It is not known whether IGF-2R activates cardiomyocyte hypertrophy during growth of the fetal heart. Fetal sheep hearts (125 ± 0.4 days gestation) were dissected, and the cardiomyocytes isolated from the left and right ventricles for culturing. Cultured cardiomyocytes were treated with either LONG R(3)IGF-1, an IGF-1R agonist; picropodophyllin, an IGF-1R autophosphorylation inhibitor; U0126, an inhibitor of extracellular signal-regulated protein kinase (ERK); Leu(27)IGF-2, an IGF-2R agonist; G?6976, a protein kinase C inhibitor; KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII); or KN-92, an L-type calcium channel inhibitor and negative control for KN-93. The cross-sectional area of cultured cardiomyocytes was determined relative to control cardiomyocytes treated with serum-free culture medium. IGF-1R and IGF-2R activation each resulted in ERK signaling, but IGF-2R activation alone induced CaMKII signaling, resulting in hypertrophy of cardiomyocytes in the late gestation sheep fetus. These data suggest that changes in the intrauterine environment that result in increased cardiac IGF-2R may also lead to cardiomyocyte hypertrophy in the fetus and potentially an increased risk of cardiovascular disease in adult life.     

Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein

DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf.     

Extracellular regulated kinase, but not protein kinase C, is an antiapoptotic signal of insulin-like growth factor-1 on cultured cardiac myocytes

This study aims to elucidate the signaling pathway for insulin-like growth factor-1 (IGF-1) in cultured neonatal rat cardiomyocytes and particularly the role of IGF-1 in cardiac apoptosis. IGF-1 stimulated polyphosphoinositide turnover, translocation of protein kinase C (PKC) isoforms (alpha, epsilon, and delta) from the soluble to the particulate fraction, activation of phospholipid-dependent and Ca(2+)-, phospholipid-dependent PKC, and activation of the extracellular-regulated kinase (ERK). IGF-1 attenuated sorbitol-induced cardiomyocyte viability and nuclear DNA fragmentation. These antiapoptotic effects of IGF-1 were blocked by PD-098059 (an MEK inhibitor) but not by bisindolylmaleimide I (BIM, a specific PKC inhibitor). The ERK pathway may therefore be an important component in the mechanism whereby IGF-1 exerts its antiapoptotic effect on the cardiomyocyte.     

Angiogenic oligosaccharides of hyaluronan induce multiple signaling pathways affecting vascular endothelial cell mitogenic and wound healing responses

Hyaluronan (HA) is a large nonsulfated glycosaminoglycan and an important regulator of angiogenesis, in particular, the growth and migration of vascular endothelial cells. We have identified some of the key intermediates responsible for induction of mitogenesis and wound recovery. Treatment of bovine aortic endothelial cells with oligosaccharides of hyaluronan (o-HA) resulted in rapid tyrosine phosphorylation and plasma membrane translocation of phospholipase Cgamma1 (PLCgamma1). Cytoplasmic loading with inhibitory antibodies to PLCgamma1, Gbeta, and Galpha(i/o/t/z) inhibited activation of extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the Galpha(i/o) inhibitor, pertussis toxin, reduced o-HA-induced PLCgamma1 tyrosine phosphorylation, protein kinase C (PKC) alpha and beta1/2 membrane translocation, ERK1/2 activation, mitogenesis, and wound recovery, suggesting a mechanism for o-HA-induced angiogenesis through G-proteins, PLCgamma1, and PKC. In particular, we demonstrated a possible role for PKCalpha in mitogenesis and PKCbeta1/2 in wound recovery. Using antisense oligonucleotides and the Ras farnesylation inhibitor FTI-277, we showed that o-HA-induced bovine aortic endothelial cell proliferation, wound recovery, and ERK1/2 activation were also partially dependent on Ras activation, and that o-HA-stimulated tyrosine phosphorylation of the adapter protein Shc, as well as its association with Sos1. Binding of Src to Shc was required for its activation and for Ras-dependent activation of ERK1/2, cell proliferation, and wound recovery. Neither Src nor Ras activation was inhibited by pertussis toxin, suggesting that their activation was independent of heterotrimeric G-proteins. However, the specific Src kinase inhibitor PP2 inhibited Gbeta subunit co-precipitation with PLCgamma1, suggesting a possible role for Src in activation of PLCgamma1 and interaction between two distinct o-HA-induced signaling pathways.     

Signal transduction pathways triggered by fibroblast growth factor receptor 1 expressed in Xenopus laevis oocytes after fibroblast growth factor 1 addition. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cgamma.

Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) were used as a biological model system to analyse the signal transduction pathways that are triggered by fibroblast growth factor 1 (FGF1). Germinal vesicle breakdown (GVBD) and phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) occured 15 h after FGF1 addition. These events were Ras-dependent as they were blocked by a Ras dominant negative form. The Ras activity was promoted by three upstream effectors, growth factor-bound protein 2 (Grb2), phosphatidylinositol 3-kinase (PI3K) and Src cytoplasmic kinase. Ras activation was inhibited by a Grb2 dominant negative form (P49L), by PI3K inhibitors, including wortmannin, LY294002, the N-SH2 domain of p85alpha PI3K and by the SH2 domain of Src. Src activation induced by FGF1 was blocked by the SH2 domain of Src and PP2, a specific inhibitor of Src. The Grb2 adaptor was recruited by the upstream Src homology 2/alpha-collagen-related (Shc) effector, as the SH2-Shc domain prevented the GVBD and the ERK2 phosphorylation induced by FGF1. The importance of another signalling pathway involving phospholipase Cgamma (PLCgamma) was also investigated. The use of the PLCgamma inhibitory peptide, neomycin and the calcium chelator BAPTA-AM on oocytes expressing FGFR1 or the stimulation by PDGF-BB of oocytes expressing PDGFR-FGFR1 mutated on the PLCgamma binding site, prevented GVBD and ERK2 phosphorylation. This study shows that the transduction cascade induced by the FGFR1-FGF1 interaction in Xenopus oocytes represents the sum of Ras-dependent and PLCgamma-dependent pathways. It emphasizes the role played by PI3K and Src and their connections with the Ras cascade in the FGFR1 signal transduction.     

EGFR and FGFR signaling through FRS2 is subject to negative feedback control by ERK1/2

Fibroblast growth factor (FGF) receptor substrate 2 (FRS2) is a membrane-anchored docking protein that has been shown to play an important role in linking FGF, nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) receptors with the Ras/mitogen-activated protein (MAP) kinase signaling cascade. Here we provide evidence that FRS2 can also play a role in epidermal growth factor (EGF) signaling. Upon EGF stimulation, FRS2 mediates enhanced MAPK activity and undergoes phosphorylation on tyrosine as well as serine/threonine residues. This involves the direct interaction of the FRS2 PTB domain with the EGFR and results in a significantly altered mobility of FRS2 in SDS-PAGE which is also observed in FGF stimulated cells. This migration shift of FRS2 is completely abrogated by U0126, a specific MAPK kinase 1 (MEK1) inhibitor, suggesting that ERK1/2 acts as serine/threonine kinase upstream of FRS2. Indeed, we show that the central portion of FRS2 constitutively associates with ERK1/2, whereas the FRS2 carboxy-terminal region serves as substrate for ERK2 phosphorylation in response to EGF and FGF stimulation. Notably, tyrosine phosphorylation of FRS2 is enhanced when ERK1/2 activation is inhibited after both EGF and FGF stimulation. These results indicate a ligand-stimulated negative regulatory feedback loop in which activated ERK1/2 phosphorylates FRS2 on serine/threonine residues thereby down-regulating its tyrosine phosphorylation. Our findings support a broader role of FRS2 in EGFR-controlled signaling pathways in A-431 cells and provide insight into a molecular mechanism for ligand-stimulated feedback regulation with FRS2 as a central regulatory switch point.     

Model analysis of difference between EGF pathway and FGF pathway

The difference in time course of Ras and mitogen activated protein kinase (MAPK) cascade by different growth factors is considered to be the cause of different cellular responses. We have developed the computer simulation of Ras-MAPK signal transduction pathway containing newly identified negative feedback system, Sprouty, and adaptor molecules. Unexpectedly, negative feedback system did not profoundly affect time course of MAPK activation. We propose the key role of fibroblast growth factor receptor substrate 2 (FRS2) in NGF/FGF pathway for sustained MAPK activation. More Grb2-SOS complexes were recruited to the plasma membrane by binding to membrane-bound FRS2 in FGF pathway than in EGF pathway and caused sustained activation of ERK. The EGF pathway with high concentration of EGF receptor also induced sustained MAPK activation, which is consistent with the results in the PC12 cell overexpressing the EGF receptors. The simulated time courses of FRS2 knock-out cells were consistent with those of the reported experimental results.     

Stimulatory effect of epidermal growth factor on the development of mouse early embryos in vitro.

Effects of epidermal growth factor (EGF) on the development of mouse 2-cell embryos cultured in vitro were investigated. The addition of EGF at a concentration of 0.5 ng/ml enhanced the development of 2-cell embryos during 24 h of incubation. As expected, EGF stimulated the synthesis of DNA in the 2-cell embryos about 4-fold over the control. The growth-promoting effect of EGF seemed to be specific in that other growth factors, such as transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), nerve growth factor (NGF) and fibroblast growth factor (FGF) had no effect on the embryonal development. The addition of EGF also increased the rate of RNA synthesis in a dose-related manner between 0.1 and 50 ng/ml. However, protein synthesis was unaffected by EGF. These results raise the possibility that EGF may participate in the process of early embryogenesis in vivo.     

A differential role of extracellular signal-regulated kinase in stimulated PC12 pheochromocytoma cell movement

Rat pheochromocytoma PC12 cells have been widely used as a cell system for study of growth factor-stimulated cell functions. We report here that nerve growth factor (NGF) stimulated both chemotaxis (directional migration) and chemokinesis (random migration) of PC12 cells. Treatment with a MEK1/2-specific inhibitor (PD98059) or expression of a dominant negative variant of Ras differentially inhibited NGF-stimulated chemotaxis but not chemokinesis of PC12 cells. Priming of PC12 cells with NGF resulted in reduced extracellular signal-regulated kinase (ERK) activation and loss of chemotactic, but not chemokinetic, response. In addition, NGF stimulation of ERK is known to involve an early transient phase of activation followed by a late sustained phase of activation; in contrast, epidermal growth factor (EGF) elicits only early transient ERK activation. We observed that like NGF, EGF also stimulated both chemotaxis and chemokinesis, and treatment with PD98059 abolished the EGF-stimulated chemotaxis. Therefore, the early transient phase of ERK activation functioned in signaling chemotaxis; the late sustained phase of ERK activation did not seem to have an essential role. In addition, our results suggested that chemotactic signaling required a threshold level of ERK activation; at below threshold level of ERK activation, chemotaxis would not occur.     

Cholecystokinin stimulates extracellular signal-regulated kinase through activation of the epidermal growth factor receptor,Yes, and protein kinase C. Signal amplification at the level of Raf by activation of protein kinase Cepsilon

Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through G(q)-coupled receptors (CCK(A) and CCK(B)) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCK(A) and CCK(B). CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cepsilon (PKCepsilon), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCepsilon, but not PKCalpha or PKCdelta, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCK(B) receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCepsilon, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level.