Tyrosyl phosphorylation of Shp2 is required for normal ERK activation in response to some,but not all,growth factors

The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the "Grb2-adapter" model of Shp2 action.     

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis

Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor β (PDGFRβ). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions.     

A novel function for the protein tyrosine phosphatase Shp2 during lung branching morphogenesis

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis.     

Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system.

Activated receptor tyrosine kinases induce a large number of tyrosine phosphorylation-dependent protein-protein interactions through which they mediate their various ligand-exerted functions including regulation of proliferation, differentiation and survival. TrkB receptor tyrosine kinase activated by binding of brain-derived neurotrophic factor (BDNF) also stimulates various protein interactions in a tyrosine phosphorylation-dependent manner in neuronal cells. To examine tyrosine phosphorylation-dependent interactions stimulated by active TrkB, we developed a modified yeast two-hybrid system, which we call the yeast two-and-a-half-hybrid system. In this system, yeast was engineered to express a tyrosine kinase domain of TrkB as an effector, in addition to two fusion proteins with GAL4 DNA-binding and GAL4 activation domains as bait and prey proteins, respectively. Using this system with Shp2 as the bait, we demonstrated that Shp2 interacts directly with BIT/SHPS-1 (also called SIRP) and Grb2 depending on tyrosine phosphorylation mediated by TrkB. Furthermore, we screened an adult human brain cDNA library with the yeast two-and-a-half-hybrid system in order to identify other Shp2-binding proteins in TrkB-stimulated tyrosine phosphorylation signaling. We found that fibroblast growth factor receptor substrate 2beta (FRS2beta), also called SNT2, interacts with Shp2 dependently on TrkB-mediated tyrosine phosphorylation of FRS2beta/SNT2. Therefore, we show that the two-and-a-half-hybrid system is a powerful tool for studying tyrosine phosphorylation-dependent protein-protein interactions in intracellular signaling pathways stimulated by TrkB receptor tyrosine kinase.     

Involvement of phosphatases in the anchorage-dependent regulation of ERK2 activation

Activation of extracellular signal-regulated kinase (ERK) is known to be regulated by cell adhesion, namely "anchorage dependence". Most studies on the anchorage-dependent regulation have focused on the upstream activating components. We previously reported that the focal adhesion protein vinexin beta can induce the anchorage-independent activation of ERK2. We show here that vinexin beta-induced anchorage-independent activation of ERK2 involves prevention of the dephosphorylation of ERK2, but not the promotion of MEK1 or Raf1 activity. Furthermore, knockdown of vinexin beta resulted in a faster dephosphorylation of ERK2 in A549 cells. Moreover, the coexpression of MKP3/rVH6, an ERK2 specific phosphatase, suppressed the anchorage-independent activation of ERK2 induced by vinexin beta. These results suggest that vinexin beta can prevent the dephosphorylation of ERK2 stimulated by cell detachment, leading to the anchorage-independent activation of ERK2. Furthermore, we found that phosphatase activity directed against activated ERK2 was higher in suspended cells than in adherent cells. In addition, orthovanadate efficiently induces anchorage-independent activation of ERK2 without marked activation of MEK1 in NIH3T3 cells. These observations suggest that the anchorage dependence of ERK1/2 activation is regulated not only by upstream kinases, Raf1 and MEK, but also by phosphatases acting against ERK1/2 and that vinexin beta can induce anchorage-independent activation of ERK by preventing the inactivation of ERK1/2.     

An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stem cell survival

Little is known about how growth factors control tissue stem cell survival and proliferation. We analyzed mice with a null mutation of Shp2 (Ptpn11), a key component of receptor tyrosine kinase signaling. Null embryos die peri-implantation, much earlier than mice that express an Shp2 truncation. Shp2 null blastocysts initially develop normally, but they subsequently exhibit inner cell mass death, diminished numbers of trophoblast giant cells, and failure to yield trophoblast stem (TS) cell lines. Molecular markers reveal that the trophoblast lineage, which requires fibroblast growth factor-4 (FGF4), is specified but fails to expand normally. Moreover, deletion of Shp2 in TS cells causes rapid apoptosis. We show that Shp2 is required for FGF4-evoked activation of the Src/Ras/Erk pathway that culminates in phosphorylation and destabilization of the proapoptotic protein Bim. Bim depletion substantially blocks apoptosis and significantly restores Shp2 null TS cell proliferation, thereby establishing a key mechanism by which FGF4 controls stem cell survival.     

Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway

Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2-Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.     

Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.     

Vinexin beta regulates the anchorage dependence of ERK2 activation stimulated by epidermal growth factor

ERK is activated by soluble growth factors in adherent cells. However, activation of ERK is barely detectable and not sufficient for cell proliferation in non-adherent cells. Here, we show that exogenous expression of vinexin beta, a novel focal adhesion protein, allows anchorage-independent ERK2 activation stimulated by epidermal growth factor. In contrast, expression of vinexin beta had no effect on ERK2 activation in adherent cells, suggesting that vinexin beta regulates the anchorage dependence of ERK2 activation. Analyses using deletion mutants demonstrated that a linker region between the second and third SH3 domains of vinexin beta, but not the SH3 domains, is required for this function of vinexin beta. To evaluate the pathway regulating the anchorage dependence of ERK2 activation, we used a dominant-negative mutant of p21-activated kinase (PAK) and a specific inhibitor (H89) of cAMP-dependent protein kinase (PKA) because PAK and PKA are known to regulate the anchorage dependence of ERK2 activation. The dominant-negative mutant of PAK suppressed the anchorage-independent ERK2 activation induced by expression of vinexin beta. The dominant-negative mutant of vinexin beta inhibited the anchorage-independent ERK2 activation induced by the PKA inhibitor. Together, these observations indicate that vinexin beta plays a key role in regulating the anchorage dependence of ERK2 activation through PKA-PAK signaling.     

Shp2, an SH2-containing protein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty

Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, we demonstrate that Sprouty (Spry) is a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 was able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry resulted in dissociation of Spry from Grb2. Furthermore, Shp2 could reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry.     

Netrin stimulates tyrosine phosphorylation of the UNC-5 family of netrin receptors and induces Shp2 binding to the RCM cytodomain

Caenorhabditis elegans UNC-5 and its mammalian homologues such as RCM are receptors for the secreted axon guidance cue UNC-6/netrin and are required to mediate the repulsive effects of UNC-6/netrin on growth cones. We find that C. elegans UNC-5 and mouse RCM are phosphorylated on tyrosine in vivo. C. elegans UNC-5 tyrosine phosphorylation is reduced in unc-6 null mutants, and RCM tyrosine phosphorylation is induced by netrin-1 in transfected HEK-293 cells, demonstrating that phosphorylation of UNC-5 proteins is enhanced by UNC-6/netrin stimulation in both worms and mammalian cells. An activated Src tyrosine kinase induces phosphorylation of RCM at multiple cytoplasmic tyrosine residues creating potential binding sites for cytoplasmic signaling proteins. Indeed, the NH2-terminal SH2 domain of the Shp2 tyrosine phosphatase bound specifically to a Tyr(568) RCM phosphopeptide. Furthermore, Shp2 associated with RCM in a netrin-dependent manner in transfected cells, and co-immunoprecipitated with RCM from an embryonic mouse brain lysate. A Y568F mutant RCM receptor failed to bind Shp2 and was more highly phosphorylated on tyrosine than the wild type receptor. These results suggest that netrin-stimulated phosphorylation of RCM Tyr(568) recruits Shp2 to the cell membrane where it can potentially modify RCM phosphorylation and function.     

Direct binding of activated c-Src to the beta 3-adrenergic receptor is required for MAP kinase activation

Both beta(2)- and beta(3)-adrenergic receptors (ARs) are able to activate the extracellular signal-regulated kinase (ERK) pathway. We previously showed that c-Src is required for ERK activation by beta(2)AR and that it is recruited to activated beta(2)AR through binding of the Src homology 3 (SH3) domain to proline-rich regions of the adapter protein beta-arrestin1. Despite the absence of sites for phosphorylation and beta-arrestin binding, ERK activation by beta(3)AR still requires c-Src. Agonist activation of beta(2)AR, but not beta(3)AR, led to redistribution of green fluorescent protein-tagged beta-arrestin to the plasma membrane. In beta-arrestin-deficient COS-7 cells, beta-agonist-dependent co-precipitation of c-Src with the beta(2)AR required exogenous beta-arrestin, but activated beta(3)AR co-precipitated c-Src in the absence or presence of beta-arrestin. ERK activation and Src co-precipitation with beta(3)AR also occurred in adipocytes in an agonist-dependent and pertussis toxin-sensitive manner. Protein interaction studies show that the beta(3)AR interacts directly with the SH3 domain of Src through proline-rich motifs (PXXP) in the third intracellular loop and the carboxyl terminus. ERK activation and Src co-precipitation were abolished in cells expressing point mutations in these PXXP motifs. Together, these data describe a novel mechanism of ERK activation by a G protein-coupled receptor in which the intracellular domains directly recruit c-Src.     

Maximal beta3-adrenergic regulation of lipolysis involves Src and epidermal growth factor receptor-dependent ERK1/2 activation

Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.     

Epidermal growth factor receptor (EGFR)-mediated positive feedback of protein-tyrosine phosphatase epsilon (PTPepsilon) on ERK1/2 and AKT protein pathways is required for survival of human breast cancer cells

Increased tyrosine phosphorylation has been correlated with human cancer, including breast cancer. In general, the activation of tyrosine kinases (TKs) can be antagonized by the action of protein-tyrosine phosphatases (PTPs). However, in some cases PTPs can potentiate the activation of TKs. In this study, we have investigated the functional role of PTPε in human breast cancer cell lines. We found the up-regulation and activation of receptor PTPε (RPTPε) in MCF-7 cells and MDA-MB-231 upon PMA, FGF, and serum stimulation, which depended on EGFR and ERK1/2 activity. Diminishing the expression of PTPε in human breast cancer cells abolished ERK1/2 and AKT activation, and decreased the viability and anchorage-independent growth of the cells. Conversely, stable MCF-7 cell lines expressing inducible high levels of ectopic PTPε displayed higher activation of ERK1/2 and anchorage-independent growth. Our results demonstrate that expression of PTPε is up-regulated and activated in breast cancer cell lines, through EGFR, by sustained activation of the ERK1/2 pathway, generating a positive feedback regulatory loop required for survival of human breast cancer cells.     

Specific inhibition of growth factor-stimulated extracellular signal-regulated kinase 1 and 2 activation in intact cells by electroporation of a growth factor receptor-binding protein 2-Src homology 2 binding peptide.

Activation of the Ras pathway is central to mitogenesis by a variety of growth factors such as the epidermal growth factor, platelet-derived growth factor, or hepatocyte growth factor. Ras activation requires the function of adaptors such as growth factor receptor-binding protein 2, which can bind either directly or indirectly through Src homology 2 domains to the activated receptor. To examine the role of the Src homology 2 domain of growth factor receptor-binding protein 2 in the mitogenic response triggered by these growth factors, we introduced a peptide (PVPE-phosphono-methylphenylalanine-INQS) that can selectively bind this domain into mouse, rat, or human cells growing on conductive indium-tin oxide-coated glass by in situ electroporation. Cells were subsequently stimulated with growth factors and assessed for activation of a downstream target, extracellular signal-regulated kinase (ERK) 1/2, by probing with antibodies specific for its activated form. Electrodes and slides were configured to provide nonelectroporated control cells side by side with the electroporated ones, both growing on the same type of indium-tin oxide-coated glass surface. The data demonstrate that the peptide can cause a dramatic inhibition of epidermal growth factor or platelet-derived growth factor-mediated ERK1/2 activation and DNA synthesis in vivo, compared with its control phenylalanine-containing counterpart. In contrast, the peptide had a very limited effect on hepatocyte growth factor-triggered ERK1/2 activation and DNA synthesis. These results demonstrate the potential of the in situ electroporation approach described here in the study of the coupling of activated receptor tyrosine kinases to the ERK1/2 cascade.     

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.     

Both Gs and Gi proteins are critically involved in isoproterenol-induced cardiomyocyte hypertrophy

Activation of beta-adrenoreceptors induces cardiomyocyte hypertrophy. In the present study, we examined isoproterenol-evoked intracellular signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs) and cardiomyocyte hypertrophy. Inhibitors for cAMP and protein kinase A (PKA) abolished isoproterenol-evoked ERK activation, suggesting that Gs protein is involved in the activation. Inhibition of Gi protein by pertussis toxin, however, also suppressed isoproterenol-induced ERK activation. Overexpression of the Gbetagamma subunit binding domain of the beta-adrenoreceptor kinase 1 and of COOH-terminal Src kinase, which inhibit functions of Gbetagamma and the Src family tyrosine kinases, respectively, also inhibited isoproterenol-induced ERK activation. Overexpression of dominant-negative mutants of Ras and Raf-1 kinase and of the beta-adrenoreceptor mutant that lacks phosphorylation sites by PKA abolished isoproterenol-stimulated ERK activation. The isoproterenol-induced increase in protein synthesis was also suppressed by inhibitors for PKA, Gi, tyrosine kinases, or Ras. These results suggest that isoproterenol induces ERK activation and cardiomyocyte hypertrophy through two different G proteins, Gs and Gi. cAMP-dependent PKA activation through Gs may phosphorylate the beta-adrenoreceptor, leading to coupling of the receptor from Gs to Gi. Activation of Gi activates ERKs through Gbetagamma, Src family tyrosine kinases, Ras, and Raf-1 kinase.