A function for phosphoinositide 3-kinase beta lipid products in coupling beta gamma to Ras activation in response to lysophosphatidic acid

Although Gbetagamma is thought to mediate mitogen-activated protein kinase (MAPK) activation in response to G protein-coupled receptor stimulation, the mechanisms involved in this pathway have not been clearly defined. Phosphoinositide 3-kinase (PI3K) has been proposed as an early intermediate in this process, but its role has remained elusive. We have observed that dominant negative mutants of p110beta, but not of p110gamma, inhibited MAPK stimulation in response to lysophosphatidic acid (LPA). The role of p110beta was located upstream from Ras. To determine which of the lipid or protein kinase activities of p110beta were important for Ras activation, we produced a mutant p110beta lacking the lipid but not the protein kinase activity. This protein displayed a dominant negative activity similar to a kinase-dead mutant, indicating that p110beta lipid kinase activity was essentially involved in Ras activation. In agreement, overexpression of the lipid phosphatase PTEN was found to specifically inhibit Ras stimulation induced by LPA. In addition, we have observed that the PH domain-containing adapter protein Gab1, which is involved in p110beta activation during LPA stimulation, is also implicated in this pathway downstream of p110beta. Indeed, both membrane redistribution and phosphorylation of Gab1 were reduced in the presence of PI3K inhibitors or dominant negative p110beta. Downstream of Gab1, the tyrosine phosphatase SHP2 was found to mediate Ras activation in response to LPA and to be recruited through PI3K and Gab1, because transfection of Gab1 mutant deficient for SHP2 binding inhibited Ras activation without interfering with PI3K activation. We conclude that LPA-induced Ras activation is mediated by a p110beta/Gab1/SHP2 pathway. Moreover, we present data indicating that p110beta is effectively the target of betagamma in this pathway, suggesting that the p110beta/Gab1/SHP2 pathway provides a novel link between betagamma and Ras by integrating two early events of LPA signaling, i.e. Gbetagamma release and tyrosine kinase receptor transactivation.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP2

A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide substrates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2DeltaN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2DeltaN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.     

Signaling of hepatocyte growth factor/scatter factor (HGF) to the small GTPase Rap1 via the large docking protein Gab1 and the adapter protein CRKL

Hepatocyte growth factor (HGF; scatter factor) is a multipotent protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF-receptor c-Met binds and phosphorylates the multisite docking protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1.GTP in the absence of HGF. In contrast, transfection of the GabDeltaYXXP mutant abolished the elevation of Rap1.GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration.     

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613–694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1_613–694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.     

Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling

The Src homology 2-containing phosphotyrosine phosphatase (SHP2) is primarily a positive effector of receptor tyrosine kinase signaling. However, the molecular mechanism by which SHP2 effects its biological function is unknown. In this report, we provide evidence that defines the molecular mechanism and site of action of SHP2 in the epidermal growth factor-induced mitogenic pathway. We demonstrate that SHP2 acts upstream of Ras and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell by interfering with the process of Ras inactivation catalyzed by Ras GTPase-activating protein (RasGAP). It does so by inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane, to its substrate (GTP-Ras) microdomain. Inhibition is achieved through the dephosphorylation of RasGAP binding sites at the level of the plasma membrane. We have identified Tyr992 of the epidermal growth factor receptor (EGFR) to be one such site, since its mutation to Phe renders the EGFR refractory to the effect of dominant-negative SHP2. To our knowledge, this is the first report to outline the site and molecular mechanism of action of SHP2 in EGFR signaling, which may also serve as a model to describe its role in other receptor tyrosine kinase signaling pathways.     

Roles of Gab1 and SHP2 in paxillin tyrosine dephosphorylation and Src activation in response to epidermal growth factor

Epidermal growth factor (EGF) induces paxillin tyrosine dephosphorylation and Src activation, but the signaling pathways that mediate these responses were largely undefined. We found that Gab1, a docking protein for the SHP2 protein-tyrosine phosphatase in EGF-stimulated cells, was associated with paxillin. SHP2 dephosphorylated paxillin and caused dissociation of Csk, a negative regulator of Src, from paxillin but had no effect on paxillin-Src association. A lower level of Src Tyr-530 phosphorylation was detected in paxillin-associated Src in EGF-stimulated cells. Expression of an SHP2 binding defective mutant of Gab1 (Gab1FF) or a catalytically inactive mutant of SHP2 (SHP2DN) prevented paxillin tyrosine dephosphorylation and Src activation induced by EGF. Importantly, Gab1FF blocked paxillin-SHP2 complex formation, Src Tyr-530 dephosphorylation, Erk activation, and cell migration induced by EGF. Inhibition of Src tyrosine kinase activity abrogated EGF-stimulated Erk activation and cell migration. Together, these results reveal that Gab1 recruits SHP2 to dephosphorylate paxillin, leading to dissociation of Csk from the paxillin-Src complex and Src activation and that Src is an SHP2 effector involved in EGF-stimulated Erk activation and cell migration.     

A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.     

Epidermal growth factor-induced DNA synthesis. Key role for Src phosphorylation of the docking protein Gab2

We have previously demonstrated that phosphatidylinositol 3-kinase (PI3-kinase) is necessary and sufficient to account for epidermal growth factor (EGF)-induced mitogenesis in rat primary hepatocytes. A cytosolic Gab2-containing complex accounts for >80% of the total EGF-induced PI3-kinase activity (Kong, M., Mounier, C., Wu, J., and Posner, B. I. (2000) J. Biol. Chem. 275, 36035-36042), suggesting a key role for Gab2 in EGF-induced mitogenesis. Here, we demonstrate that PP1, a selective inhibitor of Src family kinases, blocks the EGF-induced Gab2 tyrosine phosphorylation without inhibiting EGF-induced phosphorylation of the EGF receptor, ErbB3, or Shc. We also show that Gab2 phosphorylation is increased in Csk knockout cells in which Src family kinases are constitutively activated. Furthermore, PP1 blocks Gab2-associated downstream events including EGF-induced PI3-kinase activation, Akt phosphorylation, and DNA synthesis. We demonstrate that Gab2 and Src are constitutively associated. Since this association involves the proline-rich sequences of Gab2, it probably involves the Src homology 3 domain of Src kinase. Mutation of the proline-rich sequences in Gab2 prevented EGF-induced Gab2 phosphorylation, PI3-kinase/Akt activation, and DNA synthesis, demonstrating that Gab2 phosphorylation is critical for EGF-induced mitogenesis and is not complemented by ErbB3 or Shc phosphorylation. We also found that overexpression of a Gab2 mutant lacking SHP2 binding sites increased EGF-induced Gab2 phosphorylation and the activation of PI3-kinase but blocked activation of MAPK. In addition, we demonstrated that the Src-induced response was down-regulated by Gab2-associated SHP2. In summary, our results have defined the role for Src activation in EGF-induced hepatic mitogenesis through the phosphorylation of Gab2 and the activation of the PI3-kinase cascade.     

The scaffolding adapter Gab2, via Shp-2, regulates kit-evoked mast cell proliferation by activating the Rac/JNK pathway

The scaffolding adapter Gab2 mediates cell signaling and responses evoked by various extracellular stimuli including several growth factors. Kit, the receptor for stem cell factor (SCF), plays a critical role in the proliferation and differentiation of a variety of cell types, including mast cells. Kit, via Tyr(567) and Tyr(719), activates Src family kinases (SFK) and PI3K respectively, which converge on the activation of a Rac/JNK pathway required for mast cell proliferation. However, how Kit Tyr(567) signals to Rac/JNK is not well understood. By analyzing Gab2(-/-) mast cells, we find that Gab2 is required for SCF-evoked proliferation, activation of Rac/JNK, and Ras. Upon Kit activation in wild-type mast cells, Gab2 becomes tyrosyl-phosphorylated and associates with Kit and Shp-2. Tyr(567), an SFK binding site in Kit, and SFK activity were required for Gab2 tyrosyl phosphorylation and association with Shp-2. By re-expressing Gab2 or a Gab2 mutant that cannot bind Shp-2 in Gab2(-/-) mast cells or acutely by deleting Shp-2 in mast cells, we found that Gab2 requires Shp-2 for SCF-evoked Rac/JNK, Ras activation, and mast cell proliferation. Lastly, by analyzing mast cells from mice with compound Gab2 and Kit Y719F mutations (i.e., Gab2(-/-): KitY719F/Y719F mice), we find that Gab2, acting in a parallel pathway to PI3K from Kit Tyr(719), regulates mast cell proliferation and development in specific tissues. Our data show that Gab2 via Shp-2 is critical for transmitting signals from Kit Tyr(567) to activate the Rac/JNK pathway controlling mast cell proliferation, which likely contributes to mast cell development in specific tissues.     

Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor

Grb2-associated binder-1 (Gab1) is a multisite docking protein containing a pleckstrin homology (PH) domain, multiple potential tyrosine phosphorylation sites, and several proline-rich sequences. Gab1 becomes tyrosine-phosphorylated in cells stimulated with growth factors, cytokines, and ligands for G protein-coupled receptors. A major Gab1-binding protein detected in cells treated with extracellular stimuli is the tyrosine phosphatase, SHP2. Although the role of SHP2-Gab1 interaction in cell signaling has not yet been characterized, SHP2 is known to mediate mitogen-activated protein (MAP) kinase activation induced by the epidermal growth factor (EGF). However, the mechanism by which the SHP2 phosphatase exerts a positive signaling role remains obscure. In this study, we prepared Gab1 mutants lacking the SHP2 binding site (Gab1Y627F), the phosphatidylinositol 3-kinase (PI3K) binding sites (Gab1DeltaPI3K), and the PH domain (Gab1DeltaPH). Expression of Gab1Y627F blocked the extracellular signal-regulated kinase-2 (ERK2) activation by lysophosphatidic acid (LPA) and EGF. Conversely, expression of the wild-type Gab1 in HEK293 cells augmented the LPA receptor Edg2-mediated ERK2 activation. Whereas the PH domain was required for Gab1 mediation of ERK2 activation by LPA, it was not essential for EGF-induced ERK2 activation. Expression of Gab1DeltaPI3K had no apparent effect on ERK2 activation by LPA and EGF in the cells that we have examined. These results establish a role for Gab1 in the LPA-induced MAP kinase pathway and clearly demonstrate that Gab1-SHP2 interaction is essential for ERK2 activation by LPA and EGF. These findings also suggest that the positive role of SHP2 in the MAP kinase pathway depends on its interaction with Gab1.     

G‐CSF‐dependent neutrophil differentiation requires downregulation of MAPK activities through the Gab2 signaling pathway

Granulocyte colony‐stimulating factor (G‐CSF) stimulation of myeloid cells induced tyrosine‐phosphorylation of cellular proteins. One of the tyrosine‐phosphorylated proteins was found to be a scaffold protein, Grb2‐associated binding protein 2 (Gab2). Another member of Gab family protein, Gab3, was exogenously overexpressed in neutrophil progenitor cells to make the Gab3 protein to compete with the endogenous Gab2 for the G‐CSF‐dependent signaling. In Gab3‐overexpressed cells, the level of tyrosine phosphorylation of endogenous Gab2 by G‐CSF stimulation was markedly downregulated, while the phosphorylation of Gab3 was significantly enhanced. The Gab3‐overexpressed cells continuously proliferated in the medium containing G‐CSF and lost the ability to differentiate to the mature neutrophil, characterized by the lobulated nucleus. The G‐CSF stimulation‐dependent tyrosine phosphorylation of Gab3, the association of SHP2 to Gab3 and the following mitogen‐activated protein kinase (MAPK) activation were prolonged in the Gab3‐overexpressed cells, compared to the parental cells, where the binding of SHP2 to Gab2 protein and thereby the activation of MAPK were not sustained after G‐CSF stimulation. Inhibition of MAPK by pharmaceutical inhibitor restored the Gab3‐overexpressed cells to the ability to differentiate to mature neutrophil. Therefore, G‐CSF‐dependent Gab2 phosphorylation and following its downregulation led the short‐term MAPK activation. The downregulation of MAPK after transient Gab2 phosphorylation was necessary for the consequent neutrophil differentiation induced by G‐CSF stimulation.     

Biochemical and biological responses induced by coupling of Gab1 to phosphatidylinositol 3-kinase in RET-expressing cells

Grb2-associated binder-1 (Gab1) is a docking protein closely related to insulin receptor substrates. We previously reported that tyrosine 1062 in RET receptor tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF) represents a binding site for the Shc-Grb2-Gab1 complex, and that the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and SHP2 tyrosine phosphatase is associated with Gab1 in GDNF-treated cells. In the present study, we further analyzed the physiological roles of Gab1 downstream of RET, using Gab1 mutants that lack the binding sites for PI3K (Gab1 PI3K-m) or SHP-2 (Gab1 SHP2-m). Expression of Gab1 PI3K-m in SK-N-MC human primitive neuroectodermal tumor cells expressing wild-type RET markedly impaired Akt phosphorylation, Rac1 activation, and lamellipodia formation that were induced by GDNF whereas expression of Gab1 SHP2-m partially impaired Erk activation. Furthermore, expression of Gab1 PI3K-m, but not Gab1 SHP2-m, in TT human medullary thyroid carcinoma cells expressing RET with a multiple endocrine neoplasia 2A mutation enhanced cytochrome c release, and apoptosis induced by etoposide, suggesting that PI3K is involved in survival of TT cells via a mitochondrial pathway. These findings demonstrated that coupling of Gab1 to PI3K is important for biological responses in RET-expressing cells.     

Ras GTPase-activating protein binds to Akt and is required for its activation

RasGAP (Ras GTPase-activating protein) is a negative regulator as well as a downstream effector of Ras. To identify partners of RasGAP we used it as the bait in a yeast two-hybrid screen. This resulted in discovering its interaction with Akt. Overexpression of RasGAP or a mutant lacking the GTPase-activating domain (nGAP) enhanced phosphorylation and activity of Akt, which was dependent on the upstream integrin-linked kinase. Also, nGAP protected the cells against staurosporin-induced apoptosis through an Akt-dependent pathway. To determine the role of RasGAP in receptor-mediated activation of Akt, we used short hairpin RNA interference to knock out endogenous RasGAP expression. Although this procedure resulted in enhanced Ras activity, it inhibited Akt phosphorylation. Thus, we propose that Ras-GAP interacts with Akt and is necessary for its activation, possibly via integrin-linked kinase-mediated phosphorylation of Ser-473. The data suggest that this effect is independent of Ras activity.     

ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase

We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2.     

Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation

In epidermis, Ras can influence proliferation and differentiation; however, regulators of epidermal Ras function are not fully characterized, and Ras effects on growth and differentiation are controversial. EGF induced Ras activation in epidermal cells along with phosphorylation of the multisubstrate docking protein Gab1 and its binding to SHP-2. Expression of mutant Gab1Y627F deficient in SHP-2 binding or dominant-negative SHP-2C459S reduced basal levels of active Ras and downstream MAPK proteins and initiated differentiation. Differentiation triggered by both Gab1Y627F and SHP-2C459S could be blocked by coexpression of active Ras, consistent with Gab1 and SHP-2 action upstream of Ras in this process. To study the role of Gab1 and SHP-2 in tissue, we generated human epidermis overexpressing active Gab1 and SHP-2. Both proteins stimulated proliferation. In contrast, Gab1Y627F and SHP-2C459S inhibited epidermal proliferation and enhanced differentiation. Consistent with a role for Gab1 and SHP-2 in sustaining epidermal Ras/MAPK activity, Gab1-/- murine epidermis displayed lower levels of active Ras and MAPK with postnatal Gab1-/- epidermis, demonstrating the hypoplasia and enhanced differentiation seen previously with transgenic epidermal Ras blockade. These data provide support for a Ras role in promoting epidermal proliferation and opposing differentiation and indicate that Gab1 and SHP-2 promote the undifferentiated epidermal cell state by facilitating Ras/MAPK signaling.     

Enhanced phosphatidylinositol 3-kinase activity and high phosphorylation state of its downstream signalling molecules mediated by ret with the MEN 2B mutation.

We compared the intracellular signalling pathways through Ret tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF), multiple endocrine neoplasia (MEN) 2A, or MEN 2B mutation. Tyrosine phosphorylation of Grb2-associated binder-1 (Gab1) and activation of phosphatidylinositol 3-kinase (PI 3-kinase) were induced at higher levels by GDNF stimulation or the MEN 2B mutation than by the MEN 2A mutation. Tyrosine-phosphorylated Gab1 was a major component that interacted with the active PI 3-kinase in vivo. In addition, we found that p62Dok and PKB/Akt were phosphorylated in a PI 3-kinase-dependent manner and the levels of their phosphorylation were significantly higher in the MEN 2B transfectant than in the MEN 2A transfectant. Tyrosine phosphorylation of p62Dok resulted in its complex formation with the Ras GTPase-activating protein (RasGAP) and the Nck adaptor protein. These findings thus suggested that high levels of activation of PI 3-kinase and of phosphorylation of its downstream signalling molecules may be associated with the clinical phenotype of MEN 2B.     

New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway

Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K). Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (betac) and the IL-2 receptor beta chain (IL-2Rbeta), lack such sites, leaving the mechanism by which they activate PI-3K unclear. Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway. Analysis of Y-->F and "add-back" mutants of betac shows that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation. When fused directly to a mutant form of IL-2Rbeta that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation. Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation. Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation. The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth. Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2. Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation.