Analysis of a Shc family adaptor protein, ShcD/Shc4, that associates with muscle-specific kinase

Shc family proteins serve as phosphotyrosine adaptor molecules in various receptor-mediated signaling pathways. In mammals, three distinct Shc genes have been described that encode proteins characterized by two phosphotyrosine-interaction modules, an amino-terminal phosphotyrosine binding (PTB) domain and a carboxy-terminal Src homology 2 domain. Here, we report the analysis of an uncharacterized fourth Shc family protein, ShcD/Shc4, that is expressed in adult brain and skeletal muscle. Consistent with this expression pattern, we find that ShcD can associate via its PTB domain with the phosphorylated muscle-specific kinase (MuSK) receptor tyrosine kinase and undergo tyrosine phosphorylation downstream of activated MuSK. Interestingly, additional sites of tyrosine phosphorylation, including a novel Grb2 binding site, are present on ShcD that are not found in other Shc family proteins. Activation of MuSK upon agrin binding at the neuromuscular junction (NMJ) induces clustering and tyrosine phosphorylation of acetylcholine receptors (AChRs) required for synaptic transmission. ShcD is coexpressed with MuSK in the postsynaptic region of the NMJ, and in cultured myotubes stimulated with agrin, expression of ShcD appears to be important for early tyrosine phosphorylation of the AChR. Thus, we have characterized a new member of the Shc family of docking proteins, which may mediate a specific aspect of signaling downstream of the MuSK receptor.     

Interaction of Shc with Grb2 regulates association of Grb2 with mSOS.

The adapter protein Shc has been implicated in Ras signaling via many receptors, including the T-cell antigen receptor (TCR), B-cell antigen receptor, interleukin-2 receptor, interleukin-3 receptor, erythropoietin receptor, and insulin receptor. Moreover, transformation via polyomavirus middle T antigen is dependent on its interaction with Shc and Shc tyrosine phosphorylation. One of the mechanisms of TCR-mediated, tyrosine kinase-dependent Ras activation involves the simultaneous interaction of phosphorylated Shc with the TCR zeta chain and with a second adapter protein, Grb2. Grb2, in turn, interacts with the Ras guanine nucleotide exchange factor mSOS, thereby leading to Ras activation. Although it has been reported that in fibroblasts Grb2 and mSOS constitutively associate with each other and that growth factor stimulation does not alter the levels of Grb2:mSOS association, we show here that TCR stimulation leads to a significant increase in the levels of Grb2 associated with mSOS. This enhanced Grb2:mSOS association, which occurs through an SH3-proline-rich sequence interaction, is regulated through the SH2 domain of Grb2. The following observations support a role for Shc in regulating the Grb2:mSOS association: (i) a phosphopeptide corresponding to the sequence surrounding Tyr-317 of Shc, which displaces Shc from Grb2, abolished the enhanced association between Grb2 and mSOS; and (ii) addition of phosphorylated Shc to unactivated T cell lysates was sufficient to enhance the interaction of Grb2 with mSOS. Furthermore, using fusion proteins encoding different domains of Shc, we show that the collagen homology domain of Shc (which includes the Tyr-317 site) can mediate this effect. Thus, the Shc-mediated regulation of Grb2:mSOS association may provide a means for controlling the extent of Ras activation following receptor stimulation.     

Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras.

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.     

Signal transduction by the alpha 6 beta 4 integrin: distinct beta 4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes.

We have examined the mechanism of signal transduction by the hemidesmosomal integrin alpha 6 beta 4, a laminin receptor involved in morphogenesis and tumor progression. Immunoprecipitation and immune complex kinase assays indicated that antibody- or laminin-induced ligation of alpha 6 beta 4 causes tyrosine phosphorylation of the beta 4 subunit in intact cells and that this event is mediated by a protein kinase(s) physically associated with the integrin. Co-immunoprecipitation and GST fusion protein binding experiments showed that the adaptor protein Shc forms a complex with the tyrosine-phosphorylated beta 4 subunit. Shc is then phosphorylated on tyrosine residues and recruits the adaptor Grb2, thereby potentially linking alpha 6 beta 4 to the ras pathway. The beta 4 subunit was found to be phosphorylated at multiple tyrosine residues in vivo, including a tyrosine-based activation motif (TAM) resembling those found in T and B cell receptors. Phenylalanine substitutions at the beta 4 TAM disrupted association of alpha 6 beta 4 with hemidesmosomes, but did not interfere with tyrosine phosphorylation of Shc and recruitment of Grb2. These results indicate that signal transduction by the alpha 6 beta 4 integrin is mediated by an associated tyrosine kinase and that phosphorylation of distinct sites in the beta 4 tail mediates assembly of the hemidesmosomal cytoskeleton and recruitment of Shc/Grb2.     

Tumor Promoter Arsenite Activates Extracellular Signal-Regulated Kinase through a Signaling Pathway Mediated by Epidermal Growth Factor Receptor and Shc

Although arsenite is an established carcinogen, the mechanisms underlying its tumor-promoting properties are poorly understood. Previously, we reported that arsenite treatment leads to the activation of the extracellular signal-regulated kinase (ERK) in rat PC12 cells through a Ras-dependent pathway. To identify potential mediators of the upstream signaling cascade, we examined the tyrosine phosphorylation profile in cells exposed to arsenite. Arsenite treatment rapidly stimulated tyrosine phosphorylation of several proteins in a Ras-independent manner, with a pattern similar to that seen in response to epidermal growth factor (EGF) treatment. Among these phosphorylated proteins were three isoforms of the proto-oncoprotein Shc as well as the EGF receptor (EGFR). Tyrosine phosphorylation of Shc allowed for enhanced interactions between Shc and Grb2 as identified by coimmunoprecipitation experiments. The arsenite-induced tyrosine phosphorylation of Shc, enhancement of Shc and Grb2 interactions, and activation of ERK were all drastically reduced by treatment of cells with either the general growth factor receptor poison suramin or the EGFR-selective inhibitor tyrphostin AG1478. Down-regulation of EGFR expression through pretreatment of cells with EGF also attenuated ERK activation and Shc tyrosine phosphorylation in response to arsenite treatment. These results demonstrate that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that arsenite acts as a tumor promoter largely by usurping this growth factor signaling pathway.     

Expression of the ErbB4 receptor causes reversal regulation of PP2A in the Shc signal transduction pathway in human cancer cells

Expression of ErbB4 receptor is correlated with the incidence of non-metastatic types of human cancers, whereas the overexpression of other ErbB receptor families (ErbB1/EGFR, ErbB2 and ErbB3) is correlated to the formation of metastatic tumors. However, the molecular mechanism underlying this phenomenon has been unclear. Earlier, we demonstrated that okadaic acid (OA), an inhibitor of a serine/threonine phosphatase PP2A, stimulated the growth hormone-induced ERK phosphorylation in the wild type Chinese hamster ovary (CHO) cells and the cells expressing ErbB1 receptor, but suppressed ERK activation in CHO cells that express ErbB4 receptor. PP2A had been understood as a negative regulator of the growth hormone-stimulated signal transduction pathways, however, this observation suggested that expression of ErbB4 receptor reversed the regulation of PP2A in the ErbB4 signalling pathway. In this study, we found that OA suppressed phosphorylation of Shc at Tyr317, therefore it down-regulated ERK phosphorylation in the ErbB4 expressing CHO cells. Accordingly, basal PP2A contributed to the phosphorylation of Shc Tyr317 in ErbB4 expressing CHO cells, nevertheless it had been reported that PP2A negatively regulates Shc tyrosine phosphorylation in the EGF- or IGF-I-induced signalling pathways. By testing OA for human cancer cell lines that express different types of ErbB receptors, we found that ErbB4 receptor expression was accompanied with positive regulation of PP2A for phosphorylation of Shc Tyr317 and its downstream ERK phosphorylation in MCF-7 and SK-OV-3 cell lines, but not in LNCaP and PC-3 cells. Thus, PP2A regulates the ERK activity in a cell-specific manner, and it is speculated that distinct regulation of PP2A in the ErbB4 receptor signalling pathway may cause a difference in progression of cancer phenotypes.     

Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors.     

Neuregulins mediate calcium-induced glucose transport during muscle contraction

Neuregulin, a growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and exercise, neuregulins stimulate glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscle. Like muscle contraction, neuregulins have additive effects with insulin on glucose uptake. Therefore, we examined whether neuregulins are involved in the mechanism by which muscle contraction regulates glucose transport. We show that caffeine-induced increases in cytosolic Ca2+ mediate a metalloproteinase-dependent release of neuregulins, which stimulates tyrosine phosphorylation of ErbB4 receptors. Activation of ErbB4 is necessary for Ca2+-derived effects on glucose transport. Furthermore, blockage of ErbB4 abruptly impairs contraction-induced glucose uptake in slow twitch muscle fibers, and to a lesser extent, in fast twitch muscle fibers. In conclusion, we provide evidence that contraction-induced activation of neuregulin receptors is necessary for the stimulation of glucose transport and a key element of energetic metabolism during muscle contraction.     

The SH2 inositol 5-phosphatase Ship1 is recruited in an SH2-dependent manner to the erythropoietin receptor

Ship1 (SH2 inositol 5-phosphatase 1) has been shown to be a target of tyrosine phosphorylation downstream of cytokine and immunoregulatory receptors. In addition to its catalytic activity on phosphatidylinositol substrates, it can serve as an adaptor protein in binding Shc and Grb2. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to activate the tyrosine phosphorylation of Shc, resulting in recruitment of Grb2. However, the mechanism by which the erythropoietin receptor (EPO-R) recruits Shc remains unknown. EPO activates the tyrosine phosphorylation of Ship1, resulting in the interdependent recruitment of Shc and Grb2. Ship1 is recruited to the EPO-R in an SH2-dependent manner. Utilizing a panel of EPO-R deletion and tyrosine mutants, we have discovered remarkable redundancy in Ship1 recruitment. EPO-R Tyr(401) appears to be a major site of Ship1 binding; however, Tyr(429) and Tyr(431) can also serve to recruit Ship1. In addition, we have shown that EPO stimulates the formation of a ternary complex consisting of Ship1, Shc, and Grb2. Ship1 may modulate several discrete signal transduction pathways. EPO-dependent activation of ERK1/2 and protein kinase B (PKB)/Akt was examined utilizing a panel of EPO-R deletion mutants. Activation of ERK1/2 was observed in EPO-RDelta99, which retains only the most proximal tyrosine, Tyr(343). In contrast, EPO-dependent PKB activation was observed in EPO-RDelta43, but not in EPO-RDelta99. It appears that EPO-dependent PKB activation is downstream of a region that indirectly couples to phosphatidylinositol 3-kinase.     

Mechanism of SHIP-mediated inhibition of insulin- and platelet-derived growth factor-stimulated mitogen-activated protein kinase activity in 3T3-L1 adipocytes

The Src homology 2-containing 5' inositolphosphatases (SHIP and SHIP2) dephosphorylate 3'-phosphorylated PtdIns on the 5' position, decreasing intracellular levels of PtdIns 3,4,5-P3. In the current study, we investigated the role of SHIP in insulin and platelet-derived growth factor (PDGF) signaling by expressing wild-type (WT) and catalytically inactive SHIPDeltaIP in 3T3-L1 adipocytes, utilizing adenoviral infection. Insulin and PDGF both stimulated tyrosine phosphorylation of SHIP-WT and of SHIPDeltaIP, and tyrosine phosphorylation of SHIP-associated proteins increased after ligand stimulation. Tyrosine-phosphorylated PDGFR, IR, and insulin receptor substrate-1 all immunoprecipitated with SHIP. Expression of WT and DeltaIP mutant SHIP did not affect tyrosine phosphorylation of either the insulin or the PDGF receptor, or the expression of insulin receptor substrate-1 and Shc proteins. Both SHIP-WT and SHIPDeltaIP blocked insulin and PDGF-induced MAPK and MAPK kinase phosphorylation as well as, GTP-bound Ras activity, suggesting that the catalytic activity of SHIP is not necessary for these effects. SHIP associated with Shc upon ligand stimulation, indicating that the SHIP-Shc association is phosphorylation dependent. This association was primarily between the SHIP-SH2 domain and the phosphorylated tyrosine residues of Shc because no association was observed when the 3YF-Shc mutant was coexpressed with SHIP. The Shc*Grb2 association was not compromised by SHIP expression, despite complete inhibition of the Ras/MAPK pathway. Interestingly, son-of-sevenless (SOS) protein normally found in Grb2 complexes was markedly reduced in SHIP expressing cells, whereas the displaced SOS was recovered when the post-Grb2-IP supernatants were blotted with anti-SOS antibody. Thus, SHIP competes son-of-sevenless (SOS) away from Shc-Grb2. In summary, 1) SHIP-WT and SHIPDeltaIP expression inhibit insulin and PDGF stimulated Ras, MAPK kinase, and MAPK activities; 2) SHIP associates with tyrosine phosphorylated Shc, and the proline-rich sequences in SHIP associate with Grb2 and titrate out SOS to form Shc*Grb2*SHIP complexes; and 3) dissociation of SOS from the Shc*Grb2 complex inhibits Ras GTP loading, leading to decreased signaling through the MAPK pathway.     

Shc and Enigma Are Both Required for Mitogenic Signaling by Ret/ptc2

Ret/ptc2 is a constitutively active, oncogenic form of the c-Ret receptor tyrosine kinase. Like the other papillary thyroid carcinoma forms of Ret, Ret/ptc2 is activated through fusion of the Ret tyrosine kinase domain to the dimerization domain of another protein. Investigation of requirements for Ret/ptc2 mitogenic activity, using coexpression with dominant negative forms of Ras and Raf, indicated that these proteins are required for mitogenic signaling by Ret/ptc2. Because activation of Ras requires recruitment of Grb2 and SOS to the plasma membrane, the subcellular distribution of Ret/ptc2 was investigated, and it was found to localize to the cell periphery. This localization was mediated by association with Enigma via the Ret/ptc2 sequence containing tyrosine 586. Because Shc interacts with MEN2 forms of Ret, and because phosphorylation of Shc results in Grb2 recruitment and subsequent signaling through Ras and Raf, the potential interaction between Ret/ptc2 and Shc was investigated. The PTB domain of Shc also interacted with Ret/ptc2 at tyrosine 586, and this association resulted in tyrosine phosphorylation of Shc. Coexpression of chimeric proteins demonstrated that mitogenic signaling from Ret/ptc2 required both recruitment of Shc and subcellular localization by Enigma. Because Shc and Enigma interact with the same site on a Ret/ptc2 monomer, dimerization of Ret/ptc2 allows assembly of molecular complexes that are properly localized via Enigma and transmit mitogenic signals via Shc.     

ErbB-1 and ErbB-2 Acquire Distinct Signaling Properties Dependent upon Their Dimerization Partner

The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGF-activated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed.     

PKB-mediated negative feedback tightly regulates mitogenic signalling via Gab2.

Heregulin (HRG)-induced tyrosine phosphorylation of the Gab2 docking protein was enhanced by pretreatment with wortmannin, indicating negative regulation via a PI3-kinase-dependent pathway. This represents phosphorylation by the serine/threonine kinase protein kinase B (PKB), since PKB constitutively associates with Gab2, phosphorylates Gab2 on a consensus phosphorylation site, Ser159, in vitro and inhibits Gab2 tyrosine phosphorylation. However, expression of Gab2 mutated at this site (S159A Gab2) not only enhanced HRG-induced Gab2 tyrosine phosphorylation and association with Shc and ErbB2, but also markedly increased tyrosine phosphorylation of ErbB2 and other cellular proteins and amplified activation of the ERK and PKB pathways. The impact of this negative regulation was further emphasized by a potent transforming activity for S159A Gab2, but not wild-type Gab2, in fibroblasts. These studies establish Gab2 as a proto-oncogene, and a model in which receptor recruitment of Gab2 is tightly regulated via an intimate association with PKB. Release of this negative constraint enhances growth factor receptor signalling, possibly since Gab2 binding limits dephosphorylation and disassembly of receptor-associated signalling complexes.     

Multiple cytokines stimulate the binding of a common 145-kilodalton protein to Shc at the Grb2 recognition site of Shc.

We recently reported that interleukin-3, Steel factor, and erythropoietin all induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cell lines. We have now further characterized the proteins that become associated with Shc following stimulation with these cytokines and found that, in response to all three, the tyrosine-phosphorylated form of Shc binds to common 145- and 52-kDa proteins which also become tyrosine phosphorylated in response to these growth factors. The 145-kDa protein, which appears, from antiphosphotyrosine blots of two-dimensional O'Farrell gels, to exist in four different phosphorylation states following cytokine stimulation (with isoelectric points ranging from 7.2 to 7.8), does not appear to be immunologically related to the beta subunit of the interleukin-3 receptor, c-Kit, BCR, ABL, JAK1, JAK2, Sos1, eps15, or insulin receptor substrate 1 protein. Silver-stained sodium dodecyl sulfate gels indicate that the association of the 145-kDa protein with Shc occurs only after cytokine stimulation and that it can bind to the tyrosine-phosphorylated form of Shc in its non-tyrosine-phosphorylated state. The latter finding, in conjunction with the observations that p145 does not bind, in vitro, to the Src homology 2 (SH2) domain of Shc, that it is not present in anti-Grb2 immunoprecipitates, and that a phosphopeptide which blocks the binding of Shc to the SH2 domain of Grb2 also blocks the binding of Shc to p145, suggests that p145 contains an SH2 domain and competes with Grb2 for the same tyrosine-phosphorylated site on Shc. This implicates p145 as a potential regulator of Ras activity and, perhaps, of other as yet unidentified functions of Shc.