The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.     

Erythropoietin receptor Y479 couples to ERK1/2 activation via recruitment of phospholipase Cgamma

Red blood cell development is primarily controlled by erythropoietin (EPO). Several studies have revealed the importance of EPO-R Y343 and Y479 for erythroid cell growth, differentiation, and survival. In order to isolate critical signaling proteins that bind to EPO-R, we initiated a Cloning of Ligand Target (COLT) screen using a murine embryonic day 16 phage library and a biotinylated EPO-R Y343 phosphopeptide. One of the clones isolated encodes Phospholipase C (PLC)gamma1. PLCgamma1 is rapidly tyrosine phosphorylated upon EPO stimulation and associates with EPO-R in an SH2-domain-dependent manner. Although PLCgamma1 bound EPO-R Y343, Y401, Y429, Y431, and Y479 in the COLT screen, PLCgamma1 required Y479 for association with EPO-R in Ba/F3-EPO-R cells. Studies have identified EPO-R Y479 as important for ERK activation. Since PI3-kinase binds EPO-R Y479, one group has suggested that ERK activation downstream of PI3-kinase accounts for the importance of this residue in EPO signaling. However, we show that inhibition of PI3-kinase does not abolish ERK activation. Furthermore, we demonstrate interaction of PLCgamma1 with Grb2 and SOS2. Hence, we have identified a novel adapter function for PLCgamma1 in EPO signaling in which recruitment of PLCgamma1 to EPO-R may lead to activation of the ERK pathway.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

A dominant negative erythropoietin (EPO) receptor inhibits EPO-dependent growth and blocks F-gp55-dependent transformation.

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO- or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.     

Tyrosine phosphorylation of shc in response to B cell antigen receptor engagement depends on the SHIP inositol phosphatase

Tyrosine phosphorylation of Shc in response to B cell Ag receptor (BCR) engagement creates binding sites for the Src homology 2 (SH2) domain of Grb2. This facilitates the recruitment of both Grb2. Sos complexes and Grb2. SHIP complexes to the plasma membrane where Sos can activate Ras and SH2 domain-containing inositol phosphatase (SHIP) can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate. Given the importance of Shc phosphorylation, we investigated the mechanism by which the BCR stimulates this response. We found that both the SH2 domain and phosphotyrosine-binding (PTB) domain of Shc are important for BCR-induced tyrosine phosphorylation of Shc and the subsequent binding of Grb2 to Shc. The unexpected finding that the PTB domain of Shc is required for Shc phosphorylation was investigated further. Because the major ligand for the Shc PTB domain is SHIP, we asked whether the interaction of Shc with SHIP was required for BCR-induced tyrosine phosphorylation of Shc. Using SHIP-deficient DT40 cells, we show that SHIP is necessary for the BCR to induce significant levels of Shc tyrosine phosphorylation. BCR-induced tyrosine phosphorylation of Shc could be restored in the these cells by expressing wild-type SHIP but not by expressing a mutant form of SHIP that cannot bind to Shc. This suggests that BCR-induced tyrosine phosphorylation of Shc may depend on the binding of SHIP to the Shc PTB domain. Thus, we have described a novel role for SHIP in BCR signaling, promoting the tyrosine phosphorylation of Shc.     

Tyrosine phosphorylation of the beta 4 integrin cytoplasmic domain mediates Shc signaling to extracellular signal-regulated kinase and antagonizes formation of hemidesmosomes

Ligation of the alpha(6)beta(4) integrin induces tyrosine phosphorylation of the beta(4) cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of beta(4) that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr(1440), or secondarily Tyr(1422), interacts with the SH2 domain of Shc, whereas Tyr(1526), or secondarily Tyr(1642), interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by alpha(6)beta(4). In addition, mutation of beta(4) Tyr(1526), which binds to the PTB domain of Shc, but not of Tyr(1422) and Tyr(1440), which interact with its SH2 domain, abolishes the activation of ERK by alpha(6)beta(4). Phenylalanine substitution of the beta(4) tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of alpha(6)beta(4) in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of beta4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type beta(4). This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of beta(4) containing phenylalanine substitutions at Tyr(1422) and Tyr(1440), at Tyr(1526) and Tyr(1642), or at all four tyrosine phosphorylation sites. These results suggest that beta(4) Tyr(1526) interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.     

Role of Herpes Simplex Virus VP11/12 Tyrosine-Based Motifs in Binding and Activation of the Src Family Kinase Lck and Recruitment of p85, Grb2, and Shc

Previous studies have shown that the abundant herpes simplex virus 1 (HSV-1) tegument protein VP11/12, encoded by gene UL46, stimulates phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling: it binds the Src family kinase (SFK) Lck, is tyrosine phosphorylated, recruits the p85 subunit of PI3-kinase, and is essential for the activation of Akt during HSV-1 infection. The C-terminal region of VP11/12 contains tyrosine-based motifs predicted to bind the SH2 domains of SFKs (YETV and YEEI), p85 (YTHM), and Grb2 (YENV) and the phosphotyrosine-binding (PTB) domain of Shc (NPLY). We inactivated each of these motifs in the context of the intact viral genome and examined effects on binding and activation of Lck and recruitment of p85, Grb2, and Shc. Inactivating the p85, Grb2, or Shc motif reduced (p85) or eliminated (Grb2 and Shc) the interaction with the cognate signaling molecule without greatly affecting the other interactions or activation of Lck. Inactivating either SFK motif had only a minor effect on Lck binding and little or no effect on recruitment of p85, Grb2, or Shc. In contrast, inactivation of both SFK motifs severely reduced Lck binding and activation and tyrosine phosphorylation of VP11/12 and reduced (p85) or eliminated (Grb2 and Shc) binding of other signaling proteins. Overall, these data demonstrate the key redundant roles of the VP11/12 SFK-binding motifs in the recruitment and activation of SFKs and indicate that activated SFKs then lead (directly or indirectly) to phosphorylation of the additional motifs involved in recruiting p85, Grb2, and Shc. Thus, VP11/12 appears to mimic an activated growth factor receptor.     

Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling.

Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identified as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and fibroblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.     

Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.     

Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling.

SH2-containing inositol 5'-phosphatase (SHIP) plays a negative regulatory role in hematopoietic cells. We have now cloned the rat SHIP isozyme (SHIP2) cDNA from skeletal muscle, which is one of the most important target tissue of insulin action. Rat SHIP2 cDNA encodes a 1183-amino-acid protein that is 45% identical with rat SHIP. Rat SHIP2 contains an amino-terminal SH2 domain, a central 5'-phosphoinositol phosphatase activity domain, and a phosphotyrosine binding (PTB) consensus sequence and a proline-rich region at the carboxyl tail. Specific antibodies to SHIP2 were raised and the function of SHIP2 was studied by stably overexpressing rat SHIP2 in Rat1 fibroblasts expressing human insulin receptors (HIRc). Endogenous SHIP2 underwent insulin-mediated tyrosine phosphorylation and phosphorylation was markedly increased when SHIP2 was overexpressed. Although overexpression of SHIP2 did not affect insulin-induced tyrosine phosphorylation of the insulin receptor beta-subunit and Shc, subsequent association of Shc with Grb2 was inhibited, possibly by competition between the SH2 domains of SHIP2 and Grb2 for the Shc phosphotyrosine. As a result, insulin-stimulated MAP kinase activation was reduced in SHIP2-overexpressing cells. Insulin-induced tyrosine phosphorylation of IRS-1, IRS-1 association with the p85 subunit of PI3-kinase, and PI3-kinase activation were not affected by overexpression of SHIP2. Interestingly, although both PtdIns-(3,4,5)P3 and PtdIns(3,4)P2 have been implicated in the regulation of Akt activity in vitro, overexpression of SHIP2 inhibited insulin-induced Akt activation, presumably by its 5'-inositol phosphatase activity. Furthermore, insulin-induced thymidine incorporation was decreased by overexpression of SHIP2. These results indicate that SHIP2 plays a negative regulatory role in insulin-induced mitogenesis, and regulation of the Shc. Grb2 complex and of the downstream products of PI3-kinase provides possible mechanisms of SHIP2 action in insulin signaling.     

The two SH2-domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are coexpressed in human T lymphocytes.

The activation of many hematopoietic cells via cytokine receptors, as well as B and T cell receptors, leads to the tyrosine phosphorylation of Shc and its association with both Grb2-Sos1 complexes and with a 145 kDa protein referred to as the SH2 containing inositol 5-phosphatase (SHIP1). In a search of putative 5-phosphatase isoenzymes, we have isolated a second SH2 domain containing inositol 5-phosphatase, referred to as (SHIP2). Both SHIP1 and SHIP2 are coexpressed in human T lymphocytes. This was shown at the protein level by Western blot analysis in transformed T cell lines and in peripheral blood T lymphocytes either unstimulated or after in vitro activation through TCR-CD3 complex. SHIP1 protein level was not modulated after activation of T lymphocytes, in contrast to SHIP2, which was increased after long-term stimulation. SHIP1 was tyrosine phosphorylated in resting naive T cells. This was not observed in the transformed T cell lines. T lymphocyte is therefore a model of coexpression of the two SH2-containing inositol 5-phosphatases SHIP1 and SHIP2.     

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.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-gamma. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr(1096)) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr(1096) phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr(1096) abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.     

Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades.

The protein tyrosine kinase Pyk2 acts as an upstream regulator of mitogen-activated protein (MAP) kinase cascades in response to numerous extracellular signals. The precise molecular mechanisms by which Pyk2 activates distinct MAP kinase pathways are not yet fully understood. In this report, we provide evidence that the protein tyrosine kinase Src and adaptor proteins Grb2, Crk, and p130Cas act as downstream mediators of Pyk2 leading to the activation of extracellular signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK). Pyk2-induced activation of Src is necessary for phosphorylation of Shc and p130Cas and their association with Grb2 and Crk, respectively, and for the activation of ERK and JNK cascades. Expression of a Grb2 mutant with a deletion of the amino-terminal Src homology 3 domain or the carboxyl-terminal tail of Sos strongly reduced Pyk2-induced ERK activation, with no apparent effect on JNK activity. Grb2 with a deleted carboxyl-terminal Src homology 3 domain partially blocked Pyk2-induced ERK and JNK pathways, whereas expression of dominant interfering mutants of p130Cas or Crk specifically inhibited JNK but not ERK activation by Pyk2. Taken together, our data reveal specific pathways that couple Pyk2 with MAP kinases: the Grb2/Sos complex connects Pyk2 to the activation of ERK, whereas adaptor proteins p130Cas and Crk link Pyk2 with the JNK pathway.     

Recruitment and phosphorylation of SH2-containing inositol phosphatase and Shc to the B-cell Fc gamma immunoreceptor tyrosine-based inhibition motif peptide motif.

Recently, we and others have demonstrated that negative signaling in B cells selectively induces the tyrosine phosphorylation of a novel inositol polyphosphate phosphatase, p145SHIP. In this study, we present data indicating that p145SHIP binds directly a phosphorylated motif, immunoreceptor tyrosine-based inhibition motif (ITIM), present in the cytoplasmic domain of Fc gammaRIIB1. Using recombinant SH2 domains, we show that binding is mediated via the Src homology region 2 (SH2)-containing inositol phosphatase (SHIP) SH2 domain. SHIP also bound to a phosphopeptide derived from CD22, raising the possibility that SHIP contributes to negative signaling by this receptor as well as Fc gammaRIIB1. The association of SHIP with the ITIM phosphopeptide was activation independent, while coassociation with Shc was activation dependent. Furthermore, experiments with Fc gammaRIIB1-deficient B cells demonstrated a genetic requirement for expression of Fc gammaRIIB1 in the induction of SHIP phosphorylation and its interaction with Shc. Based on these results, we propose a model of negative signaling in which co-cross-linking of surface immunoglobulin and Fc gammaRIIB1 results in sequential tyrosine phosphorylation of the ITIM, recruitment and phosphorylation of p145SHIP, and subsequent binding of Shc.