Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.     

Calmodulin dependence of NFkappaB activation

TF-1/TPO cells are derived from an erythroleukemia cell line, TF-1, and are absolutely dependent on either TPO or granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-3 (IL3) for their continuous growth and survival. To gain insight into the molecular basis of hemopoietic activities shared by TPO and GM-CSF/IL3 in TF-1/TPO cells, we studied the cross-talk between signal transduction pathways elicited by these cytokines. Stimulation of TF-1/TPO cells with TPO resulted in tyrosine phosphorylation of the TPO receptor (c-Mpl) as well as the common beta subunit (beta c) of GM-CSF/IL3 receptor complex. GM-CSF, however, induced tyrosine phosphorylation of beta c but not c-Mpl. TPO-induced tyrosine phosphorylation of beta c was time- and dose-dependent. We next examined whether or not TPO-induced tyrosine phosphorylation of beta c led to recruitment of SH2-containing molecules such as Stat5 and Shc. While GM-CSF caused association of Stat5 and Shc with beta c, TPO caused association of Stat5, but not Shc, with beta c, suggesting that TPO and GM-CSF may not induce phosphorylation of the same sets of tyrosine residues in beta c. These results suggest that activation of c-Mpl affects the signaling pathway of GM-CSF/IL3 but not vice versa.     

Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high-affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high-affinity; the cytoplasmic domain of beta was unnecessary for this high-affinity conversion. Proliferation assays showed that the membrane-proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.     

Tyrosine 474 of ZAP-70 is required for association with the Shc adaptor and for T-cell antigen receptor-dependent gene activation

The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation. Following receptor engagement, ZAP-70 is recruited to the phosphorylated subunits of the T-cell antigen receptor (TCR). This event results in ZAP-70 activation and in association of ZAP-70 with a number of signaling proteins. Among these is the Shc adaptor, which couples the activated TCR to Ras. Shc interaction with ZAP-70 is mediated by the Shc PTB domain. The inhibitory effect of a Shc mutant containing the isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling. Here, we show that a point mutation (Phe474) of the putative Shc binding site on ZAP-70, spanning tyrosine 474, prevented ZAP-70 interaction with Shc and the subsequent binding of Shc to phospho-zeta. Neither ZAP-70 catalytic activity nor the pattern of protein phosphorylation induced by TCR triggering was affected by this mutation. However expression of the Phe474 ZAP-70 mutant resulted in impaired TCR-dependent gene activation. ZAP-70 could effectively phosphorylate Shc in vitro. Only the CH domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb2 binding sites were excellent substrates for ZAP-70. The data show that Tyr474 on ZAP-70 is required for TCR signaling and suggest that Shc association with ZAP-70 and the resulting phosphorylation of Shc might be an obligatory step in linking the activated TCR to the Ras pathway.     

Interleukin-6 induces tyrosine phosphorylation of the Ras activating protein Shc, and its complex formation with Grb2 in the human multiple myeloma cell line LP-1

Like many other cytokines and growth factors, interleukin-6 (IL-6) activates p21ras. However, the precise biochemical mechanisms inducing this activation are unknown. Therefore, we investigated the effects of IL-6 on some recently identified signaling intermediates, Shc (Src homology and collagen) and Grb2 (growth factor receptor bound protein 2), known to activate p21ras. In the multiple myeloma cell line LP-1, IL-6 stimulated the tyrosine phosphorylation of Shc in a time- and concentration-dependent manner. This led to the complex formulation of Shc with Grb2, an adaptor protein known to relocate a p21ras-GDP exchange factor. Sos1 (Son-of-sevenless), to the cell membrane. Taken together, these findings suggest that IL-6 might activate the Ras signaling pathway via tyrosine phosphorylation of Shc and subsequent recruitment of Grb2. Further studies will elucidate which of the IL-6 receptor associated non-receptor tyrosine kinases of the Src kinase or Janus kinase family, mediate these effects.     

Gads is a novel SH2 and SH3 domain-containing adaptor protein that binds to tyrosine-phosphorylated Shc

Shc proteins are important substrates of receptor and cytoplasmic tyrosine kinases that couple activated receptors to downstream signaling enzymes. Phosphorylation of Shc tyrosine residues 239 and 317 leads to recruitment of the Grb2-Sos complex, thus linking Shc phosphorylation to Ras activation. We have used phosphorylated peptides corresponding to the regions spanning tyrosine 239/240 and 317 of Shc in an expression library screen to identify additional downstream targets of Shc. Here we report the identification of Gads, a novel adaptor protein most similar to Grb2 and Grap that contains amino and carboxy terminal SH3 domains flanking a central SH2 domain and a 120 amino acid unique region. Gads is most highly expressed in the thymus and spleen of adult animals and in human leukemic cell lines. The binding specificity of the Gads SH2 domain is similar to Grb2 and mediates the interaction of Gads with Shc, Bcr-Abl and c-kit. Gads does not interact with Sos, Cbl or Sam68, although the isolated carboxy terminal Gads SH3 domain is able to bind these molecules in vitro. Our results suggest that the unique structure of Gads regulates its interaction with downstream SH3 domain-binding proteins and that Gads may function to couple tyrosine-phosphorylated proteins such as Shc, Bcr-Abl and activated receptor tyrosine kinases to downstream effectors distinct from Sos and Ras.     

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.     

Roles of Lck, Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were able to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a synergy between the syk-family and scr-family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.     

Activation of Vav and Ras through the nerve growth factor and B cell receptors by different kinases

Engagement of the B-cell antigen receptor (BCR) or the nerve growth factor receptor (NGFR/TrkA) induces activation of multiple tyrosine kinases, resulting in phosphorylation of numerous intracellular substrates. We show that addition of NGF or anti-IgM antibody leads to the early tyrosine phosphorylation of p95(vav), which is expressed exclusively in hematopoietic cells; NGF, similar to crosslinking the BCR, also results in the rapid activation of Ras. The phosphorylation of Vav and activation of Ras triggered by NGF is mediated through Trk tyrosine kinase, whereas signaling through the BCR uses a different tyrosine kinase. We also show that NGF induces tyrosine phosphorylation of Shc and its association with Grb2. Vav and Ras with the adaptor proteins Shc and Grb2 appear to serve as a link between different receptor-mediated signaling pathways and, in human B cells, may play an important regulatory role in neuroimmune interactions.     

The kinase domain of Jak2 mediates induction of bcl-2 and delays cell death in hematopoietic cells

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 stimulate DNA synthesis and proliferation and inhibit apoptosis in hematopoietic cells. Multiple signal pathways are activated by binding of these ligands to their receptors, which share a common beta subunit. Janus protein kinase 2 (Jak2) binds to the membrane proximal domain of the beta chain and is phosphorylated on receptor ligation. To explore the role of Jak2 in the regulation of specific signal transduction pathways, we constructed fusion proteins with a CD16 external domain, a CD7 transmembrane region, and a Jak2 cytoplasmic domain. This cytoplasmic domain consisted either of wild type Jak2 (CD16/Jak2-W) or Jak2 mutations with deletions of (a) the amino terminus (CD16/Jak2-N), (b) kinase-like domain (CD16/Jak2-B), (c) kinase domain (CD16/Jak2-C), or (d) amino-terminal and kinase-like domains, leaving the kinase domain (CD16/Jak-K) intact. In contrast to the CD16/Jak2-W fusion protein, which requires cross-linking for activation, CD16/Jak2-N, CD16/Jak2-B, and CD16/Jak2-K were constitutively phosphorylated, and they stimulated Shc phosphorylation and increased binding of STAT to DNA in Ba/F3 cells. Cell lines derived from IL-3-dependent Ba/F3 cells stably transfected with CD16/Jak2-W, CD16/Jak2-N, or CD16/Jak2-B mammalian expression vectors died at a rate similar to that of the parental cells on IL-3 deprivation. In contrast, CD16/Jak2-K cell lines exhibited increased expression of bcl-2 and pim-1 mRNA and maintained their viability when compared with control cell lines. Thus, activation of tyrosine phosphorylation by creating a CD16/Jak2-K fusion is sufficient to activate pathways that prevent cell death.     

Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.     

Evidence for a direct interaction between insulin receptor substrate-1 and Shc

Insulin receptor substrate-1 (IRS-1) and Shc are two proteins implicated in intracellular signal transduction. They are activated by an increasing number of extracellular signals, mediated by receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors. In this study we demonstrate that Shc interacts directly with IRS-1, using the yeast two-hybrid system and an in vitro interaction assay. Deletion analysis of the proteins to map the domains implicated in this interaction shows that the phosphotyrosine binding domain of Shc binds to the region of IRS-1 comprising amino acids 583-661. An in vitro association assay, performed with or without activation of tyrosine kinases, gives evidence that tyrosine phosphorylation of IRS-1 and Shc drastically improves the interaction. Site-directed mutagenesis on IRS-1 583-693 shows that the asparagine, but not the tyrosine residue of the N625GDY628motif domain, is implicated in the IRS-1-Shc-phosphotyrosine binding interaction. Mutation of another tyrosine residue, Tyr608, also induced a 40% decrease in the interaction. This study, describing a phosphotyrosine-dependent interaction between IRS-1 and Shc, suggests that this association might be important in signal transduction.     

The low-density lipoprotein receptor-related protein (LRP) mediates clearance of coagulation factor Xa in vivo

In response to fibroblast growth factor (FGF), FGF receptor-1 (FGFR-1) (flg) becomes tyrosine phosphorylated and associates with phospholipase C gamma (PLC gamma) and a 90 kDa protein. We report here that in cells transformed by v-Src, FGFR-1 becomes phosphorylated on tyrosine; however, neither PLC gamma nor p90 was found to be associated with tyrosine-phosphorylated FGFR-1. Instead, there was a strong constitutive association of FGFR-1 with the adaptor proteins Shc and Grb2 and the Ras guanine nucleotide exchange factor Sos. Association with Shc and Grb2 and Sos was not observed in response to FGF. Suramin did not prevent either tyrosine phosphorylation or Shc/Grb2/Sos association, indicating a non-autocrine mechanism. Thus, in cells transformed by v-Src, tyrosine phosphorylation of FGFR-1 results not in the expected association with PLC gamma and p90, but rather in the recruitment of the Ras activating Shc/Grb2/Sos complex. These data suggest a mechanism for Ras activation by v-Src involving phosphorylation of novel tyrosine(s) on FGFR-1.     

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.     

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.     

Cholecystokinin stimulates formation of shc-grb2 complex in rat pancreatic acinar cells through a protein kinase C-dependent mechanism

Cholecystokinin (CCK) has recently been shown to activate the mitogen-activated protein kinase (MAPK) cascade (Ras-Raf-MAPK kinase-MAPK) in pancreatic acini. The mechanism by which the Gq protein-coupled CCK receptor activates Ras, however, is currently unknown. Growth factor receptors are known to activate Ras by means of adaptor proteins that bind to phosphotyrosine domains. We therefore compared the effects of CCK and epidermal growth factor (EGF) on Tyr phosphorylation of the adaptor proteins Shc and its association with Grb2 and the guanine nucleotide exchange factor SOS. Three major isoforms of Shc (p46, p52, p66) were detected in isolated rat pancreatic acini with p52 Shc being the predominant form. CCK and EGF increased tyrosyl phosphorylation of Shc (251 and 337% of control, respectively). CCK-stimulated tyrosyl phosphorylation of Shc as well as Shc-Grb2 complex formation was significant at 2.5 min, maximal at 5 min, and persisted for at least 30 min. Finally, SOS was found to be associated with Grb2 as assessed by probing of anti-Grb2 immunoprecipitates with anti-SOS. Since MAPK in pancreatic acini is activated via protein kinase C (PKC), we studied the effect of phorbol esters on Shc phosphorylation and found 12-O-tetradecanoylphorbol-13-acetate to be as potent as CCK. Furthermore, GF-109203X, a PKC inhibitor, abolished the effect of 12-O-tetradecanoylphorbol-13-acetate and also the effect of CCK but not the effect of EGF on Shc tyrosyl phosphorylation. CCK-induced tyrosyl phosphorylation of Shc was found to be phosphatidylinositol 3-kinase-independent, and CCK did not cause EGF receptor activation. These results suggest that formation of an Shc-Grb2-SOS complex via a PKC-dependent mechanism may provide the link between Gq protein-coupled CCK receptor stimulation and Ras activation in these cells.     

Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival

Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5-stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with beta common (betac) chain of the IL-5 receptor (IL-5betacR). The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated betac receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.     

Differing rate dependence and temporal distribution of repolarization alternans in patients with and without ventricular tachycardia

The Shc protein helps to transmit signals from receptor and cytoplasmic tyrosine kinases to Ras. We have shown that several breast cancer cell lines (MDA-MB-453, BT474, MDA-MB-361, and SKBR3), which overexpress the ErbB2 receptor tyrosine kinase, contain constitutively tyrosine phosphorylated Shc. To investigate the role of Shc in these cells, we transfected them with a Shc-Y317F dominant-negative mutant defective in signaling to Ras. The transfectants were unable to form stable colonies, suggesting a critical role for Shc in the proliferation of these cells. In contrast, dominant-negative Shc transfectants of the nontransformed breast epithelial cell line HBL-100 grew normally. Surprisingly, cell cycle analysis of transfected SKBR3 cells suggested that the cells were blocked not only in G0-G1, but also in G2-M. The G2-M block was unexpected because Shc-Y317 is downstream of receptor tyrosine kinases that drive the early events in the cell cycle. Both the G0-G1 and G2-M arrest were rescued by transfection with wild-type Shc or oncogenic Ras 12V. Rescue by Ras suggests that Shc Y317 signals upstream of Ras, and that Shc to Ras effector pathways are involved in G2-M, although confirmation awaits a detailed molecular analysis. Most importantly, this work provides the first evidence for Shc involvement in G2-M.     

Shc interaction with Src homology 2 domain containing inositol phosphatase (SHIP) in vivo requires the Shc-phosphotyrosine binding domain and two specific phosphotyrosines on SHIP

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, cytokine receptors, and antigen receptors on lymphocytes. Besides the well characterized interaction of Shc with molecules involved in Ras activation, Shc also associates with a 145-kDa tyrosine-phosphorylated protein upon triggering via antigen receptors and many cytokine receptors. This 145-kDa protein has been recently identified as an SH2 domain containing 5'-inositol phosphatase (SHIP) and has been implicated in the regulation of growth and differentiation in hematopoietic cells. In this report, we have addressed the molecular details of the interaction between Shc and SHIP in vivo. During T cell receptor signaling, tyrosine phosphorylation of SHIP and its association with Shc occurred only upon activation. We demonstrate that the phosphotyrosine binding domain of Shc is necessary and sufficient for its association with tyrosine-phosphorylated SHIP. Through site-directed mutagenesis, we have identified two tyrosines on SHIP, Tyr-917, and Tyr-1020, as the principal contact sites for the Shc-phosphotyrosine binding domain. Our data also suggest a role for the tyrosine kinase Lck in phosphorylation of SHIP. We also show that the SH2 domain of SHIP is dispensable for the Shc-SHIP interaction in vivo. These data have implications for the localization of the Shc.SHIP complex and regulation of SHIP function during T cell receptor signaling.