Association Of Shc Research Articles

Mutation of a Shc Binding Site Tyrosine Residue in ErbB3/HER3 Blocks Heregulin-dependent Activation of Mitogen-activated Protein Kinase

The ErbB2 and ErbB3 proteins together constitute a functional coreceptor for heregulin (neuregulin). Heregulin stimulates the phosphorylation of both coreceptor constituents and initiates a variety of other signaling events, which include phosphorylation of the Shc protein. The role of Shc in heregulin-stimulated signal transduction through the ErbB2.ErbB3 coreceptor was investigated here. Heregulin was found to promote ErbB3/Shc association in NIH-3T3 cells expressing endogenous ErbB2 and recombinant ErbB3. A mutant ErbB3 protein was generated in which Tyr-1325 in a consensus Shc phosphotyrosine-binding domain recognition site was mutated to Phe (ErbB3-Y/F). This mutation abolished the association of Shc with ErbB3 and blocked the activation of mitogen-activated protein kinase by heregulin. Whereas heregulin induced mitogenesis in NIH-3T3 cells transfected with wild-type ErbB3 cDNA, this mitogenic response was markedly attenuated in NIH-3T3 cells transfected with the ErbB3-Y/F cDNA. These results showed a specific interaction of Shc with the ErbB3 receptor protein and demonstrated the importance of this interaction in the activation of mitogenic responses by the ErbB2. ErbB3 heregulin coreceptor complex.

Insulin-like Growth Factor I (IGF-I)-stimulated Pancreatic β-Cell Growth Is Glucose-dependent: SYNERGISTIC ACTIVATION OF INSULIN RECEPTOR SUBSTRATE-MEDIATED SIGNAL TRANSDUCTION PATHWAYS BY GLUCOSE AND IGF-I IN INS-1 CELLS

Nutrients and certain growth factors stimulate pancreatic beta-cell mitogenesis, however, the appropriate mitogenic signal transduction pathways have not been defined. In the glucose-sensitive pancreatic beta-cell line, INS-1, it was found that glucose (6-18 mM) independently increased INS-1 cell proliferation (>20-fold at 15 mM glucose). Insulin-like growth factor I (IGF-I)-induced INS-1 cell proliferation was glucose-dependent only in the physiologically relevant concentration range (6-18 mM glucose). The combination of IGF-I and glucose was synergistic, increasing INS-1 cell proliferation >50-fold at 15 mM glucose + 10 nM IGF-I. Glucose metabolism and phosphatidylinositol 3'-kinase (PI 3'-kinase) activation were necessary for both glucose and IGF-I-stimulated INS-1 cell proliferation. IGF-I and 15 mM glucose increased tyrosine phosphorylation mediated recruitment of Grb2/mSOS and PI 3'-kinase to IRS-2 and pp60. Glucose and IGF-I also induced Shc association with Grb2/mSOS. Glucose (3-18 mM) and IGF-I, independently of glucose, activated mitogen-activated protein kinase but this did not correlate with IGF-I-induced beta-cell proliferation. In contrast, p70(S6K) was activated with increasing glucose concentration (between 6 and 18 mM), and potentiated by IGF-I in the same glucose concentration range which correlated with INS-1 cell proliferation rate. Thus, glucose and IGF-I-induced beta-cell proliferation were mediated via a signaling mechanism that was facilitated by mitogen-activated protein kinase but dependent on IRS-mediated induction of PI 3'-kinase activity and downstream activation of p70(S6K). The glucose dependence of IGF-I mediated INS-1 cell proliferation emphasizes beta-cell signaling mechanisms are rather unique in being tightly linked to glycolytic metabolic flux.

The CBL-related Protein CBLB Participates in FLT3 and Interleukin-7 Receptor Signal Transduction in Pro-B Cells

The FLT3 receptor tyrosine kinase and its ligand, FL, play an important role in early hematopoietic development. We have found that CBLB, a recently characterized molecule closely related to the CBL protooncogene product, is phosphorylated on tyrosine(s) following FL treatment of JEA2 human pro-B cells and THP1 monocytic cells. Treatment of JEA2 cells with interleukin (IL)-7 induces CBLB phosphorylation as well. FL and IL-7, respectively, induce and increase association of tyrosine-phosphorylated SHC and the p85 subunit of phosphatidylinositol 3'-kinase with CBLB. In these cells, CBLB constitutively binds the GRB2 adaptor predominantly through its N-terminal SH3 domain, to form a complex that is distinct from the GRB2.CBL and GRB2.SOS1 complexes. Together with the fact that CBLB is consistently found in blast cells from acute leukemias and in peripheral blood mononuclear cells, this suggests that CBLB has a role in tyrosine kinase-regulated signaling pathways in many hematolymphoid cells.

Thrombopoietin Induces Tyrosine Phosphorylation of a Common β Subunit of GM-CSF Receptor and Its Association with Stat5 in TF-1/TPO Cells

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 β subunit (βc) of GM-CSF/IL3 receptor complex. GM-CSF, however, induced tyrosine phosphorylation of βc but not c-Mpl. TPO-induced tyrosine phosphorylation of βc was time- and dose-dependent. We next examined whether or not TPO-induced tyrosine phosphorylation of βc led to recruitment of SH2-containing molecules such as Stat5 and Shc. While GM-CSF caused association of Stat5 and Shc with βc, TPO caused association of Stat5, but not Shc, with βc, suggesting that TPO and GM-CSF may not induce phosphorylation of the same sets of tyrosine residues in βc. These results suggest that activation of c-Mpl affects the signaling pathway of GM-CSF/IL3 but notvice versa.

Cell type-specific angiotensin II-evoked signal transduction pathways: critical roles of Gbetagamma subunit, Src family, and Ras in cardiac fibroblasts.

Angiotensin II (Ang II) induces hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. To determine the molecular mechanism by which Ang II displayed different effects on cardiac myocytes and fibroblasts, we examined signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs). Ang II-induced ERK activation was abolished by pretreatment with pertussis toxin and by overexpression of the Gbetagamma subunit-binding domain of the beta-adrenergic receptor kinase 1 in cardiac fibroblasts but not in cardiac myocytes. Inhibition of protein kinase C strongly inhibited activation of ERKs by Ang II in cardiac myocytes, whereas inhibitors of tyrosine kinases but not of protein kinase C abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of C-terminal Src kinase (Csk), which inactivates Src family tyrosine kinases, suppressed the activation of transfected ERK in cardiac fibroblasts. Ang II rapidly induced phosphorylation of Shc and association of Shc with Grb2. Cotransfection of the dominant-negative mutant of Ras or Raf-1 kinase abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of Csk or the dominant-negative mutant of Ras had no effects on Ang II-induced ERK activation in cardiac myocytes. These findings suggest that Ang II-evoked signal transduction pathways differ among cell types. In cardiac fibroblasts, Ang II activates ERKs through a pathway including the Gbetagamma subunit of Gi protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1 kinase, whereas Gq and protein kinase C are important in cardiac myocytes.

Activation of P2Y2 Receptors by UTP and ATP Stimulates Mitogen-activated Kinase Activity through a Pathway That Involves Related Adhesion Focal Tyrosine Kinase and Protein Kinase C

We examined downstream signaling events that followed the exposure of PC12 cells to extracellular ATP and UTP, and we compared the effects of these P2 receptor agonists with those of growth factors and other stimuli. Based on early findings, we focused particular attention on the mitogen-activated protein (MAP) kinase pathway. ATP and/or UTP produced increases in tyrosine phosphorylation of multiple proteins, including p42 MAP (ERK2) kinase, related adhesion focal tyrosine kinase (RAFTK) (PYK2, CAKbeta), focal adhesion kinase (FAK), Shc, and protein kinase Cdelta (PKCdelta). MAP (ERK2) kinase activity (quantified by substrate phosphorylation) was increased by UTP, ATP, phorbol 12-myristate 13-acetate, ionomycin, and growth factors. UTP and ATP were equipotent (EC50 approximately 25 microM) in stimulating MAP kinase activity, suggesting that these effects were mediated via the Gi-linked P2Y2 (P2U) receptor. Consistent with this, the UTP- and ATP-promoted activation of MAP kinase was diminished in pertussis toxin-treated cells. Treatment of cells with pertussis toxin also reduced both the UTP-dependent increases in intracellular calcium ion concentration ([Ca2+]i) and the tyrosine phosphorylation of RAFTK. Similarly, when [Ca2+]i elevation was prevented using BAPTA and EGTA, the activation of MAP kinase by UTP and ionomycin was blocked, and the tyrosine phosphorylation of RAFTK was reduced. The UTP-promoted increase in MAP kinase activity was partially reduced in cells in which PKC was down-regulated, suggesting that both PKC-dependent and PKC-independent pathways were involved. PKCdelta, which increases MAP kinase activity in some systems, became tyrosine-phosphorylated within 15 s of exposure of cells to ATP or UTP; but epidermal growth factor, nerve growth factor, and insulin had little effect. UTP also promoted the association of Shc with Grb2. These results suggest that the P2Y2 receptor-initiated activation of MAP kinase was dependent on the elevation of [Ca2+]i, involved the recruitment of Shc and Grb2, and was mediated by RAFTK and PKC.

Nerve growth factor signal transduction in mature pig oligodendrocytes.

It has previously been shown that nerve growth factor (NGF) is of functional significance for mature pig oligodendrocytes (OLs) in culture. The present data give evidence for the expression of TrkA, the so-called high-affinity NGF receptor, and of p75NTR, the so-called low-affinity NGF receptor. TrkA is upregulated during culturing, in contrast to the p75 receptor. Exposure of OLs to NGF induces an autophosphorylation of TrkA via its intrinsic tyrosine kinase. K-252a inhibits the TrkA autophosphorylation, which reduces the OL process formation to control levels. To the tyrosine-phosphorylated sites of TrkA several proteins, such as phospholipase C-gamma1, the adaptor protein SHC, the phosphotyrosine phosphatase SH-PTP2 (SYP) associate via their SH2 phosphotase SH-PTP2 domain. The association of SHC to TrkA is shown by co-immunoprecipitation. Indirect evidence for a possible activation of PLC-gamma1 is given by an NGF-induced increase of oligodendroglial [Ca2+]i. Downstream from TrkA, a mitogen-activated protein kinase cascade, which includes Erk1 and Erk2, is operating. An in-gel myelin basic protein kinase assay revealed that NGF activates predominantly Erk1. Finally, it is shown that NGF stimulates expression of c-fos.

12-O-Tetradecanoylphorbol-13acetate Activates the Ras/ Extracellular Signal-regulated Kinase (ERK) Signaling Pathway Upstream of SOS Involving Serine Phosphorylation of Shc in NIH3T3 Cells

We investigated the activation of the Ras/ERK signaling pathway by 12-O-tetradecanoylphorbol-13-acetate (TPA) in NIH3T3 fibroblasts. Interestingly, the activation was suppressed not only by dominant negative Raf-1 but also by dominant negative Ras and SOS. Further analysis revealed that TPA treatment induced, dependently on protein kinase C, the mobility shift of p66(shc) in SDS-polyacrylamide gel electrophoresis, which could be prevented by treatment of the Shc immunoprecipitate with serine/threonine-specific protein phosphatase 1 (PP1) or 2A (PP2A). Phosphoamino acid analysis of Shc showed that unlike growth factor-induced Shc phosphorylation, where Shc is mainly phosphorylated at tyrosine residues, TPA-induced phosphorylation was only at serine residues. Like growth factor-induced Shc phosphorylation, which leads to the association of Shc with Grb2, TPA also induced this association, but, correspondingly to the above results, the TPA-induced association was disrupted by in vitro treatment of the Shc immunoprecipitate with PP1. Taken together, these results suggest that the TPA signal was fed at or upstream of Shc to activate the Ras/ERK signaling pathway involving serine phosphorylation of Shc.

Selective binding of shc-SH2 domain to tyrosine-phosphorylated zeta but not gamma-chain upon CD16 ligation on human NK cells.

Fc gamma RIII (CD16) is a hetero-oligomeric receptor composed of a ligand-binding alpha subunit associated with homo- or heterodimers of the TCR zeta- and Fc epsilon RI gamma-chains. We have previously demonstrated that CD16 ligation promotes complex formation between tyrosine-phosphorylated shc and Grb2, leading to activation of ras signaling pathway in human NK cells. Here we report that CD16 engagement induces rapid shc association with the tyrosine-phosphorylated receptor complex in human NK cells. In vitro binding studies demonstrate that this interaction is mediated by the shc-SH2 domain, and immunodepletion experiments indicate that the zeta- but not the gamma-chain has the capability to mediate this association. Jurkat cell clones expressing CD16-zeta or -gamma homodimers have been used to gain more information about the mechanism of shc/CD16 association. Our data show that, while engagement of both receptors induces tyrosine phosphorylation of shc and Grb2 recruitment, shc-SH2/receptor complex association is evident only in CD16-zeta but not in CD16-gamma transfectants. Overall, our data demonstrate that the adaptor protein shc can be recruited to the activated CD16 complex by interaction with tyrosine-phosphorylated zeta-chain in a SH2-dependent manner. These results also provide further support to the notion that zeta- and gamma-chains might couple to different biochemical pathways.

Signaling molecules involved in coupling growth hormone receptor to mitogen-activated protein kinase activation.

We have shown previously that GH stimulates the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal-regulated kinases) 1 and 2. To examine pathways coupling GH receptor (GHR) to MAP kinase activation, we have determined the effects of GH on SHC-growth factor receptor bound 2-son of Sevenless (SHC-Grb2-SOS) association and activation of Ras, Raf, and MAP-ERK kinase (MEK). GH promoted the rapid, transient association of SHC with the Grb2-SOS complex, which correlated with the time course of Ras, Raf, and MEK activation. Despite the continuous presence of GH, these activation events were transient with Ras, Raf, and MEK returning to near basal activity by 15 or 30 min. The inactivation of Ras, Raf, and MEK directly correlated with the serine/threonine phosphorylation of SOS and dissociation of SOS from Grb2 but not Grb2 from tyrosine-phosphorylated SHC. Phosphorylation was blocked by the MEK inhibitor, PD98059. Based upon the established functions of the MAP kinase pathway, these data indicate that GH stimulation results in the assembly of a SHC-Grb2-SOS complex that serves to activate Ras and thereby engage the Raf-MEK-ERK pathway. Activation of this pathway generates a feedback kinase cascade that phosphorylates SOS resulting in the dissociation of SHC-Grb2 complexes from SOS, thereby causing a more rapid termination of the signaling pathway than would result from SHC dephosphorylation.

Cytoskeletal association of epidermal growth factor receptor and associated signaling proteins is regulated by cell density in IEC-6 intestinal cells.

Epidermal growth factor (EGF) mediates a variety of physiologic responses in rat intestine. EGF receptor (EGFR) responsiveness to EGF is mediated by the surface expression of high affinity EGFR, which is associated with the cytoskeleton (CSK). EGFR signal transduction appears to be mediated by the CSK association of EGFR and related signaling proteins. In the nontransformed intestinal cell line IEC-6, expression of EGFR, Src homology and collagen protein (SHC), phospholipase C gamma 1 (PLC gamma), and their tyrosine phosphorylation in response to EGF was assayed by immunoblot. The distribution of EGFR and tyrosine-phosphorylated EGFR was regulated by cell density. At confluence, EGFR and tyrosine-phosphorylated EGFR were predominantly associated with the Triton X-100-insoluble CSK at confluence, while predominantly Triton X-100-soluble at subconfluence. PLC gamma was predominantly soluble at both states of confluence. Confluent but not subconfluent IEC-6 cells demonstrated a cascade of EGF-mediated events consisting of a transient CSK association of PLC gamma with EGFR, a brief expression of tyrosine-phosphorylated PLC gamma, a brief increase in PLC gamma CSK association, and a prolonged soluble association of PLC gamma with the EGFR. EGF led to an increase in the CSK association of SHC at both states of confluence and was greater at confluence. EGFR association with SHC was primarily soluble at subconfluence, while at confluence EGFR association was markedly increased and predominantly in the CSK. Thus, cell density regulates the CSK association of the EGFR and its ability to associate and activate signaling pathways in intestinal cells.

Functional Importance of Shc Tyrosine 317 on Insulin Signaling in Rat1 Fibroblasts Expressing Insulin Receptors

Shc is phosphorylated on Tyr-317, which serves as a docking site for Grb2. To investigate the specific role of Shc phosphorylation and Shc.Grb2 coupling on insulin signaling, we generated expression vectors for wild-type (WT-Shc) and a mutant Shc with a Tyr-317 --> Phe substitution (317Y/F-Shc) and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). From different clonal cell lines, cells expressing 10 times greater amounts of WT-Shc or 317Y/F-Shc compared with endogenous Shc were chosen for analysis of insulin signaling. Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in WT-Shc cells. Because of competition between Shc and IRS-1 for interaction with the insulin receptor, insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in WT-Shc cells compared with that in HIRc cells. Likewise, reduction of endogenous Shc expression by antisense Shc mRNA resulted in increased insulin stimulation of IRS-1 phosphorylation. Although 317Y/F-Shc was also able to interact with insulin receptor, decreased amounts of Shc phosphorylation and Shc association with Grb2 were observed in 317Y/F-Shc cells, indicating that 317Y/F-Shc functions as a dominant-negative mutant. The kinetics of mitogen-activated protein (MAP) kinase activation closely paralleled the kinetics of Shc phosphorylation. Thus, insulin stimulation of MAP kinase activation occurred more rapidly and was prolonged in WT-Shc cells, while the activation was delayed and transient in 317Y/F-Shc cells compared with that in HIRc cells. Importantly, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine and bromodeoxyuridine incorporation, whereas the sensitivity was decreased in 317Y/F-Shc cells. These results indicate that Shc Tyr-317 phosphorylation plays an important role, via coupling with Grb2 and competition with IRS-1, in signal transduction to MAP kinase by insulin, ultimately leading to mitogenesis in Rat1 fibroblasts.

The Src Homology 2 (SH2) Domain of SH2-containing Inositol Phosphatase (SHIP) Is Essential for Tyrosine Phosphorylation of SHIP, Its Association with Shc, and Its Induction of Apoptosis

In this study we have investigated the role that the Src homology 2 domain (SH2) of the 145-kDa 5-phosphatase, SH2-containing inositol phosphatase (SHIP), plays in three of the properties that have been associated with this protein following cytokine stimulation: its association with Shc, its tyrosine phosphorylation, and its inhibition of hemopoietic cell growth. In vitro studies using this SH2 domain revealed that it was capable of binding directly to the Tyr(P)317 motif of Shc with a KD of approximately 290 nM, in keeping with other specific SH2/Tyr(P) interactions. In vivo analysis revealed the SH2 and NPXpY motifs of SHIP acted together, with the Tyr(P)317 and phosphotyrosine binding (PTB) domains of Shc, respectively, to ensure a high affinity SHIP.Shc complex. Expression of cDNAs encoding hemagglutinin-tagged wild type and SH2-inactivated forms of SHIP in the murine hemopoietic cell line DA-ER revealed that wild type SHIP becomes both tyrosine-phosphorylated and associated with Shc following interleukin-3 stimulation, as expected, but the SH2-inactivated SHIPs do neither. Moreover, while the growth rates of parental DA-ER cells and cells expressing these various SHIP constructs are identical, the wild type SHIP-expressing cells die, via programmed cell death, far more rapidly than parental cells. Cells expressing SH2-inactivated SHIPs, on the other hand, show either a reduced or no effect on apoptosis. These results suggest that the SH2 domain of SHIP is required not only for the tyrosine phosphorylation of SHIP and Shc association following cytokine stimulation but also for its induction of apoptosis.

ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.

We have analyzed ErbB receptor interplay induced by the epidermal growth factor (EGF)-related peptides in cell lines naturally expressing the four ErbB receptors. Down-regulation of cell surface ErbB-1 or ErbB-2 by intracellular expression of specific antibodies has allowed us to delineate the role of these receptors during signaling elicited by: EGF and heparin binding EGF (HB-EGF), ligands of ErbB-1; betacellulin (BTC), a ligand of ErbB-1 and ErbB-4; and neu differentiation factor (NDF), a ligand of ErbB-3 and ErbB-4. Ligand-induced ErbB receptor heterodimerization follows a strict hierarchy and ErbB-2 is the preferred heterodimerization partner of all ErbB proteins. NDF-activated ErbB-3 or ErbB-4 heterodimerize with ErbB-1 only when no ErbB-2 is available. If all ErbB receptors are present, NDF receptors preferentially dimerize with ErbB-2. Furthermore, EGF- and BTC-induced activation of ErbB-3 is impaired in the absence of ErbB-2, suggesting that ErbB-2 has a role in the lateral transmission of signals between other ErbB receptors. Finally, ErbB-1 activated by all EGF-related peptides (EGF, HB-EGF, BTC and NDF) couples to SHC, whereas only ErbB-1 activated by its own ligands associates with and phosphorylates Cbl. These results provide the first biochemical evidence that a given ErbB receptor has distinct signaling properties depending on its dimerization.

Roles of insulin receptor substrate-1 and Shc on insulin-like growth factor I receptor signaling in early passages of cultured human fibroblasts.

Insulin-like growth factor-I (IGF-I) improves glucose metabolism and growth in patients with leprechaunism. We investigated signal transduction through IGF-I receptor in comparison with epidermal growth factor (EGF) receptor in early passages of cultured skin fibroblasts from a normal subject and a patient with leprechaunism whose insulin receptor tyrosine kinase was almost nonexistent. Insulin receptor substrate-1 (IRS-1) became tyrosine-phosphorylated and bound growth factor receptor-bound protein 2 (GRB2) quickly by IGF-I. The association of Shc with GRB2 by IGF-I was detected by immunoblot with anti-Shc antibody but was hardly visible with antiphosphotyrosine antibody, which was in marked contrast to efficient tyrosine phosphorylation of Shc by EGF. However, the potency of IGF-I for DNA synthesis was far stronger than EGF, which was not parallel with the potency of these growth factors to activate Shc or MAP kinase. Rather, phosphatidylinositol (PI) 3-kinase activity, which was activated by IGF-I about 5- to 10-fold more strongly than EGF, appeared to correlate with mitogenesis. Signal transduction pathways following IGF-I receptor or EGF receptor activation were indistinguishable between the normal subject and the patient. Our results strongly suggest that in human skin fibroblasts, which represent a more physiological cell culture: 1) IRS-1, rather than Shc, is the major tyrosine-phosphorylated protein binding GRB2 in initial phase of IGF-I signaling; 2) mitogenic potency of receptor tyrosine kinases such as IGF-I receptor and EGF receptor may not be determined solely by the amount of Shc-GRB2 complex or the activity of MAP kinase; and 3) in contrast to previous reports, IGF-I and EGF receptor signalings are not defective in leprechaunism.

Negative signaling in B cells causes reduced Ras activity by reducing Shc-Grb2 interactions.

To elucidate the molecular basis for inhibition of B cell proliferation and differentiation by the Fc receptor for IgG (Fc(gamma)RII), we compared the signaling events in B cells stimulated by cross-linking surface Ig alone (positive signaling), or by co-cross-linking surface Ig and Fc(gamma)RII (negative signaling). Both modes of stimulation induced tyrosine kinase activation. Positive signaling induced activation of Ras, Raf-1 kinase, and mitogen-activated protein kinase; these events were significantly attenuated during negative signaling. Since Ras is activated by SOS and Vav, two known guanine nucleotide exchange factors, activation events associated with these molecules using the two different stimuli were examined. Results of these experiments indicated that tyrosine phosphorylation of Vav did not change upon co-cross-linking. In contrast, the association of Shc and Grb2 was abrogated under negative and induced under positive signaling conditions. Concomitantly, Shc was observed to associate with a tyrosine-phosphorylated 145-kDa protein, previously identified as Src homology 2-containing inositol phosphatase, only under conditions of negative signaling. Based on these results, we hypothesize that negative signaling via the Fc(gamma)RII in B cells is at least partly the result of a block in Ras activation, and that SOS, but not Vav, is the major guanine nucleotide exchange factor in B cells for Ras activation.

Angiotensin II signaling in vascular smooth muscle. New concepts.

Angiotensin II is a multifunctional hormone that affects both contraction and growth of vascular smooth muscle cells through a complex series of intracellular signaling events initiated by the interaction of angiotensin II with the AT1 receptor. The cellular response to angiotensin II is multiphasic, involving stimulation within seconds of phospholipase C and Ca2+ mobilization; activation within minutes of phospholipase D, A2, protein kinase C, and MAP kinase; and stimulation after a period of hours of gene transcription and NADH/NADPH oxidase activity. Angiotensin II also activates numerous intracellular tyrosine kinases. In this respect, it shares some aspects of signaling with growth factor and cytokine receptors, including activation of phospholipase C-gamma, src, and ras; association of shc with grb2; and stimulation of the Jak/STAT pathway. The cellular events responsible for this unique series of events may involve receptor movement and the creation of a signaling domain. Elucidation of these pathways is important to our understanding of AT1 receptor function as a final effector of the renin-angiotensin system.

Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation.

Interaction of thrombopoietin (TPO) with its receptor, c-Mpl, triggers cell growth and differentiation responses controlling primitive haemopoietic cell production and megakaryocytopoiesis. To examine the important receptor domains and signal transduction pathways involved in these cellular responses, c-Mpl cytoplasmic domain truncation and tyrosine substitution mutants were generated. In the myelomonocytic leukaemia cell lines WEHI3B-D+ and M1, ectopic expression of the wild-type c-Mpl receptor induced TPO-dependent cellular differentiation characterized by increased cell migration through agar and acquisition of the morphology and molecular markers of macrophages. Consistent with the concept that proliferative and differentiation signals emanate from distinct receptor domains, the C-terminal 33 amino acids of c-Mpl were dispensable for a proliferative response in Ba/F3 cells but proved critical for WEHI3B-D+ and M1 differentiation. Finer mapping revealed that substitution of Tyr599 by phenylalanine within this c-Mpl domain was sufficient to abolish the normal differentiation response. Moreover, in contrast to the normal c-Mpl receptor, this same mplY599F mutant was also incapable of stimulating TPO-dependent Shc phosphorylation, the association of Shc with Grb2 or c-Mpl and of inducing c-fos expression. Thus activation of components of the Ras signalling cascade, initiated by interaction of Shc with c-Mpl Tyr599, may play a decisive role in specific differentiation signals emanating from the c-Mpl receptor.

The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein–protein interactions

Background Signal transduction initiated by a wide variety of extracellular signals involves the activation of protein-tyrosine kinases. Phosphorylated tyrosine residues in activated receptors or docking proteins then function as binding sites for the Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains of cytoplasmic signalling proteins. Shc is an adaptor protein that contains both PTB and SH2 domains and becomes phosphorylated on tyrosine in response to many different extracellular stimuli. These results have suggested that Shc is a prominent effector of protein-tyrosine kinase signalling. Thus far, only a single Shc phosphorylation site, the tyrosine at position 317 (Y317) has been identified. Phosphorylation of Y317 has been implicated in Grb2 binding and activation of the Ras pathway.Results Here, we report the identification of two major and novel Shc tyrosine phosphorylation sites, Y239 and Y240. These residues are present in the central proline-rich (CH1) region and are conserved in all isoforms of Shc. Y239/240 are co-ordinately phosphorylated by the Src protein-tyrosine kinase in vitro, and in response to epidermal growth factor stimulation or in v-src-transformed cells in vivo. Mutagenesis studies indicate that Y239/240 make an important contribution to the association of Shc with Grb2. Phosphopeptide-binding studies suggest that these two tyrosine residues may be involved in interactions with a number of cellular proteins.Conclusions Shc is the most prominent general substrate for protein-tyrosine kinases in vivo. The identification of two novel Shc phosphorylation sites indicates that Shc has the potential to interact with multiple downstream effectors. Shc Y239/240 are highly conserved in evolution, suggesting that the phosphorylation of these residues is of fundamental importance. We propose that distinct Shc phosphorylation isomers form different signalling complexes and thereby activate separate downstream signalling cascades.

Role of c-Src Tyrosine Kinase in G Protein-coupled Receptorand Gβγ 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.