Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612

Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.     

Insulin signal transduction by a mutant human insulin receptor lacking the NPEY sequence. Evidence for an alternate mitogenic signaling pathway that is independent of Shc phosphorylation

The cytoplasmic juxtamembrane domain of the human insulin receptor (hIR) contains a single copy of the tetrameric amino acid sequence Asn-Pro-Glu-Tyr (NPEY) (residues 969-972 in the exon 11-containing B-isoform), which exists in the context of NPXY. In this study, we examined the role of NPEY972 in mediating insulin signal transduction and cellular biological effects. Transfected Chinese hamster ovary cell lines expressing either the wild-type hIR-B isoform (hIR.WT) or a mutant receptor lacking the NPEY972 sequence (hIRDeltaNPEY) and control Chinese hamster ovary.Neo cells were used to comparatively analyze the following insulin effects: in vivo receptor tyrosine autophosphorylation and kinase activity, signal transduction to downstream signaling molecules, and stimulation of glycogen and DNA synthesis. The results showed that in comparison to hIR.WT, the hIRDeltaNPEY mutant demonstrated the following: (a) normal insulin-mediated receptor tyrosine phosphorylation, but approximately 50% reduction in phosphorylation of p185-(insulin receptor substrate-1) and binding of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase), (b) an enhanced stimulation of PI 3-kinase enzymatic activity, (c) a complete inability to phosphorylate Shc, (d) minimal impairment of insulin sensitivity for glycogen synthesis, and (e) an augmented response to insulin-stimulated DNA synthesis via a high capacity, low sensitivity pathway. These results demonstrate the following: 1) the NPEY972 sequence is important but not absolutely essential for coupling of hIR kinase to insulin receptor substrate-1 and p85 or for mediating insulin's metabolic and mitogenic effects, 2) the NPEY972 sequence is necessary for Shc phosphorylation, and 3) the absence of Shc phosphorylation releases the constraints on maximal insulin-stimulated mitogenic response, thus indicating that alternate signaling pathway(s) exist for this insulin action. This alternate pathway appears to be associated with enhanced activation of PI 3-kinase and is of high capacity and low sensitivity.     

Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase

We examined the role of 185-kDa insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) in the signaling pathway of insulin-stimulated GLUT4 translocation. We had already developed a novel cell line to detect GLUT4 on the cell surface, directly and sensitively (Kanai, F., Nishioka, Y., Hayashi, H., Kamohara, S., Todaka, M., and Ebina, Y. (1993) J. Biol. Chem. 268, 14523-14526). We stably expressed a mutant insulin receptor in which Tyr972 was replaced with phenylalanine. Insulin-stimulated tyrosyl phosphorylation of IRS-1 and GLUT4 translocation were decreased in cells expressing the mutant receptor, as compared to findings in cells expressing the normal receptor. Wortmannin, an inhibitor of PI3-kinase, inhibits the insulin-stimulated PI3-kinase activity and GLUT4 translocation at 50 nM, but not the NaF-stimulated GLUT4 translocation. These results suggest that the tyrosine phosphorylation of IRS-1 and activation of PI3-kinase may be involved in the signaling pathway of the insulin-stimulated GLUT4 translocation.     

Effect of pp120 on receptor-mediated insulin endocytosis is regulated by the juxtamembrane domain of the insulin receptor

pp120, a substrate of the insulin receptor tyrosine kinase, does not undergo ligand-stimulated phosphorylation by the insulin-like growth factor-1 (IGF-1) receptor. However, replacement of the C-terminal domain of the IGF-1 receptor beta-subunit with the corresponding segment of the insulin receptor restored pp120 phosphorylation by the chimeric receptor. Since pp120 stimulates receptor-mediated insulin endocytosis when it is phosphorylated, we examined whether pp120 regulates IGF-1 receptor endocytosis in transfected NIH 3T3 cells. pp120 failed to alter IGF-1 receptor endocytosis via either wild-type or chimeric IGF-1 receptors. Thus, the effect of pp120 on hormone endocytosis is specific to insulin, and the C-terminal domain of the beta-subunit of the insulin receptor does not regulate the effect of pp120 on insulin endocytosis. Mutation of Tyr960 in the juxtamembrane domain of the insulin receptor abolished the effect of pp120 to stimulate receptor endocytosis, without affecting pp120 phosphorylation by the insulin receptor. These findings suggest that pp120 interacts with two separate domains of the insulin receptor as follows: a C-terminal domain required for pp120 phosphorylation and a juxtamembrane domain required for internalization. We propose that the interaction of pp120 with the juxtamembrane domain is indirect and requires one or more substrates that bind to Tyr960 in the insulin receptor.     

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulation of GLUT4 translocation is tyrosine kinase-dependent

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) treatment of permeabilized adipocytes results in GLUT4 translocation similar to that elicited by insulin treatment. However, although the selective phosphatidylinositol 3-kinase inhibitor, wortmannin, completely prevented insulin-stimulated GLUT4 translocation, it was without effect on GTPgammaS-stimulated GLUT4 translocation. In addition, insulin was an effective stimulant, whereas GTPgammaS was a very weak activator of the downstream Akt serine/threonine kinase. Consistent with an Akt-independent mechanism, guanosine 5'-O-2-(thio)diphosphate inhibited insulin-stimulated GLUT4 translocation without any effect on the Akt kinase. Surprisingly, two functionally distinct tyrosine kinase inhibitors, genistein and herbimycin A, as well as microinjection of a monoclonal phosphotyrosine specific antibody, inhibited both GTPgammaS- and insulin-stimulated GLUT4 translocation. Phosphotyrosine immunoblotting and specific immunoprecipitation demonstrated that GTPgammaS did not elicit tyrosine phosphorylation of insulin receptor or insulin receptor substrate-1. In contrast to insulin, proteins in the 120-130-kDa and 55-75-kDa range were tyrosine-phosphorylated following GTPgammaS stimulation. Several of these proteins were identified and include protein-tyrosine kinase 2 (also known as CAKbeta, RAFTK, and CADTK), pp125 focal adhesion tyrosine kinase, pp130 Crk-associated substrate, paxillin, and Cbl. These data demonstrate that the GTPgammaS-stimulated GLUT4 translocation utilizes a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha

Insulin resistance is an important metabolic abnormality often associated with infections, cancer, obesity, and especially non-insulin-dependent diabetes mellitus (NIDDM). We have previously demonstrated that tumor necrosis factor-alpha produced by adipose tissue is a key mediator of insulin resistance in animal models of obesity-diabetes. However, the mechanism by which TNF-alpha interferes with insulin action is not known. Since a defective insulin receptor (IR) tyrosine kinase activity has been observed in obesity and NIDDM, we measured the IR tyrosine kinase activity in the Zucker (fa/fa) rat model of obesity and insulin resistance after neutralizing TNF-alpha with a soluble TNF receptor (TNFR)-lgG fusion protein. This neutralization resulted in a marked increase in insulin-stimulated autophosphorylation of the IR, as well as phosphorylation of insulin receptor substrate 1 (IRS-1) in muscle and fat tissues of the fa/fa rats, restoring them to near control (lean) levels. In contrast, no significant changes were observed in insulin-stimulated tyrosine phosphorylations of IR and IRS-1 in liver. The physiological significance of the improvements in IR signaling was indicated by a concurrent reduction in plasma glucose, insulin, and free fatty acid levels. These results demonstrate that TNF-alpha participates in obesity-related systemic insulin resistance by inhibiting the IR tyrosine kinase in the two tissues mainly responsible for insulin-stimulated glucose uptake: muscle and fat.     

Coordinated regulation of the tyrosine phosphorylation of Cbl by Fyn and Syk tyrosine kinases

Cross-linking of the T cell antigen receptor (TCR)-CD3 complex induces rapid tyrosine phosphorylation and activation of Src (Lck and Fyn) and Syk (Syk and Zap-70) family protein tyrosine kinases (PTKs) which, in turn, phosphorylate multiple intracellular substrates. Cbl is a prominent PTK substrate suggesting a pivotal role for it in early signal transduction events. However, the regulation of Cbl function and tyrosine phosphorylation in T cells by upstream PTKs remains poorly understood. In the present study, we used genetic and biochemical approaches to demonstrate that Cbl directly interacts with Syk and Fyn via its N-terminal and C-terminal regions, respectively. Tyr-316 of Syk was required for the interaction with Cbl as well as for the maximal tyrosine phosphorylation of Cbl. However, both wild-type Syk and Y316F-mutated Syk phosphorylated equally well the C-terminal fragment of Cbl in vivo, suggesting the existence of an alternative, N terminus-independent mechanism for the Syk-induced tyrosine phosphorylation of Cbl. This mechanism appears to involve Fyn, since, in addition to its association with the C-terminal region of Cbl, Fyn also associated with Syk and enhanced the Syk-induced tyrosine phosphorylation of Cbl. These findings implicate Fyn as an adaptor protein that facilitates the interaction between Syk and Cbl, and suggest that Src and Syk family PTKs coordinately regulate the tyrosine phosphorylation of Cbl.     

Replacement of tyrosine 1251 in the carboxyl terminus of the insulin-like growth factor-I receptor disrupts the actin cytoskeleton and inhibits proliferation and anchorage-independent growth

Insulin-like growth factor (IGF)-I signaling through the IGF-I receptor modulates cellular adhesion and proliferation and the transforming ability of cells overexpressing the IGF-I receptor. Tyrosine phosphorylation of intracellular proteins is essential for this transduction of the IGF-I-induced mitogenic and tumorigenic signals. IGF-I induces specific cytoskeletal structure and the phosphorylation of proteins in the associated focal adhesion complexes. The determination of the exact pathways emanating from the IGF-I receptor that are involved in mediating these signals will contribute greatly to the understanding of IGF-I action. We have previously shown that replacement of tyrosine residues 1250 and 1251 in the carboxyl terminus of the IGF-I receptor abrogates IGF-I-induced cellular proliferation and tumor formation in nude mice. In this study, replacement of either tyrosine 1250 or 1251 similarly reduces the cells ability to grow in an anchorage-independent manner. The actin cytoskeleton and cellular localization of vinculin are disrupted by replacement of tyrosine 1251. Tyrosine residues 1250 and 1251 are not essential for tyrosine phosphorylation of two known substrates; insulin receptor substrate-1 and SHC, nor association of known downstream adaptor proteins to these substrates. In addition, these mutant IGF-I receptors do not affect IGF-I-stimulated p42/p44 mitogen-activated protein kinase activation or phosphatidylinositol (PI) 3'-kinase activity. Thus, it appears that in fibroblasts expressing tyrosine 1250 and 1251 mutant IGF-I receptors, the signal transduction pathways impacting on mitogenesis and tumorigenesis do not occur exclusively through the PI 3'-kinase or mitogen-activated protein kinase pathways.     

T cell receptor engagement induces tyrosine phosphorylation of FAK and Pyk2 and their association with Lck

Stimulation through the TCR is known to induce tyrosine phosphorylation of a number of proteins, which leads to functional activation of T cells. Identification of the substrates that become phosphorylated and defining their interactions with other signaling molecules will provide insight into the mechanisms controlling T cell activation. Focal adhesion kinase (FAK) and the recently described Pyk2 kinase are homologous members of a non-receptor protein tyrosine kinase family. FAK has been shown to become phosphorylated upon TCR stimulation, but its role, if any, in T cell activation remains to be defined. Although Pyk2 has been shown to play a role in neuronal cell activation stimulated through G-protein-coupled receptors, a role in T cell activation has not been described. In this study we show that FAK and Pyk2 are two of the major 115-to-120-kDa proteins that become tyrosine phosphorylated in T cells following TCR complex stimulation. Furthermore, coincident with the increase in tyrosine phosphorylation, we show an association of these kinases with the SH2 domain of the tyrosine kinase Lck in vivo. The increase in tyrosine phosphorylation of both FAK and Pyk2, however, occurs in Lck-deficient cells suggesting that phosphorylation of both of these kinases does not require Lck. Taken together, these results suggest that FAK and Pyk2, perhaps in coordination with Lck, play a role in T cell activation.     

Throwing injury of the elbow: assessment with gradient three-dimensional, fourier transform gradient-echo and short tau inversion recovery images

We have recently purified and cloned a new member of the insulin receptor substrate family, designated insulin receptor substrate 3 (IRS-3), from rat adipocytes. The amino acid sequence of IRS-3 shows multiple potential sites for tyrosine phosphorylation in motifs which engage specific SH2 domain-containing proteins. In order to determine which SH2 domain proteins complex with IRS-3, we have searched for coimmunoprecipitation from lysates of untreated and insulin-stimulated adipocytes. Phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP-2 complexed with the tyrosine phosphorylated form of IRS-3, whereas the phospholipase Cgamma did not, and the adaptor Grb2 did so to a much lesser extent. These findings complete the survey of SH2 domain proteins associated with each of the four known members of the IRS family and provide the framework for further analysis of the role of IRS-3 in insulin signaling.     

Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine following treatment of UT-7 cells with erythropoietin. We have investigated the expression of IRS-1 and IRS-2 in several cell lines with erythroid and/or megakaryocytic features, and we observed that IRS-2 was expressed in all cell lines tested. In contrast, we did not detect the expression of IRS-1 in these cells. In response to erythropoietin, IRS-2 was immediately phosphorylated on tyrosine, with maximal phosphorylation between 1 and 5 min. Tyrosine-phosphorylated IRS-2 was associated with phosphatidylinositol 3-kinase and with a 140-kDa protein that comigrated with the phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase, SHIP. Moreover, IRS-2 was constitutively associated with the erythropoietin receptor. We did not observe the association of IRS-2 with JAK2, Grb2, or PTP1D. Using BaF3 cells transfected with mutated erythropoietin receptors, we demonstrate that neither the tyrosine residues of the intracellular domain nor the last 109 amino acids of the erythropoietin receptor are required for erythropoietin-induced IRS-2 tyrosine phosphorylation. Altogether, our results indicate that erythropoietin-induced IRS-2 tyrosine phosphorylation could account for the previously reported activation of phosphatidylinositol 3-kinase mediated by erythropoietin receptors mutated in the phosphatidylinositol 3-kinase-binding site (Damen, J., Cutler, R. L., Jiao, H., Yi, T., and Krystal, G. (1995) J. Biol. Chem. 270, 23402-23406; Gobert, S., Porteu, F., Pallu, S., Muller, O., Sabbah, M., Dusanter-Fourt, I., Courtois, G., Lacombe, C., Gisselbrecht, S., and Mayeux, P. (1995) Blood 86, 598-606).     

p125Fak focal adhesion kinase is a substrate for the insulin and insulin-like growth factor-I tyrosine kinase receptors

The focal adhesion kinase p125(Fak) is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125(Fak) phosphorylation, insulin has been shown to promote its dephosphorylation. In this study, we compared p125(Fak) phosphorylation in insulin-stimulated cells maintained in suspension or in an adhesion state. We found that, in nonattached cells, insulin promotes p125(Fak) phosphorylation, whereas dephosphorylation occurred in attached cells. This was observed in Rat-1 fibroblasts overexpressing the insulin receptor, as well as in Hep G2 hepatocytes and in 3T3-L1 adipocytes expressing more natural levels of insulin receptors. Insulin-induced p125(Fak) phosphorylation correlated with an increase in paxillin phosphorylation, indicating that p125(Fak) kinase activity may be stimulated by insulin. Mixing of purified insulin or insulin-like growth factor-I (IGF-I) receptors with p125(Fak) resulted in an increase in p125(Fak) phosphorylation. Using a kinase-deficient p125(Fak) mutant, we found that this protein is a direct substrate of the insulin and IGF-I receptor tyrosine kinases. This view is supported by two additional findings. (i) A peptide corresponding to p125(Fak) sequence comprising amino acids 568-582, which contains tyrosines 576 and 577 of the kinase domain regulatory loop, is phosphorylated by the insulin receptor; and (ii) p125(Fak) phosphorylation by the insulin receptor is prevented by addition of this peptide. Finally, we observed that p125(Fak) phosphorylation by the receptor results in its activation. Our results show that the nature of the cross-talk between the insulin/IGF-I receptors and p125(Fak) is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly.     

Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk- B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.     

CD50 (ICAM-3) is phosphorylated on tyrosine and is associated with tyrosine kinase activity in human neutrophils

CD50 (ICAM-3) is expressed at a high level on resting blood granulocytes, monocytes, and lymphocytes. The constitutive high expression of CD50 on resting leukocytes suggests that it is an important LFA-1 ligand in the initiation of the immune/inflammatory response. Using a radiolabeling technique initially designed to detect ecto-protein kinase activity, we found that CD50 mAbs immunoprecipitated a approximately 125- to 170-kDa phosphoprotein from human neutrophils. Phosphorylation was increased after stimulation with the chemotactic agent FMLP, platelet-activating factor, 12-O-tetradecanoyl-phorbol-13-acetate, and the calcium ionophore A23187. This increase in phosphorylation was transient with the maximal phosphorylation, being observed by 1 min. Phosphoamino acid analysis revealed that CD50 contained predominantly phosphotyrosine. Although this assay system was designed initially to detect ecto-protein kinase activity, subsequent studies have shown that membrane proteins can be phosphorylated on the cytoplasmic domain under these conditions. When CD50 was immunoprecipitated from solubilized neutrophils, protein tyrosine kinase activity associated with CD50 was detected in the immunoprecipitate. The data suggest that phosphorylation of CD50 on tyrosine by an associated tyrosine kinase plays a role in the function of CD50.     

The synapse-associated protein rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.     

Effects of tyrosine kinase inhibitors on tyrosine phosphorylations and the insulin-like effects in response to human growth hormone in isolated rat adipocytes

Recent data suggest involvement of the Janus tyrosine kinase-2 (JAK2) in human GH-induced tyrosine phosphorylation of the GH receptor and the insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), leading to activation of the phosphatidylinositol 3-kinase and the acute insulin-like effects in primary rat adipocytes. To investigate the functional role of this kinase, we screened a number of tyrosine kinase inhibitors for their ability to inhibit three rapid effects of GH on primary adipocytes: increased lipogenesis, inhibition of noradrenaline-induced lipolysis, and promotion of JAK2 tyrosine phosphorylation. Only staurosporine was found to inhibit all three effects. The inhibition of lipogenesis and antilipolysis exhibited the same staurosporine dose dependency (IC50, approximately 40 nM) as inhibition of JAK2 and IRS-1 tyrosine phosphorylation as well as binding of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2. The unidentified cytosolic tyrosine-phosphorylated protein pp95, in contrast, was not affected, suggesting that it is not phosphorylated primarily by JAK2. Protein kinase C does not seem to be directly involved in the insulin-like effects, because the selective protein kinase C inhibitor calphostin C had no effect at levels up to 100 nM above which unspecific cellular effects occurred. Methyl-2,5-dihydroxy cinnamate inhibited GH-induced lipogenesis from [3-3H]glucose and nonstimulated lipogenesis from [2-14C]-pyruvate and [3H]acetate, but was without effect on GH-induced 2-deoxy-D-[1-3H]glucose uptake, JAK2 phosphorylation and antilipolysis, suggesting unspecific effects on mitochondrial metabolism rather than a direct effect on the GH-mediated signal. Tyrphostin 25 and herbimycin A had no effect on any of the parameters studied, except for a slight increase in JAK2 phosphorylation in response to tyrphostin 25. In summary, these data support the role for JAK2 in mediating the insulin-like effects of GH in adipocytes.     

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.