The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner.

The cellular homologs of the v-Crk oncogene product are composed exclusively of Src homology region 2 (SH2) and SH3 domains. v-Crk overexpression in fibroblasts causes cell transformation and elevated tyrosine phosphorylation of specific cellular proteins. Among these proteins is a 130-kDa protein, identified as p130cas, that forms a stable complex in vivo with v-Crk. We have explored the role of endogenous Crk proteins in Bcr-Abl-transformed cells. In the K562 human chronic myelogenous leukemia cell line, p130cas is not tyrosine phosphorylated or bound to Crk. Instead, Crk proteins predominantly associate with the tyrosine-phosphorylated proto-oncogene product of Cbl. In vitro analysis showed that this interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. In NIH 3T3 cells transformed by Bcr-Abl, c-Cbl becomes strongly tyrosine phosphorylated and associates with c-Crk. The complex between c-Crk and c-Cbl is also seen upon T-cell receptor cross-linking or with the transforming, tyrosine-phosphorylated c-Cbl. These results indicate that Crk binds to c-Cbl in a tyrosine phosphorylation-dependent manner, suggesting a physiological role for the Crk-c-Cbl complex in Bcr-Abl tyrosine phosphorylation-mediated transformation.

A novel, multifuntional c-Cbl binding protein in insulin receptor signaling in 3T3-L1 adipocytes.

The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.

Divergence of signaling pathways for insulin in PC-12 pheochromocytoma cells.

A PC-12 pheochromocytoma cell line is described with roughly equivalent levels of functional receptors for nerve growth factor (NGF), epidermal growth factor (EGF), and insulin. Each of these receptors undergoes autophosphorylation upon binding of their respective ligands, and causes the activation of phosphatidylinositol-3 kinase via a mechanism involving tyrosine phosphorylation. In the case of insulin, this activation is due to the tyrosine phosphorylation of its major cellular substrate, IRS-1. Despite the presence of functional receptors in these cells, insulin does not stimulate the activity of the mitogen-activated protein (MAP) kinase, despite a 5- to 8-fold activation observed with both NGF and EGF under the same conditions. This failure to activate MAP kinase was not due to the insulin-dependent dephosphorylation of the enzyme, but correlated with the lack of activation of the MAP kinase kinase, although this enzyme was also activated by NGF and EGF. Similarly, the activation of the raf and ras protooncogenes in these cells was not observed with insulin, whereas NGF and EGF produced marked activation. In addition, insulin-dependent induction of the c-fos protein was impaired, in comparison to NGF. In contrast to a lack of effect on the MAP kinase pathway, these PC-12 cells were metabolically responsive to insulin, exhibiting increases in glucose, lipid, and protein synthesis in response to the hormone. The differential responses of phosphorylation events to insulin, NGF, and EGF in these cells indicates that divergence of signaling pathways may occur at or near the insulin receptor.

A cAMP-triggered release of a hormone-like peptide.

Preparations of the catalytic subunit of cAMP-dependent protein kinase from rabbit skeletal muscle, which appear to be homogeneous by SDS-polyacrylamide gel electrophoresis, were often found to contain a hormone-like factor (HLF) which causes an immediate rise, then a decline of intracellular cAMP in a B-lymphoma cell line. Active HLF is released when the fractions that contain it in an inactive form are incubated with cAMP prior to chromatography, or passed through an immobilized cAMP column. HLF seems to be a peptide: it loses its cell-stimulating capability after proteolysis and has an apparent molecular mass of 2.2-2.5 kDa.

Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-Cbl in 3T3-L1 adipocytes.

We report here that the product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl reaches a maximum within 1-2 min after stimulation by insulin and gradually declines thereafter. The tyrosine phosphorylation of c-Cbl was also observed after treatment of 3T3-L1 adipocytes with epidermal growth factor, whereas platelet-derived growth factor had no effect. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with fusion proteins containing the Src homology 2 (SH2) domains of Crk and the Fyn tyrosine kinase, but not with fusion proteins containing the SH2 domains of either the p85 subunit of phosphatidylinositol 3'-kinase or the tyrosine phosphatase SHPTP2/Syp. Furthermore insulin stimulates the association of c-Cbl with endogenous c-Crk and Fyn in intact 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl is regulated during adipocyte differentiation. Neither insulin-like growth factor 1 nor insulin stimulated the tyrosine phosphorylation of c-Cbl in 3T3-L1 fibroblasts. Moreover, c-Cbl is not tyrosine phosphorylated in response to insulin in cells expressing high levels of the human insulin receptor, or in hepatocytes, despite comparable levels of c-Cbl expression. These results suggest that c-Cbl might have a novel function in the regulation of insulin receptor intracellular signalling in 3T3-L1 adipocytes.

Nerve growth factor stimulates the tyrosine phosphorylation of endogenous Crk-II and augments its association with p130Cas in PC-12 cells.

The cellular homologs of the v-Crk oncogene product consist primarily of Src homology region 2 (SH2) and 3 (SH3) domains. v-Crk overexpression causes cell transformation and elevation of tyrosine phosphorylation in fibroblasts and accelerates differentiation of PC-12 cells in response to nerve growth factor (NGF). To further explore the role of Crk in NGF-induced PC-12 cell differentiation, we found that both NGF and epidermal growth factor stimulate the tyrosine phosphorylation of endogenous Crk II. Moreover, hormone stimulation enhanced the specific association of Crk proteins with the tyrosine-phosphorylated p130Cas, the major phosphotyrosine-containing protein in cells transformed with v-Crk. This interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. Furthermore, the Crk-SH2 domain binds tyrosine-phosphorylated paxillin, a cytoskeletal protein, following treatment of PC-12 cells with NGF or epidermal growth factor. These data suggest that Crk functions in a number of signaling processes in PC-12 cells.

c-Myc does not require max for transcriptional activity in PC-12 cells.

The c-Myc proto-oncogene is a basic helix-loop-helix leucine zipper (b/HLH/LZ) protein that participates in cellular growth and differentiation. The expression of c-Myc mRNA is rapidly induced by nerve growth factor (NGF) and epidermal growth factor (EGF) in PC-12 pheochromocytoma cells. In most cell types, c-Myc forms a sequence-specific DNA binding complex with the stable, constitutively expressed Max. This complex can function as a transcriptional regulator. We show here that the expression of Max mRNA or protein was not detected in PC-12 cells. Nevertheless, treatment of PC-12 cells with NGF and serum caused an increase in the expression of the c-Myc protein and the transcription of a reporter gene linked to the Myc/Max DNA binding site. Transcription from the same reporter gene is stimulated by over-expression of c-Myc. These results suggest that c-Myc protein functions as a transcriptional regulator in PC-12 cells despite the lack of Max protein. Therefore, Myc/Max complexes may not be an absolute requirement for Myc-dependent gene expression.

Cloning and characterization of a functional peroxisome proliferator activator receptor-gamma-responsive element in the promoter of the CAP gene.

c-Cbl-associating protein (CAP) is a multifunctional signaling protein that interacts with c-Cbl, facilitating the tyrosine phosphorylation of c-Cbl in response to insulin. In 3T3-L1 adipocytes and diabetic rodents, CAP gene expression is stimulated by activators of peroxisome proliferator activator receptor gamma (PPARgamma), such as thiazolidinediones (TZDs), resulting in increased insulin-stimulated c-Cbl phosphorylation. Sequence analysis of 2.5 kilobases of the 5'-flanking region of the CAP gene reveals a predicted peroxisome proliferator response element (PPRE) from -1085 to -1097. The isolated promoter was functional in 3T3 fibroblasts and adipocytes. Co-transfection of the CAP promoter with PPARgamma and retinoic acid X receptor alpha caused fold stimulation of promoter activity. The TZD rosiglitazone produced an additional 2-3-fold stimulation of the promoter. Deletion of the predicted PPRE from the CAP promoter abolished its ability to respond to rosiglitazone. Gel shift analysis of the putative PPARgamma site demonstrates direct binding of PPAR/retinoid X receptor heterodimers to the PPRE in the CAP gene. These data demonstrate that TZDs directly stimulate transcription of the CAP gene through activation of PPARgamma.

Thiazolidinediones and insulin resistance: peroxisome proliferatoractivated receptor gamma activation stimulates expression of the CAP gene.

c-Cbl-associated protein (CAP) is a signaling protein that interacts with both c-Cbl and the insulin receptor that may be involved in the specific insulin-stimulated tyrosine phosphorylation of c-Cbl. The restricted expression of CAP in cells metabolically sensitive to insulin suggests an important potential role in insulin action. The expression of CAP mRNA and proteins are increased in 3T3-L1 adipocytes by the insulin sensitizing thiazolidinedione drugs, which are activators of the peroxisome proliferator-activated receptor gamma (PPARgamma). The stimulation of CAP expression by PPARgamma activators results from increased transcription. This increased expression of CAP was accompanied by a potentiation of insulin-stimulated c-Cbl tyrosine phosphorylation. Administration of the thiazolidinedione troglitazone to Zucker (fa/fa) rats markedly increased the expression of the major CAP isoform in adipose tissue. This effect was sustained for up to 12 weeks of treatment and accompanied the ability of troglitazone to prevent the onset of diabetes and its complications. Thus, CAP is the first PPARgamma-sensitive gene identified that participates in insulin signaling and may play a role in thiazolidinedione-induced insulin sensitization.

Identification of the major SHPTP2-binding protein that is tyrosine-phosphorylated in response to insulin.

Immunoprecipitation of the cytosolic Src homology 2 domain-containing protein-tyrosine phosphatase, SHPTP2, from insulin-stimulated 3T3L1 adipocytes or Chinese hamster ovary cells expressing the human insulin receptor resulted in the coimmunoprecipitation of a diffuse tyrosine-phosphorylated band in the 115-kDa protein region on SDS-polyacrylamide gels. Although platelet-derived growth factor induced the tyrosine phosphorylation of the platelet-derived growth factor receptor and SHPTP2, there was no significant increase in the coimmunoprecipitation of tyrosine-phosphorylated pp115 with SHPTP2. SHPTP2 was also associated with tyrosine-phosphorylated insulin receptor substrate-1, but this only accounted for < 2% of the total immunoreactive SHPTP2 protein. Similarly, only a small fraction of the total amount of tyrosine-phosphorylated insulin receptor substrate-1 (< 4%) was associated with SHPTP2. Expression and immunoprecipitation of a Myc epitope-tagged wild-type SHPTP2 (Myc-WT-SHPTP2) and a catalytically inactive point mutant of SHPTP2 (Myc-C/S-SHPTP2) also demonstrated an insulin-dependent association of SHPTP2 with tyrosine-phosphorylated pp115. Furthermore, expression of the catalytically inactive SHPTP2 mutant resulted in a marked enhancement in the amount of coimmunoprecipitated tyrosine-phosphorylated pp115 compared with the expression of wild-type SHPTP2. These data indicate that the insulin-stimulated tyrosine-phosphorylated 115-kDa protein is the predominant in vivo SHPTP2-binding protein and that pp115 may function as a physiological substrate for the SHPTP2 protein-tyrosine phosphatase.

A role for CAP, a novel, multifunctional Src homology 3 domain-containing protein in formation of actin stress fibers and focal adhesions.

c-Cbl-associated protein, CAP, was originally cloned from a 3T3-L1 adipocyte cDNA expression library using full-length c-Cbl as a bait. CAP contains a unique structure, with three adjacent Src homology-3 (SH3) domains in the COOH terminus and a region sharing significant sequence similarity with the peptide hormone sorbin. Expression of CAP in NIH-3T3 cells overexpressing the insulin receptor induced the formation of stress fibers and focal adhesions. This effect of CAP expression on the organization of the actin-based cytoskeleton was independent of the type of integrin receptors engaged with extracellular matrix, whereas membrane ruffling and decreased actin stress fibers induced by insulin were not affected by expression of CAP. Immunofluorescence microscopy demonstrated that CAP colocalized with actin stress fibers. Moreover, CAP interacted with the focal adhesion kinase, p125FAK, both in vitro and in vivo through one of the SH3 domains of CAP. The increased formation of stress fibers and focal adhesions in CAP-expressing cells was correlated with decreased tyrosine phosphorylation of p125FAK in growing cells or upon integrin-mediated cell adhesion. These results suggest that CAP may mediate signals for the formation of stress fibers and focal adhesions.

Spatial determinants of specificity in insulin action.

Insulin is a potent stimulator of intermediary metabolism, however the basis for the remarkable specificity of insulin's stimulation of these pathways remains largely unknown. This review focuses on the role compartmentalization plays in insulin action, both in signal initiation and in signal reception. Two examples are discussed: (1) a novel signalling pathway leading to the phosphorylation of the caveolar coat protein caveolin, and (2) a recently identified scaffolding protein, PTG, involved directly in the regulation of enzymes controlling glycogen metabolism.

The tyrosine kinase inhibitor tyrphostin blocks the cellular actions of nerve growth factor.

A series of the synthetic protein kinase inhibitors known as tyrphostins were examined for their effects on the tyrosine autophosphorylation of the pp140c-trk, nerve growth factor (NGF) receptor. One of the tyrphostins, AG879, inhibited NGF-dependent pp140c-trk tyrosine phosphorylation, but did not affect tyrosine phosphorylation of epidermal growth factor or platelet-derived growth factor receptors. In addition, the tyrosine phosphorylation of the receptor-associated protein pp38 was also attenuated by the tyrphostin. This effect was time- and dose-dependent, although inhibition of pp38 phosphorylation occurred earlier and at lower concentrations of the compound. AG879 also inhibited NGF-induced PLC-gamma 1 phosphorylation, phosphatidylinositol-3 (PI3) kinase activation, the association of the tyrosine-phosphorylated proteins pp100 and pp110 with the p85 subunit of PI-3 kinase, mitogen activated protein and raf-1 kinases, and c-fos induction. In addition, AG879 inhibited NGF-induced neurite outgrowth in PC12 cells. These data indicate that tyrosine kinase activity of the pp140c-trk NGF receptor is essential for the cellular actions of this growth factor.

Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases.

Steroid hormones have rapid nongenomic effects on cell-signaling pathways, but the receptor mechanisms responsible for this are not understood. We have identified a specific polyproline motif in the amino-terminal domain of conventional progesterone receptor (PR) that mediates direct progestin-dependent interaction of PR with SH3 domains of various cytoplasmic signaling molecules, including c-Src tyrosine kinases. Through this interaction, PR is a potent activator of Src kinases working by an SH3 domain displacement mechanism. By mutagenesis, we also show that rapid progestin-induced activation of Src and downstream MAP kinase in mammalian cells is dependent on PR-SH3 domain interaction, but not on the transcriptional activity of PR. Preliminary evidence for the biological significance of this PR signaling pathway through regulatory SH3 domains was shown with respect to an influence on progestin-induced growth arrest of breast epithelial cells and induction of Xenopus oocyte maturation.

CAP defines a second signalling pathway required for insulin-stimulated glucose transport.

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.