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.

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.

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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.

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The Crk proteins, originally isolated as an oncogene product in an avian sarcoma virus (26), belong to a class of adapter proteins containing Src homology 2 (SH2) and SH3 domains. Other members of this group include Grb2/ASH (11) and Nck (13). These bifunctional proteins are thought to couple tyrosine-phosphorylated receptors or their substrates via the SH2 domain to downstream effectors via the SH3 domain. Three cellular homologs of v-Crk have been identified: Crk-I, a 21-kDa protein with only one SH2 and one SH3 domain (24); Crk-II, 40-and 42-kDa proteins consisting of one SH2 and two SH3 domains (36); and CrkL, a 36-kDa Crk-like protein with one SH2 and two SH3 domains (49). Despite their lack of demonstrated enzymatic activity, expression of v-Crk or Crk-I but not Crk-II leads to cell transformation and increased tyrosine phosphorylation (24,27). Furthermore, v-Crk binds directly to the major tyrosine-phosphorylated proteins present in v-Crk-transformed cells, presumably via its SH2 domain (22, 23). These phosphoproteins include paxillin, a focal adhesionassociated protein (3), and the newly identified p130 cas (39). p130cas has no known catalytic domain but contains a cluster of predicted high-affinity Crk-SH2 binding motifs and forms a stable complex in vivo with v-Crk. The Crk-SH3 domain has been shown to interact with several proteins, including Abl (8), Sos (9, 21), C3G (9, 15, 21, 46), and, more recently, Eps15 (42). Since Sos and C3G contain a guanine nucleotide exchange activity, Crk proteins have been hypothesized to play a role in the regulation of p21 ras -GTP formation. Both the SH2 and SH3 domains of v-Crk are required for the transformation of chicken embryo fibroblasts (27). In addition, Crk mutants with dysfunctional SH2 or SH3 domains inhibited neuronal differentiation of PC-12 cells and nerve growth factor-induced activation of Ras (21, 45).The c-Abl oncogene product is a member of the nonreceptor class of tyrosine kinases. Like c-Crk-II, c-Abl overexpression does not lead to cellular transformation (10,14). However, the chromosomal translocation of c-abl to the bcr gene in Philadelphia chromosome-positive human leukemias produced the 210-and 190-kDa chimeric Bcr-Abl proteins (6, 16). These fusion proteins have elevated tyrosine kinase activity and transforming properties (20,32). Although the precise mechanisms involved in cellular transformation by Bcr-Abl remain unclear, several key signaling proteins are known to be tyrosine phosphorylated in cells bearing this oncogene, including Bcr-Abl itself (32) as well as Shc (35), CrkL (48), paxillin (40), and the proto-oncogene product of cbl (1). Moreover, activation of Ras has also been implicated in Bcr-Abl-mediated cellular transformation (34,35).To evaluate the potential role of Crk proteins in the transforming functions of Bcr-Abl, we have examined their interactions in the human chronic myelogenous leukemia cell line K562. We report here that, unlike observations in v-Crk-and v-Src-transformed cells, p130cas is not tyrosine phosphorylated...

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.