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Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in

APS (adapter protein with Pleckstrin homology and Src homology 2 domains) is recruited by the autophosphorylated insulin receptor and is essential for Glut4 translocation. Although both APS and CAP (c-CblInsulin increases glucose transport into target tissues by promoting the exocytosis of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (1, 2). In recent years, it has emerged that there are two pathways required for insulin-stimulated glucose transport, namely an insulin receptor substrate/PI 1 3-kinase pathway and a PI 3-kinase independent pathway involving the c-Cbl-associated protein (CAP) and the tyrosine phosphorylation of c-Cbl (2). Although c-Cbl was thought to be recruited by CAP, it has also emerged that APS (adapter protein with a PH and SH2 domain), a member of a larger adapter protein family, can also recruit c-Cbl following insulin-stimulation (3, 4). Following the binding of insulin, the insulin receptor undergoes autophosphorylation in the activation loop of the kinase domain. This provides docking sites for the binding of several proteins, including SH2-B and the APS adapter protein (5-8).The resulting recruitment of the APS adapter protein to the kinase domain allows it to undergo tyrosine phosphorylation on Tyr-618 (7). The phosphorylated Tyr-618 then allows APS to bind to the variant SH2 domain of c-Cbl (8). This is followed by the tyrosine phosphorylation of c-Cbl on tyrosines 700 and 774, resulting in phosphorylated c-Cbl binding to the SH2 domain of Crk (4, 9). It is also thought that the adapter protein CAP could facilitate interaction with the insulin receptor, because CAP was also identified as a c-Cbl binding protein using c-Cbl as a bait in a yeast two-hybrid screen and was shown to interact with the insulin receptor in intact cells (10). CAP contains an N-terminal region homologous to Sorbin (Sorbin homology/ SOHO domain) and three SH3 domains in the C terminus (10). CAP is constitutively bound to a proline-rich region in Cbl through its C-terminal SH3 domain (11). Recently, a number of new splice variants of CAP have been described (12). Following the insulin-stimulated phosphorylation of c-Cbl, the CAP/Cbl complex migrates to the caveolin-rich lipid rafts, a movement facilitated by the interaction of the CAP SOHO domain with flotillin, a protein in lipid rafts (13). This allows the Crk/C3G complex to be recruited to this microdomain, where C3G activates the small G protein TC10 (14). The activation of TC10 occurs independently of PI 3-kinase and is crucial for insulinstimulated Glut4 translocation (2).Since APS and CAP both interact with c-Cbl (3, 4, 8, 10), the relative individual importance and the temporal sequence of these proteins in recruiting c-Cbl has not been clear. In this study, we sought to determine whether both proteins were required to recruit c-Cbl to the insulin receptor or whether they were mutually exclusive. We addressed this question by coexpression of APS and CAP in cells that do not express these proteins and by silencing of th...

The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.Insulin promotes glucose transport into target tissues by causing the exocytosis of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (1). There are thought to be two pathways required for insulin-stimulated glucose transport: an insulin receptor substrate-PI 1 3-kinase pathway and a PI 3-kinase-independent pathway involving APS, CAP, and c-Cbl. The PI 3-kinase-independent pathway is initiated by the binding of APS to the activation loop of the insulin receptor (2, 3). APS is constitutively bound to CAP and undergoes tyrosine phosphorylation on Tyr 618 allowing it to bind to the variant SH2 domain of c-Cbl (3, 4). This is followed by the tyrosine phosphorylation of c-Cbl on tyrosines 700 and 774 resulting in the phosphorylated c-Cbl binding to the SH2 domain of Crk (3, 5). Following the insulin-stimulated phosphorylation of c-Cbl, the CAP/Cbl complex migrates to the caveolin-rich lipid rafts, a movement facilitated by the interaction of the CAP SOHO domain with flotillin, a protein in lipid rafts (6). This allows the Crk/C3G complex to be recruited to this microdomain, where C3G activates the small G protein TC10 (7-10). The activation of TC10 occurs independently of PI 3-kinase and is crucial for insulin-stimulated Glut4 translocation (1).The domain structure of APS includes proline-rich regions and a n...

Background: The ras family of proto-oncogenes encodes for small GTPases that play critical roles in cell-cycle progression and cellular transformation. ERK1/2 MAP kinases are major ras effectors. Tumors in chemically treated mouse skin contain mutations in the Ha-ras protooncogene. Amplification and mutation of Ha-ras has been shown to correlate with malignant progression of these tumors. Cell lines isolated from mouse skin tumors represent the stages of tumor development, such as the PDV:PDVC57 cell line pair and B9 squamous carcinoma and A5 spindle cells. PDVC57 cells were selected from PDV cells, which were transformed with dimethylbenzanthracene (DMBA) in vitro and then transplanted in adult syngeneic mice. The PDV:PDVC57 pair contains ratio of normal:mutant Ha-ras 2:1 and 1:2, respectively. This genetic alteration correlates with more advanced tumorigenic characteristics of PDVC57 compared to PDV. The squamous carcinoma B9 cell clone was isolated from the same primary tumor as A5 spindle cell line. The mutant Ha-ras allele, also present in B9, is amplified and overexpressed in A5 cells. Therefore these cell line pairs represent an in vivo model for studies of Ha-ras and ERK1/2 signaling in mouse tumorigenesis.