c-Cbl-associated Protein Research Articles

CDNA cloning and functional characterization of a novel splice variant of c-Cbl-associated protein from mouse skeletal muscle

c-Cbl-associated protein (CAP) is an SH3-containing adapter protein that binds to the proto-oncogene c-Cbl. Recent work suggests that signaling through these molecules is involved in the regulation of insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Skeletal muscle is the major site of insulin-stimulated glucose disposal but there have been no reports of CAP function in this tissue. Using RT-PCR of mouse skeletal muscle RNA, we discovered a novel splice variant of CAP (CAP SM; GenBank Accession No. AF521593) that is different from the adipocyte form by inclusion of a novel 168 bp fragment. This fragment encodes a peptide sequence that shows very high similarity with exon 25 of the human homologue of CAP (SORBS1). To understand the function of CAP SM in glucose uptake regulation, L6 myotubes were transfected with either CAP SM or a truncated CAP SM devoid of all three SH3-binding domains (CAPΔSH3), which prevents CAP association with c-Cbl. Transfection with CAPΔSH3 decreased insulin-stimulated 2-deoxyglucose (2-DG) uptake and reduced c-Cbl phosphorylation. In contrast, transfection of L6 myotubes with CAPΔSH3 had no effect on dinitrophenol (DNP)- or hypoxia-stimulated glucose uptake, stimuli that work through insulin-independent mechanisms for the regulation of glucose uptake. These data demonstrate the existence of a novel CAP isoform expressed in skeletal muscle, and suggest the involvement of the CAP/Cbl pathway in the regulation of insulin-stimulated glucose uptake in L6 myotubes.

Insulin Action on Expression of Novel Adipose Genes in Healthy and Type 2 Diabetic Subjects

Adipose tissue secretes several molecules that may participate in metabolic cross-talk to other insulin-sensitive tissues. Thus, adipose tissue is a key endocrine organ that regulates insulin sensitivity in other peripheral insulin target tissues. We have studied the expression and acute insulin regulation of novel genes expressed in adipose tissue that are implicated in the control of whole body insulin sensitivity. Expression of adiponectin, c-Cbl-associated protein (CAP), 11-beta hydroxysteroid dehydrogenase type 1 (11beta-HSD-1), and sterol regulatory element binding protein (SREBP)-1c was determined in subcutaneous adipose tissue from type 2 diabetic and age- and BMI-matched healthy men by real-time polymerase chain reaction analysis. Expression of adiponectin, CAP, 11beta-HSD-1, and SREBP-1c was similar between healthy and type 2 diabetic subjects. Insulin infusion for 3 hours did not affect expression of CAP, 11beta-HSD-1, or adiponectin mRNA in either group. However, insulin infusion increased SREBP-1c expression by 80% in healthy, but not in type 2 diabetic, subjects. Our results provide evidence that insulin action on SREBP-1c is dysregulated in adipose tissue from type 2 diabetic subjects. Impaired insulin regulation on gene expression of select targets in adipose tissue may contribute to the pathogenesis of type 2 diabetes.

Frequency of the T228A polymorphism in the SORBS1 gene in children with premature pubarche and in adolescent girls with hyperandrogenism

Objective Because the metabolic actions of insulin are more impaired than the mitogenic pathways in polycystic ovary syndrome (PCOS), genes coding for proteins involved in insulin-mediated glucose transport can be considered as candidate genes. The sorbin and SH3-domain-containing-1 ( SORBS1) gene codes for c-Cbl–associated protein (CAP) involved in insulin-mediated glucose uptake. An association study showed that a missense variant of the SORBS1 gene is protective against obesity and diabetes. We tested the hypothesis that the frequency of the protective allele would be decreased in children with premature pubarche and adolescent girls with hyperandrogenism. Design Association study. Setting Academic research environment. Patient(s) Children referred for the evaluation of premature pubarche (n = 79), adolescent girls with hyperandrogenism (n = 56), and healthy nondiabetic controls (n = 50). Intervention(s) None. Main outcome measure(s) Frequency of the T228A allele in our patients and the relationship of body mass index to presence or absence of the T228A variant in our patient population. Result(s) Using allele-specific restriction fragment length polymorphism, allele frequencies were found to be similar among the premature pubarche, hyperandrogenism, and control groups (6.0%, 4.6%, and 8.0%, respectively). No statistically significant relationships were found between the SORBS1 genotypes and body mass index or hormone status. Conclusion(s) This SORBS1 polymorphism does not play a major role in premature pubarche, hyperandrogenism, and/or polycystic ovary syndrome in our patient population.

APS Facilitates c-Cbl Tyrosine Phosphorylation and GLUT4 Translocation in Response to Insulin in 3T3-L1 Adipocytes

APS is a Cbl-binding protein that is tyrosine phosphorylated by the insulin receptor kinase. Insulin-stimulated phosphorylation of tyrosine 618 in APS is necessary for its association with c-Cbl and the subsequent tyrosine phosphorylation of Cbl by the insulin receptor in both 3T3-L1 adipocytes and CHO-IR cells. When overexpressed in these cells, wild-type APS but not an APS/Y(618)F mutant facilitated the tyrosine phosphorylation of coexpressed Cbl and its association with Crk upon insulin stimulation. APS-facilitated phosphorylation occurred on tyrosines 371, 700, and 774 in the Cbl protein. APS also interacted directly with the c-Cbl-associated protein (CAP) and colocalized with the protein in cells. The association was dependent on the SH3 domains of CAP and was independent of insulin treatment. Overexpression of the APS/Y(618)F mutant in 3T3-L1 adipocytes blocked the insulin-stimulated tyrosine phosphorylation of endogenous Cbl and binding to Crk. Moreover, the translocation of GLUT4 from intracellular vesicles to the plasma membrane was also inhibited by overexpression of the APS/Y(618)F mutant. These data suggest that APS serves as an adapter protein linking the CAP/Cbl pathway to the insulin receptor and, further, that APS-facilitated Cbl tyrosine phosphorylation catalyzed by the insulin receptor is a crucial event in the stimulation of glucose transport by insulin.

Molecular scanning of the human sorbin and SH3-domain-containing-1 (SORBS1) gene: positive association of the T228A polymorphism with obesity and type 2 diabetes.

In the mouse, the SH3P12 or the c-Cbl-associated protein (CAP) has been shown as an important signaling molecule in insulin-stimulated glucose uptake. The human homolog for the sorbin and SH3-domain-containing-1 gene, termed SORBS1, might play a role in human disorders with insulin resistance. To explore the genetic role of SORBS1 in human obesity and type 2 diabetes, we investigated the nucleotide polymorphisms in the SORBS1 gene with molecular scanning. After scanning for a total of 13,136 bp in each of 40 chromosomes, we have identified 14 single nucleotide polymorphisms (SNPs) in the human SORBS1 gene. Among them, two SNPs affected amino acid coding (R74W and T228A), four occurred within exons but did not affect amino acid coding, and the remaining eight occurred within introns, which were located outside of the consensus region of the splicing mechanism. Further studies in 202 non-obese, 113 obese and 455 subjects with type 2 diabetes revealed that the A-allele of the T228A polymorphism in exon 7 exerted a protective role for both obesity [relative risk 0.466; 95% confidence interval (95% CI) 0.265-0.821] and diabetes (relative risk 0.668; 95% CI 0.472-0.945). Neither allele of the R74W polymorphism was associated with either obesity or diabetes. In conclusion, our results suggest that the A228 allele of the T228A polymorphism of the SORBS1 gene is a protective factor for both obesity and diabetes, and also imply that the SORBS1 gene plays an important role in the pathogenesis of human disorders with insulin resistance.

The sorbin homology domain: a motif for the targeting of proteins to lipid rafts.

On phosphorylation of Cbl, the c-Cbl-associated protein (CAP)/Cbl complex dissociates from the insulin receptor and translocates to a lipid raft membrane fraction to form a ternary complex with flotillin. Deletion analyses of the CAP gene identified a 115-aa region responsible for flotillin binding. This region is homologous to the peptide sorbin and is referred to as the sorbin homology (SoHo) domain. This domain is present in two other proteins, vinexin and ArgBP2. Vinexin also interacted with flotillin, and deletion of its SoHo domain similarly blocked flotillin binding. The overexpression of a CAP mutant in which the SoHo domain had been deleted (CAPDeltaSoHo) prevented the translocation of Cbl to lipid rafts and subsequently blocked the recruitment of CrkII and C3G. Moreover, overexpression of CAPDeltaSoHo prevented the stimulation of glucose transport and GLUT4 translocation by insulin. These results suggest a mechanism for localization of signaling proteins to the lipid raft that mediates the compartmentalization of crucial signal transduction pathways.

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.