c-Cbl-associated Protein Research Articles

STAP-2 facilitates insulin signaling through binding to CAP/c-Cbl and regulates adipocyte differentiation

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor molecule involved in several cellular signaling cascades. Here, we attempted to identify novel STAP-2 interacting molecules, and identified c-Cbl associated protein (CAP) as a binding protein through the C-terminal proline-rich region of STAP-2. Expression of STAP-2 increased the interaction between CAP and c-Cbl, suggesting that STAP-2 bridges these proteins and enhances complex formation. CAP/c-Cbl complex is known to regulate GLUT4 translocation in insulin signaling. STAP-2 overexpressed human hepatocyte Hep3B cells showed enhanced GLUT4 translocation after insulin treatment. Elevated levels of Stap2 mRNA have been observed in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs) during adipocyte differentiation. The differentiation of 3T3-L1 cells into adipocytes was highly promoted by retroviral overexpression of STAP-2. In contrast, STAP-2 knockout (KO) MEFs exhibited suppressed adipogenesis. The increase in body weight with high-fat diet feeding was significantly decreased in STAP-2 KO mice compared to WT animals. These data suggest that the expression of STAP-2 correlates with adipogenesis. Thus, STAP-2 is a novel regulatory molecule that controls insulin signal transduction by forming a c-Cbl/STAP-2/CAP ternary complex.

The adaptor protein c-Cbl-associated protein (CAP) limits pro-inflammatory cytokine expression by inhibiting the NF-κB pathway

C-Cbl-associated protein (CAP), also known as Sorbin and SH3 domain-containing protein 1 (Sorbs1) or ponsin, an adaptor protein of the insulin-signalling pathway, mediates anti-viral and anti-cytotoxic protection in acute viral heart disease. In the present study we describe a novel protective immuno-modulatory function of CAP in inflammation.Among the three members of the Sorbs family of adapter molecules, which include CAP (Sorbs1), ArgBP2 (Sorbs2), and Vinexin (Sorbs3), CAP consistently down-regulated the expression of pro-inflammatory cytokines in mouse fibroblasts, cardiomyocytes, and myeloid-derived leukocytes, after Toll-like receptor (TLR) stimulation. Upon the same TLR stimulation, ArgBP2 partially down-regulated pro-inflammatory cytokine production in mouse fibroblasts and cardiomyocytes, while Vinexin rather promoted their production. Mechanistically, CAP limited pro-inflammatory cytokine expression by suppressing the phosphorylation of Inhibitor of kappa B (IκB) kinase (Iκκ)-α and Iκκ-β and their downstream NF-κB-dependent signalling pathway. Molecular affinity between CAP and Iκκ-α/ Iκκ-β was necessary to block the NF-κB pathway. The CAP-dependent inhibitory mechanism - in vivo exclusively IL-6 inhibition - was confirmed after collecting blood from mice with systemic inflammation induced by lipopolysaccharide (LPS) and in the heart tissue collected from mice infected with the cardiotropic Coxsackievirus B3 (CVB3).Taken together, CAP down-regulates pro-inflammatory cytokines by interfering with the normal function of the NF-κB pathway. The promotion of CAP production could support the development of new strategies aiming to limit excessive and detrimental activation of the immune system.

Polysaccharides from Cyclocarya paliurus: Chemical composition and lipid-lowering effect on rats challenged with high-fat diet

The present study was performed to analyse the chemical composition of Cyclocarya paliurus polysaccharides (CPP) and investigate their lipid-lowering effects on high-fat diet (HFD)-induced hyperlipidaemic rats. CPP mainly consisted of two polysaccharides with an average molecular weight (MW) of 190.1 and 2.1kDa, as well as 67.8% total sugar, 19.23% uronic acid, 6.56% proteins, and six monosaccharides. CPP treatment markedly reduced the plasma glucose level, restored lipid profile changes, modulated the activities of hepatic lipid-related enzymes, suppressed hypertrophy of visceral adipose tissue (VAT), and improved hepatic steatosis. In addition, CPP ameliorated glucose tolerance and insulin resistance, which could be attributed to the regulation of CCAAT/enhancer binding protein-α (C/EBPα), peroxisome proliferator-activated receptor (PPAR)-γ, PPARδ, and c-Cbl-associated protein (CAP) expression. The present findings suggest that CPP exhibit a lipid-lowering effect by regulating the activities of hepatic lipid metabolism-related enzymes and alleviating insulin resistance.

Skeletal Muscle CAP Expression Increases after Dietary Restriction and Aerobic Training in Women with a History of Gestational Diabetes.

The purpose is to determine the effects of 6 months caloric restriction and aerobic training (3x/wk) (CR+AEX) on c-CBL associated protein (CAP) gene expression in women with a history of GDM. CAP is involved in cell signaling and protein ubiquitination, and is linked to the development of insulin resistance. Obese (BMI=32 ± 1 kg/m2, % fat=46 ± 2, X ± SEM), sedentary (VO2 max=21.2 ± 1.2 ml/kg/min), women aged 52 ± 2 years participated in 6 months D+WL (n=10) with body composition, fitness (VO2 max), and glucose tolerance testing. Insulin sensitivity was assessed during the last 30 min of 2-hour hyperinsulinemic-euglycemic clamps (40 mU.m-2.min-1) pre and post interventions. Vastus lateralis skeletal muscle biopsies (n=7) were conducted and CAP, GLUT4 and glycogen synthase (GS) gene expression measured by RT-PCR. No change in FFM by DXA was observed, but body weight decreased 8% with losses of total body fat mass (P<0.05) and a 10% increase in VO2 max (P<0.01). Glucose and insulin areas under the curve by OGTT decreased (P<0.05). Glucose utilization during the clamp increased 27% (23.1 ± 3.8 vs. 29.4 ± 3.6 umol.kg.min-1, P<0.05). Vastus lateralis skeletal muscle CAP expression increased 21% (P<0.05) but GLUT4 did not. Results suggest that changes in CAP could be involved in the improvement in glucose metabolism with caloric restriction and aerobic training in women with a history of gestational diabetes.

Structural investigation of the interaction between the tandem SH3 domains of c-Cbl-associated protein and vinculin.

c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807(SH3a) and D881(SH3b) are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.

The adapter protein c-Cbl-associated protein (CAP) protects from acute CVB3-mediated myocarditis through stabilization of type I interferon production and reduced cytotoxicity.

c-Cbl-associated protein (CAP), also called Sorbs1 or ponsin, has been described as an essential adapter protein in the insulin-signalling pathway. Here, we describe for the first time a unique protective role for CAP in viral myocarditis. Mortality and heart failure development were increased in CAP(-/-) mice compared to CAP(+/+) littermates after Coxsackievirus (CVB3) infection. Mechanistically, CAP protected from tissue apoptosis because of reduced CD8(+) T and natural killer cell cytotoxicity. Despite reduced cytotoxic elimination of CVB3-infected cells in CAP(+/+) hearts, however, CAP enhanced interferon regulatory factor 3 (IRF3)-dependent antiviral type I interferon production and decreased viral proliferation in vitro by binding to the cytoplasmic RIG-I-like receptor melanoma differentiation-associated protein 5 (MDA5). Taken together, these findings reveal a novel modulatory role for CAP in the heart as a key protein stabilizing antiviral type I interferon production, while protecting from excessive cytotoxic responses. Our study will help to define future strategies to develop treatments to limit detrimental responses during viral heart inflammation.

Effects of ursolic acid on c-Cbl-associated protein expression in 3T3-L1 adipocytes with insulin resistance

To observe the effects of ursolic acid (UA) on insulin resistance and cell differentiation in 3T3-L1 adipocytes and to explore the mechanisms. 3T3-L1 adipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with glucose (25 mmol/L) and insulin (10(-6) mol/L) to induce insulin resistance. After culture, glucose consumption of the adipocytes was detected by glucose oxidase method and glucose uptake was detected by using tritium-marked glucose. Drug concentration for following test was determined through detecting the effects of different concentrations of UA on the activity of 3T3-L1 adipocytes with insulin resistance by methyl thiazolyl tetrazolium (MTT) staining. 3T3-L1 adipocytes with insulin resistance were cultured with DMEM, rosiglitazone, and low- and high-dose UA, and then, glucose uptake and differentiation of 3T3-L1 adipocytes were detected. Finally, real-time fluorescence quantitative polymerase chain reaction and Western blot methods were used to detect the effects of UA on expressions of adipocyte lipid binding protein (aP2), c-Cbl-associated protein (CAP) and matrix metalloproteinase-1 (MMP-1) in 3T3-L1 cells with insulin resistance. After dealing with high glucose/hyperinsulin for 24 h, insulin resistance was induced successfully in the 3T3-L1 adipocytes. The concentrations of UA were defined to be 4 to 20 μmol/L. Compared with the model group, the glucose uptake was significantly increased in the rosiglitazone group and groups treated with low- and high-dose UA (P<0.01). The differentiation levels of 3T3-L1 adipocytes in the UA groups were lower than those in the control group and the rosiglitazone group. Effects of UA on the expressions of aP2 and MMP-1 were not obvious, but UA could up-regulate expression of CAP both in mRNA and protein levels (P<0.01). Low- and high-dose UA can improve the glycometabolism and differentiation of 3T3-L1 adipocytes with insulin resistance by up-regulating the expression of CAP.

Whole body C‐cbl Associated Protein (CAP) deleted mice display impaired endothelium dependent dilation and nitric oxide bioavailability

In endothelial cells, high glucose impairs insulin‐stimulated endothelial nitric oxide synthase (eNOS) activity in a c‐casitas B lymphoma (c‐cbl) associated protein (CAP)‐dependent manner. In adipocytes, the CAP/cbl complex associates with lipid rafts and participates in GLUT4 trafficking upon insulin stimulation. Together, these findings raise the possibility that CAP/cbl participate in eNOS activation/trafficking. We tested the hypothesis that compared to wild‐type (CAP+/+) littermates, arterial function is impaired in mice with whole body CAP deletion (CAP−/−). Maximal ex vivo carotid artery endothelium dependent dilation (EDD) to acetylcholine (ACh) was reduced in CAP−/− versus CAP+/+ (72.2 ± 7.3 v 99.84 ± 0.8%, p &lt; 0.05). Impaired EDD was associated with reduced nitric oxide (NO), evidenced by the abolition of differences in EDD after incubation with a NOS inhibitor. This does not appear to be associated with increased superoxide, as incubation with a superoxide dismutase mimetic failed to improve dilation in CAP−/− mice. Endothelium independent dilation was not different between groups. CAP deletion was without effect on aortic stiffness (pulse wave velocity) or mean arterial pressure (p's &gt; 0.19). CAP deletion results in impaired EDD via a superoxide‐independent reduction in NO. Although the mechanism is unknown, dysregulation of eNOS trafficking is a possibility requiring examination.

Arg/Abl‐binding protein, a Z‐body and Z‐band protein, binds sarcomeric, costameric, and signaling molecules

ArgBP2 (Arg/Abl-Binding Protein) is expressed at high levels in the heart and is localized in the Z-bands of mature myofibrils. ArgBP2 is a member of a small family of proteins that also includes vinexin and CAP (c-Cbl-associated protein), all characterized by having one sorbin homology (SOHO) domain and three C-terminal SH3 domains. Antibodies directed against ArgBP2 also react with the Z-bodies of myofibril precursors: premyofibrils and nascent myofibrils. Expression in cardiomyocytes of plasmids encoding Yellow Fluorescent Protein (YFP) fused to either full length ArgBP2, the SOHO, mid-ArgBP or the SH3 domains of ArgBP2 led to Z-band targeting of the fusion proteins, whereas an N-terminal fragment lacking these domains did not target to Z-bands. Although ArgBP2 is not found in skeletal muscle cells, YFP-ArgBP2 did target to Z-bodies and Z-bands in cultured myotubes. GST-ArgBP2-SH3 bound actin, α-actinin and vinculin proteins in blot overlays, cosedimentation assays, and EM negative staining techniques. Over-expression of ArgBP2 and ArgBP2-SH3 domains, but not YFP alone, led to loss of myofibrils in cardiomyocytes. Fluorescence recovery after photobleaching was used to measure the rapid dynamics of both the full length and some truncated versions of ArgBP2. Our results indicate that ArgBP2 may play an important role in the assembly and maintenance of myofibrils in cardiomyocytes.

Cbl-associated protein is tyrosine phosphorylated by c-Abl and c-Src kinases

BackgroundThe c-Cbl-associated protein (CAP), also known as ponsin, localizes to focal adhesions and stress fibers and is involved in signaling events. Phosphorylation has been described for the other two members of the sorbin homology family, vinexin and ArgBP2, but no data exist about the putative phosphorylation of CAP. According to previous findings, CAP binds to tyrosine kinase c-Abl. However, it is not known if CAP is a substrate of c-Abl or other tyrosine kinases or if phosphorylation regulates its localization.ResultsWe here show that CAP is Tyr phosphorylated by and interacts with both c-Abl and c-Src. One major phosphorylation site, Tyr360, and two minor contributors Tyr326 and Tyr632 were identified as Abl phosphorylation sites, whereas Src preferentially phosphorylates Tyr326 and Tyr360. Phosphorylation of CAP was not necessary for its localization to focal adhesions and stress fibers, but Tyr326Phe substitution alters the function of CAP during cell spreading.ConclusionThis is the first demonstration of phosphorylation of CAP by any kinase. Our findings suggest that coordinated action of Src and Abl might regulate the function of CAP and reveal a functional role especially for the Src-mediated Tyr phosphorylation of CAP in cell spreading.

Effects of chromium picolinate on glucose uptake in insulin-resistant 3T3-L1 adipocytes involve activation of p38 MAPK

Chromium picolinate (CrPic) has been discovered as a supplemental or alternative medication for type 2 diabetes, but its mechanism of action is not well understood. The purpose of this study was to explore the possible anti-diabetic mechanisms of CrPic in insulin-resistant 3T3-L1 adipocytes; the insulin resistance was induced by treatment with high glucose and insulin for 24 h. The effects of CrPic on glucose metabolism and the glucose uptake-inducing activity of CrPic were investigated. Meanwhile, the effects of CrPic on glucose transporter 4 (GLUT4) translocation were visualized by immonofluorescence microscopy. In addition, its effects on insulin signaling pathways and mitogen-activated protein kinase (MAPK) signaling cascades were assessed by immunoblotting analysis and real-time PCR. The results showed that CrPic induced glucose metabolism and uptake, as well as GLUT4 translocation to plasma membrane (PM) in both control and insulin-resistant 3T3-L1 adipocytes without any changes in insulin receptor β (IR-β), protein kinase B (AKt), c-Cbl, extracellular signal-regulated kinase (ERK), c-Jun phosphorylation and c-Cbl-associated protein (CAP) mRNA levels. Interestingly, CrPic was able to increase the basal and insulin-stimulated levels of p38 MAPK activation in the control and insulin-resistant cells. Pretreatment with the specific p38 MAPK inhibitor SB203580 partially inhibited the CrPic-induced glucose transport, but CrPic-activated translocation of GLUT4 was not inhibited by SB203580. This study provides an experimental evidence of the effects of CrPic on glucose uptake through the activation of p38 MAPK and it is independent of the effect on GLUT4 translocation. The findings also suggest exciting new insights into the role of p38 MAPK in glucose uptake and GLUT4 translocation.

Activation of atypical protein kinase C ζ toward TC10 is regulated by high-fat diet and aerobic exercise in skeletal muscle

We determined whether sustained aerobic exercise reverses high-fat diet–induced impairments in the c-Cbl associated protein (CAP)/Casitas b-lineage lymphoma (c-Cbl) signaling cascade in rodent skeletal muscle. Sprague-Dawley rats were placed into either control (n = 16) or high-fat–fed (n = 32) diet groups for 4 weeks. During a subsequent 4-week experimental period, 16 high-fat–fed rats remained sedentary, 16 high-fat–fed rats completed 4 weeks of exercise training, and control animals were sedentary and remained on the control diet. After the intervention period, animals were subjected to hind limb perfusions in the presence (n = 8 per group) or absence (n = 8 per group) of insulin. In the plasma membrane fractions, neither high-fat feeding nor exercise training altered adaptor protein with PH and SH2 domains, (APS), c-Cbl, or TC10 protein concentrations. In contrast, CAP protein concentration and insulin-stimulated plasma membrane c-Cbl tyrosine phosphorylation were reduced by high-fat feeding; but exercise training reversed these impairments. Of note was that insulin-stimulated atypical protein kinase C ζ kinase activity toward TC10 was reduced by high-fat feeding but normalized by exercise training. We conclude that sustained (4 weeks) exercise training can reverse high-fat diet–induced impairments on the CAP/c-Cbl pathway in high-fat–fed rodent skeletal muscle. We also provide the first evidence that the CAP/c-Cbl insulin signaling cascade in skeletal muscle may directly interact with components of the classic (phosphoinositide 3-kinase dependent) insulin signaling cascade.

Resistin is expressed in human hepatocytes and induces insulin resistance

Resistin, known as an adipocyte-specific secretory factor (ADSF), is implicated to modulate insulin resistance in rodents. However, the precise role of this factor for human insulin resistance has remained elusive. Here, we investigate the relationship between human resistin and insulin resistance in hepatocytes and the effect of Metformin on resistin. In this study, the expression of resistin in human hepatocytes and hepatic tissues was examined, and the human resistin eukaryotic expression vector was constructed and stably transfected in HepG2 cells. Data showed that resistin is expressed in human hepatocytes and hepatic tissues. Overexpression of human resistin impaired significantly insulin-stimulated glucose uptake and glycogen synthesis in HepG2 cells. It also decreased the expression of insulin receptor substrate 2 (IRS-2) and c-cbl associated protein (CAP), whereas increased the expression of glycogen synthetase kinase 3beta (GSK-3beta). The result suggested that human resistin induced insulin resistance in hepatocytes by blocking the two insulin signal transduction pathways of PI-3K/Akt and of CAP/c-cbl. We also concluded that Metformin reversed the effect of resistin and downregulated the expression of resistin in hepatocytes.

A single nucleotide polymorphism in exon 7 of sorbin and SH3-domain-containing-1 (SORBS1) in Korean PCOS patients

Patients with polycystic ovarian syndrome (PCOS) are characterized by high levels of androgens, irregular or no menstrual cycle and increased hair growth. In addition, insulin resistance and glucose tolerance are caused in patients with PCOS. It was recently reported that PCOS in women is associated with a single nucleotide polymorphism (SNP) of genes, including the sorbin and SH3-domain-containing-1 (SORBS1) gene involved in insulin resistance and glucose uptake. SORBS1, which belongs to the sorbin homology (SoHo) family of proteins, becomes protein-protein interaction through three SH3 domains. In addition, SORBS1 is related to c-Cbl-associated protein (CAP) that has interacted with the downstream signaling components of glucose uptake. We therefore studied the association between the SNP of the SORBS1 gene and PCOS in a Korean population. SORBS1 raises glucose uptake and insulin resistance. The frequency of G allele was slightly higher in the patient group (n=233) than in the control group (n=105). A statistically significant relationship was found between the SNP in exon 7 of the SORBS1 gene and PCOS in a Korean population group.

Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein

Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation.

The association between the SH2‐containing inositol polyphosphate 5‐Phosphatase 2 (SHIP2) and the adaptor protein APS has an impact on biochemical properties of both partners

SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is a phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase containing various motifs susceptible to mediate protein-protein interaction. In cell models, SHIP2 negatively regulates insulin signalling through its catalytic PtdIns(3,4,5)P(3) 5-phosphatase activity. We have previously reported that SHIP2 interacts with the c-Cbl associated protein (CAP) and c-Cbl, proteins implicated in the insulin cellular response regulating the small G protein TC10. The first steps of the TC10 pathway are the recruitment and tyrosine phosphorylation by the insulin receptor of the adaptor protein with Pleckstrin Homology and Src Homology 2 domains (APS). Herein, we show that SHIP2 can directly interact with APS in 3T3-L1 adipocytes and in transfected CHO-IR cells (Chinese hamster ovary cells stably transfected with the insulin receptor). Upon insulin stimulation, APS and SHIP2 are recruited to cell membranes as seen by immunofluorescence studies, which is consistent with their interaction. We also observed that SHIP2 negatively regulates APS insulin-induced tyrosine phosphorylation and consequently inhibits APS association with c-Cbl. APS, which specifically interacts with SHIP2, but not PTEN, in turn, increases the PtdIns(3,4,5)P(3) 5-phosphatase activity of SHIP2 in an inositol phosphatase assay. Co-transfection of SHIP2 and APS in CHO-IR cells further increases the inhibitory effect of SHIP2 on Akt insulin-induced phosphorylation. Therefore, the interaction between APS and SHIP2 provides to both proteins potential negative regulatory mechanisms to act on the insulin cascade.

Cytokines Promote Wnt Signaling and Inflammation and Impair the Normal Differentiation and Lipid Accumulation in 3T3-L1 Preadipocytes

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFalpha impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein alpha, in contrast to peroxisome proliferator-activated receptor gamma, was markedly reduced. TNFalpha also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFalpha maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and beta-catenin levels. TNFalpha, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFalpha prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.

Primary and Essential Role of the Adaptor Protein APS for Recruitment of Both c-Cbl and Its Associated Protein CAP in Insulin Signaling

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-Cbl-associated protein) interact with c-Cbl during insulin signaling, the relative importance of each protein in recruiting c-Cbl has not been clear. We performed a side-by-side comparison by ectopic expression of APS or Src homology 2-Balpha (SH2-Balpha) and CAP in Chinese hamster ovary (CHO) cells. In cells co-expressing insulin receptor and CAP, without APS, no association of the insulin receptor and CAP could be detected and no insulin-stimulated phosphorylation of Cbl was observed. Insulin-stimulated Cbl phosphorylation was reconstituted when APS was co-expressed with insulin receptor, with or without CAP. APS or SH2-Balpha and CAP interacted in the basal state, and in the case of APS this interaction was mediated by the C terminus of APS. Insulin stimulation resulted in the dissociation of APS and CAP. Similarly, insulin stimulation also resulted in the dissociation of SH2-Balpha and CAP in CHO cells. CAP was localized to the membrane in the presence of APS. Insulin stimulation resulted in the re-localization of CAP to the cytosol only when APS was co-expressed. In 3T3-L1 adipocytes, small interfering RNA-mediated knockdown of the mouse APS gene abolished the insulin-stimulated phosphorylation of c-Cbl. Taken together, these results indicate that APS plays a central role in recruiting both CAP and c-Cbl to the insulin receptor after insulin stimulation and is necessary and sufficient for the insulin-stimulated phosphorylation of c-Cbl, whereas SH2-Balpha may provide an alternative pathway for the recruitment of CAP.

Effects of Rexinoids on Glucose Transport and Insulin-mediated Signaling in Skeletal Muscles of Diabetic (db/db) Mice

Rexinoids and thiazolidinediones (TZDs) are two classes of nuclear receptor ligands that induce insulin sensitization in diabetic rodents. TZDs are peroxisome proliferator-activated receptor gamma (PPARgamma) activators, whereas rexinoids are selective ligands for the retinoid X receptors (RXRs). Activation of both the insulin receptor substrates (IRSs)/Akt and the c-Cbl-associated protein (CAP)/c-Cbl pathways are important in regulating insulin-stimulated glucose transport. We have compared the effects of a rexinoid (LG268) and a TZD (rosiglitazone) on these two signal pathways in skeletal muscle of diabetic (db/db) mice. The results we have obtained show that treatment of db/db mice with either LG268 or rosiglitazone for 2 weeks results in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Treatment with LG268 increases insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation in skeletal muscle without affecting the activity of the CAP/c-Cbl pathway. In contrast, rosiglitazone increases the levels of CAP expression and insulin-stimulated c-Cbl phosphorylation without affecting the IRS-1/Akt pathway. The effects of LG268 on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Ser(307) phosphorylation. Taken together, these data suggest that rexinoids improve insulin sensitivity via changes in skeletal muscle metabolism that are distinct from those induced by TZDs. Rexinoids represent a novel class of insulin sensitizers with potential applications in the treatment of insulin resistance.

Endogenous SHIP2 does not localize in lipid rafts in 3T3-L1 adipocytes

SH2 domain containing inositol polyphosphate 5-phosphatase (SHIP2) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3) into phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2). SHIP2 knock-out mice demonstrated that SHIP2 acts as a negative regulator of insulin cascade in vivo. Our two-hybrid study showed that SHIP2 interacts with c-Cbl associated protein (CAP) and c-Cbl, implicated in the insulin signaling. As some proteins implicated in insulin signaling, like insulin receptor, CAP, c-Cbl or TC10, were reported to localize in lipid rafts, we addressed the same question for SHIP2. SHIP2 was detected in the non-raft fraction in CHO-IR, C2C12 myotubes and 3T3-L1 adipocytes except when it is overexpressed in CHO-IR, where we detected SHIP2 in the raft fraction.