Cbl-associated Protein Research Articles

The interactions of CAP and LYN with the insulin signaling transducer CBL play an important role in polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS) is a hormonal disorder characterized by hyperandrogenism, ovulatory dysfunction, and insulin resistance. Evidence suggests that aberrations in insulin signaling-associated pathways may underlie PCOS pathogenesis. Our aim was to investigate the molecular mechanisms underlying PCOS and associated insulin resistance using in silico analyses, in vitro cell models, and in vivo murine models. MethodsR-based bioinformatics analysis was performed on granulosa cell microarray data from three human cohorts: healthy control, PCOS patients without insulin resistance, and PCOS patients with insulin resistance. Transgenic human granulosa cell models were utilized for in vitro studies. Transgenic murine models of dehydroepiandrosterone (DHEA)-induced PCOS were utilized for in vivo studies. ResultsSorbin and SH3 Domain Containing 1 (SORBS1), the parent gene of the insulin receptor-associated Casitas B-lineage lymphoma protein (CBL)-associated protein (CAP), is a key downregulated gene in PCOS patients with insulin resistance. CAP binding to CBL reduced CBLY731 phosphorylation, CBL-phosphoinositide 3-kinase (PI3K) p85α interactivity, protein kinase B (Akt)S473 phosphorylation, and NFκB-induced inflammatory marker expression but enhanced CRKII-mediated membrane GLUT4 translocation in granulosa cells. In contrast, the tyrosine kinase Lck/Yes-Related Novel Protein (LYN) is upregulated in PCOS patients with insulin resistance. LYN binding to CBL enhanced CBLY731 phosphorylation, CBL-PI3K p85α interactivity, AktS473 phosphorylation, and NFκB-induced inflammatory marker expression but did not impact membrane GLUT4 translocation. In PCOS mice, Cap overexpression, Cap transactivation by metformin, or enhancing Cbl-CrkII binding improved insulin sensitivity and ovarian dysfunction (i.e., estrous cycle disruption, cyst-like follicle formation, and sex hormone dysregulation). In contrast, Lyn knockdown, Lyn inhibition by PP2, or CBL-PI3K p85α blockade improved only ovarian dysfunction. Cbl3YF phosphomutant overexpression (which enhances Cbl-CrkII binding but blocks Cbl-PI3K p85α binding) ameliorated both ovarian dysfunction and insulin resistance. ConclusionsThe interactions of CAP and LYN with CBL, and the resulting effects on CBL phosphorylation and activity, may play an important role in PCOS pathogenesis. Targeting these players may be a viable therapeutic strategy for PCOS.

Concussion/Mild Traumatic Brain Injury (TBI) Induces Brain Insulin Resistance: A Positron Emission Tomography (PET) Scanning Study.

Brain injury/concussion is a growing epidemic throughout the world. Although evidence supports association between traumatic brain injury (TBI) and disturbance in brain glucose metabolism, the underlying molecular mechanisms are not well established. Previously, we reported the release of cellular prion protein (PrPc) from the brain to circulation following TBI. The PrPc level was also found to be decreased in insulin-resistant rat brains. In the present study, we investigated the molecular link between PrPc and brain insulin resistance in a single and repeated mild TBI-induced mouse model. Mild TBI was induced in mice by dropping a weight (~95 g at 1 m high) on the right side of the head. The procedure was performed once and thrice (once daily) for single (SI) and repeated induction (RI), respectively. Micro PET/CT imaging revealed that RI mice showed significant reduction in cortical, hippocampal and cerebellum glucose uptake compared to SI and control. Mice that received RI also showed significant motor and cognitive deficits. In co-immunoprecipitation, the interaction between PrPc, flotillin and Cbl-associated protein (CAP) observed in the control mice brains was disrupted by RI. Lipid raft isolation showed decreased levels of PrPc, flotillin and CAP in the RI mice brains. Based on observation, it is clear that PrPc has an interaction with CAP and the dislodgment of PrPc from cell membranes may lead to brain insulin resistance in a mild TBI mouse model. The present study generated a new insight into the pathogenesis of brain injury, which may result in the development of novel therapy.

Cbl-Associated Protein CAP contributes to correct formation and robust function of the Drosophila heart tube.

The formation of a tube-like structure is a basic step in the making of functional hearts in vertebrates and invertebrates and therefore, its understanding provides important information on heart development and function. In Drosophila, the cardiac tube originates from two bilateral rows of dorsally migrating cells. On meeting at the dorsal midline, coordinated changes in cell shape and adhesive properties transform the two sheets of cells into a linear tube. ECM and transmembrane proteins linked to the cytoskeleton play an important role during these dynamic processes. Here we characterize the requirement of Cbl-Associated Protein (CAP) in Drosophila heart formation. In embryos, CAP is expressed in late migrating cardioblasts and is located preferentially at their luminal and abluminal periphery. CAP mutations result in irregular cardioblast alignment and imprecisely controlled cardioblast numbers. Furthermore, CAP mutant embryos show a strongly reduced heart lumen and an aberrant shape of lumen forming cardioblasts. Analysis of double heterozygous animals reveals a genetic interaction of CAP with Integrin- and Talin-encoding genes. In post-embryonic stages, CAP closely colocalizes with Integrin near Z-bands and at cell–cell contact sites. CAP mutants exhibit a reduced contractility in larval hearts and show a locally disrupted morphology, which correlates with a reduced pumping efficiency. Our observations imply a function of CAP in linking Integrin signaling with the actin cytoskeleton. As a modulator of the cytoskeleton, CAP is involved in the establishment of proper cell shapes during cardioblast alignment and cardiac lumen formation in the Drosophila embryo. Furthermore, CAP is required for correct heart function throughout development.

SORBS1 serves a metastatic role via suppression of AHNAK in colorectal cancer cell lines.

Cbl-associated protein (CAP) is encoded by the sorbin and SH3 domain-containing 1 (SORBS1) gene. CAP has been reported to be associated with the actin cytoskeleton, receptor tyrosine kinase signaling and cell adhesion through interactions with various proteins. It may be hypothesized that SORBS1 has numerous unknown functions, which may include providing a favorable condition for metastasis. Although CAP has been demonstrated to possess a number of functions, the role of this protein has only been reported in metabolic signaling pathways and its function in cancer remains to be elucidated. In the present study, SORBS1 expression was detected in colorectal cancer cell lines divided into the primary group and the metastatic group by reverse transcription-quantitative PCR and western blot analysis. In addition, SORBS1 expression was manipulated by vector transfection and lentivirus transduction. The metastatic role of SORBS1, as determined by assessing its effects on cell proliferation and migration, was determined by colony formation assay, cell cycle analysis and Boyden chamber assay. To elucidate the SORBS1-binding protein, immunoprecipitation was performed. Co-localization of SORBS1 and AHNAK nucleoprotein (AHNAK) was identified by confocal microscopy. Notably, the protein expression levels of CAP were higher in SNU-769A and SW480 cells than in SNU-769B and SW620 cells. In addition, the number of colonies in the SORBS1-overexpressing group was significantly increased compared with that of the control group, as determined using the colony formation assay; the SORBS1 overexpression group formed >8-fold more colonies than the control group. The proliferative ability of the SORBS1 overexpression group was also significantly increased compared with the control group over the entire incubation period. Cell migration assays revealed that the number of migrated SORBS1-knockdown cells was reduced compared with the control in both HCT-116 and SNU-C4 cell lines; migration area was decreased to 31 and 26% in HCT-116 and SNU-C4 cell lines, respectively. Consequently, it was confirmed that SORBS1 could form a complex with AHNAK, which functions as a tumor suppressor through inhibition of phosphorylated-ERK and Rho-associated coiled-coil containing protein kinase 1. In conclusion, SORBS1 may serve a crucial role in cancer growth and migration via inhibition of AHNAK expression.

Abstract 1640: SORBS1-related multiprotein complex regulates metastasis of cancer

Abstract Purpose: CAP (Cbl-associated protein), also known as Sorbin and SH3 domain-containing protein 1 is encoded by the SORBS1 gene. These proteins contain a conserved sorbin homology (SOHO) domain and three SH3 domains. CAP is an adaptor protein that has been associated with the actin cytoskeleton, signaling through receptor tyrosine kinases and cell adhesion. Although, CAP is proven to have many known functions, this protein is only reported in specific field of signaling pathway. Cell adhesion and actin dynamics which have been supposed to participate in CAP are crucial factors of cancer metastasis. The role of CAP has been described in the literature, although their specific roles remain to be clarified in cancer. Methods and materials: Colorectal cancer cell lines were transduced by lentivirus containing shRNA plasmid (shSORBS1 plasmid). To elucidate function of SORBS1 as regulator of cancer metastasis, western blotting, cell migration assay and immunoprecipitation assays were performed. Conclusion: According to the several reports of SORBS1 function, phosphorylation of ERK and mTOR was identified. Although ERK phosphorylation were not changed in normal condition, there were different aspects between control group and SORBS1 knockdown group with insulin treatment. Moreover, mTOR phosphorylation of SORBS1 knockdown group was less in the control group than in the normal condition. Immunoprecipitation of SORBS1 was performed to search for the binding compounds. We chose the AHNAK protein as the convincing candidate protein related with SORBS1. Several studies reported that AHNAK protein is related to proliferation and EMT by binding of specific signaling molecules. Indeed, there were remarkable differences of invasiveness ability between control group and knockdown group. Citation Format: Woo-Cheol Cho, Ja-Lok Ku. SORBS1-related multiprotein complex regulates metastasis of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1640.

Effect of dietary fat modification on subcutaneous white adipose tissue insulin sensitivity in patients with metabolic syndrome.

To determine whether the insulin resistance that exists in metabolic syndrome (MetS) patients is modulated by dietary fat composition. Seventy-five patients were randomly assigned to one of four diets for 12 wk: high-saturated fatty acids (HSFAs), high-MUFA (HMUFA), and two low-fat, high-complex carbohydrate (LFHCC) diets supplemented with long-chain n-3 (LFHCC n-3) PUFA or placebo. At the end of intervention, the LFHCC n-3 diet reduced plasma insulin, homeostasis model assessment of insulin resistance, and nonsterified fatty acid concentration (p < 0.05) as compared to baseline Spanish habitual (BSH) diet. Subcutaneous white adipose tissue (WAT) analysis revealed decreased EH-domain containing-2 mRNA levels and increased cbl-associated protein gene expression with the LFHCC n-3 compared to HSFA and HMUFA diets, respectively (p < 0.05). Moreover, the LFHCC n-3 decreased gene expression of glyceraldehyde-3-phosphate dehydrogenase with respect to HMUFA and BSH diets (p < 0.05). Finally, proteomic characterization of subcutaneous WAT identified three proteins of glucose metabolism downregulated by the LFHCC n-3 diet, including annexin A2. RT-PCR analysis confirmed the decrease of annexin A2 (p = 0.027) after this diet. Our data suggest that the LFHCC n-3 diet reduces systemic insulin resistance and improves insulin signaling in subcutaneous WAT of MetS patients compared to HSFA and BSH diets consumption.

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

Metformin Regulates Glucose Transporter 4 (GLUT4) Translocation through AMP-activated Protein Kinase (AMPK)-mediated Cbl/CAP Signaling in 3T3-L1 Preadipocyte Cells

Metformin is a leading oral anti-diabetes mellitus medication and is known to stimulate GLUT4 translocation. However, the mechanism by which metformin acts is still largely unknown. Here, we showed that short time treatment with metformin rapidly increased phosphorylation of Cbl in an AMP-activated protein kinase (AMPK)-dependent fashion in 3T3-L1 preadipocytes. Metformin also increased phosphorylation of Src in an AMPK-dependent manner. Src inhibition blocked metformin-mediated Cbl phosphorylation, suggesting that metformin stimulates AMPK-Src-Cbl axis pathway. In addition, long term treatment with metformin stimulated the expression of Cbl-associated protein (CAP) mRNA and protein. Long term treatment with metformin stimulated phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream molecule c-Jun, which is a critical molecule for CAP transcription. Knockdown of AMPK and JNK blocked metformin-induced expression of CAP, implying that metformin stimulates AMPK-JNK-CAP axis pathway. Moreover, AMPK knockdown attenuated metformin-induced Cbl/CAP multicomplex formation, which is critical for GLUT4 translocation. A colorimetric absorbance assay demonstrated that metformin-induced translocation of GLUT4 was suppressed in CAP or Cbl knockdown cells. Furthermore, the promoter activity of CAP was increased by metformin in an AMPK/JNK-dependent fashion. In summary, these results demonstrate that metformin modulates GLUT4 translocation by regulating Cbl and CAP signals via AMPK.

Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.

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.

Nuclear factor-κB decoy oligodeoxynucleotides ameliorate impaired glucose tolerance and insulin resistance in mice with cecal ligation and puncture-induced sepsis*

Insulin-resistant hyperglycemia is commonly observed in septic patients and may actually lead to some of adverse outcomes. We examined the changes in insulin signaling and glucose uptake regulation in sepsis and the involvement of the nuclear factor-kappaB pathway. Controlled animal study. University research laboratory. One hundred fifty-four BALB/c mice (8-12 wks of age). The following four experimental groups were studied: sham-operated control, cecal ligation and puncture-induced sepsis, sepsis + nuclear factor-kappaB decoy oligodeoxynucleotide treatment, and sepsis + scrambled decoy oligodeoxynucleotide treatment. Septic mice were markedly hyperinsulinemic with apparently normal blood glucose levels in the fasted state, suggesting they are insulin-resistant. In fact, glucose clearance in response to insulin was markedly impaired in septic mice. They had impaired GLUT4 membrane translocation resulting from impaired insulin signaling as indicated by the decreased amount of insulin receptor substrate protein and the reduced activation of phosphatidylinositol 3-kinase and Akt. Interestingly, injection of nuclear factor-kappaB decoy oligodeoxynucleotide into the skeletal muscle dramatically improved all of the changes, including glucose clearance and insulin signaling. We also found that the Cbl-associated protein to TC10 pathway, another pathway regulating GLUT4 translocation, was up-regulated in septic mice in a nuclear factor-kappaB-dependent manner. This pathway may be one of the compensatory mechanisms to translocate GLUT4 because silencing of the individual components of the pathway with small interfering RNAs further reduced GLUT4 translocation in muscles of septic mice. In sepsis, skeletal muscle GLUT4 translocation is impaired as a result of the reduced phosphatidylinositol 3-kinase/Akt pathway associated with insulin receptor substrate down-regulation through nuclear factor-kappaB activation.

CAP (Cbl associated protein) regulates receptor-mediated endocytosis

CAP (c-Cbl associated protein)/ponsin belongs to a family of adaptor proteins implicated in cell adhesion and signaling. Here we show that CAP binds to and co-localizes with the essential endocytic factor dynamin. We demonstrate that CAP promotes the formation of dynamin-decorated tubule like structures, which are also coated with actin filaments. Accordingly, we found that the expression of CAP leads to the inhibition of dynamin-mediated endocytosis and increases EGFR stability. Thus, we suggest that CAP may coordinate the function of dynamin with the regulation of the actin cytoskeleton during endocytosis. Structured summary: MINT- 6804322: CAP (uniprotkb: Q9BX66) physically interacts (MI: 0218) with Cbl (uniprotkb: Q8K4S7) and dynamin 2 (uniprotkb: P39052) by pull down (MI: 0096) MINT- 6804285: CAP (uniprotkb: Q9BX66) physically interacts (MI: 0218) with FAK (uniprotkb: O35346), vinculin (uniprotkb: P85972) and dynamin 2 (uniprotkb: P39052) by pull down (MI: 0096) MINT- 6804245, MINT- 6804259, MINT- 6804272: CAP (uniprotkb: Q9BX66) physically interacts (MI: 0218) with dynamin 2 (uniprotkb: P39052) by pull down (MI: 0096) MINT- 6804344: CAP (uniprotkb: Q9BX66) physically interacts (MI: 0218) with dynamin 2 (uniprotkb: P50570) by anti tag coimmunoprecipitation (MI: 0007) MINT- 6804371: dynamin 1 (uniprotkb: P21575) physically interacts (MI: 0218) with CAP (uniprotkb: O35413) by anti bait coimmunoprecipitation (MI: 0006) MINT- 6804446, MINT- 6804464: F-actin (uniprotkb: P60709), CAP (uniprotkb: Q9BX66) and dynamin 2 (uniprotkb: P50570) colocalize (MI: 0403) by fluorescence microscopy (MI: 0416)

Identification of CAP as a Costameric Protein that Interacts with Filamin C

Cbl-associated protein (CAP) is an adaptor protein that interacts with both signaling and cytoskeletal proteins. Here, we characterize the expression, localization and potential function of CAP in striated muscle. CAP is markedly induced during myoblast differentiation, and colocalizes with vinculin during costamerogenesis. In adult mice, CAP is enriched in oxidative muscle fibers, and it is found in membrane anchorage complexes, including intercalated discs, costameres, and myotendinous junctions. Using both yeast two-hybrid and proteomic approaches, we identified the sarcomeric protein filamin C (FLNc) as a binding partner for CAP. When overexpressed, CAP recruits FLNc to cell-extracellular matrix adhesions, where the two proteins cooperatively regulate actin reorganization. Moreover, overexpression of CAP inhibits FLNc-induced cell spreading on fibronectin. In dystrophin-deficient mdx mice, the expression and membrane localization of CAP is increased, concomitant with the elevated plasma membrane content of FLNc, suggesting that CAP may compensate for the reduced membrane linkage of the myofibrils due to the loss of the dystroglycan-sarcoglycan complex in these mice. Thus, through its interaction with FLNc, CAP provides another link between the myofibril cytoskeleton and the plasma membrane of muscle cells, and it may play a dynamic role in the regulation and maintenance of muscle structural integrity.

Bone marrow–specific Cap gene deletion protects against high-fat diet–induced insulin resistance

Cbl-associated protein (Cap) is a member of a phosphatidylinositol 3-kinase-independent pathway for insulin-stimulated translocation of the glucose transporter GLUT4. Despite this positive role of Cap in glucose uptake, here we show that deletion of the gene encoding Cap (official gene name: Sorbs1) protects against high-fat diet (HFD)-induced insulin resistance in mice while also having an opposite, insulin-sensitizing effect, accompanied by reduced tissue markers of inflammation. Given the emerging role of chronic inflammation in insulin resistance and the macrophage in initiating this inflammatory process, we considered that Sorbs1 deletion from macrophages may have resulted in the observed protection from HFD-induced insulin resistance. Using bone marrow transplantation to generate functional Sorbs1-null macrophages, we show that the insulin-sensitive phenotype can be transferred to wild-type mice by transplantation of Sorbs1-null bone marrow. These studies show that macrophages are an important cell type in the induction of insulin resistance and that Cap has a modulatory role in this function.

Coincidence of Actin Filaments and Talin Is Required to Activate Vinculin

Vinculin regulates cell adhesion by strengthening contacts between extracellular matrix and the cytoskeleton. Binding of the integrin ligand, talin, to the head domain of vinculin and F-actin to its tail domain is a potential mechanism for this function, but vinculin is autoinhibited by intramolecular interactions between its head and tail domain and must be activated to bind talin and actin. Because autoinhibition of vinculin occurs by synergism between two head and tail interfaces, one hypothesis is that activation could occur by two ligands that coordinately disrupt both interfaces. To test this idea we use a fluorescence resonance energy transfer probe that reports directly on activation of vinculin. Neither talin rod, VBS3 (a talin peptide that mimics a postulated activated state of talin), nor F-actin alone can activate vinculin. But in the presence of F-actin either talin rod or VBS3 induces dose-dependent activation of vinculin. The activation data are supported by solution phase binding studies, which show that talin rod or VBS3 fails to bind vinculin, whereas the same two ligands bind tightly to vinculin head domain (K(d) approximately 100 nM). These data strongly support a combinatorial mechanism of vinculin activation; moreover, they are inconsistent with a model in which talin or activated talin is sufficient to activate vinculin. Combinatorial activation implies that at cell adhesion sites vinculin is a coincidence detector awaiting simultaneous signals from talin and actin polymerization to unleash its scaffolding activity.

Low‐intensity exercise increases skeletal muscle protein expression of PPARδ and UCP3 in type 2 diabetic patients

Physical exercise provides health benefits for people with type 2 diabetes mellitus, partly by enhancing skeletal muscle insulin action. We tested the hypothesis that changes in expression of key genes in skeletal muscles relate to exercise-induced improvements in type 2 diabetic patients. We determined mRNA expression of 20 selected genes following a self-supervised program of walking (> 150 min per week) over a 4-month period. This level of physical activity improved clinical parameters in approximately half the participants, as determined by reduced hypertension and enhanced insulin sensitivity (defined by reduced plasma-insulin levels and improved homeostasis model assessment (HOMA)). Skeletal muscle mRNA expression of Cbl-associated protein (CAP), diacylglycerol kinase (DGK)delta, uncoupling protein (UCP) 3, nuclear respiratory factor (NRF)-1, and peroxisome proliferator-activated receptor (PPAR)delta tended to increase in type 2 diabetic patients with an improved clinical profile. Skeletal muscle protein expression of PPARdelta and UCP3 was increased significantly after physical exercise in patients with an improved clinical profile, but were unchanged in patients who did not show exercise-mediated improvements in clinical parameters. This study provides clinical evidence that improvements in insulin sensitivity can be achieved in type 2 diabetic patients after individually executed low-intensity exercise training. Moreover, the positive clinical response to exercise is correlated with changes in skeletal muscle proteins involved in the regulation of mitochondrial biogenesis and metabolism. These changes in skeletal muscle gene expression offer a possible molecular explanation for the improvements in clinical outcomes.

Hormonal Control of Reversible Translocation of Perilipin B to the Plasma Membrane in Primary Human Adipocytes

In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.

High-fat feeding effects on components of the CAP/Cbl signaling cascade in Sprague-Dawley rat skeletal muscle

The aim of this investigation was to determine whether the CAP (Cbl-associated protein)/Cbl signaling cascade is present and responsive to insulin in skeletal muscle and if high-fat feeding impairs insulin-stimulated activation of this signaling cascade. Sprague-Dawley rats were assigned to either control (n = 16) or high fat–fed (n = 16) dietary groups. After a 12-week dietary period, animals were subjected to hind limb perfusions in the presence (n = 8 per group) or absence (n = 8 per group) of insulin. High-fat feeding reduced rates of insulin-stimulated skeletal muscle phosphatidylinositol 3-kinase activity and 3- O-methylglucose transport. In plasma membrane fractions, neither the high-fat diet nor insulin altered the insulin receptor β subunit (IR- β), APS (adaptor protein containing PH and SH2 domains), c-Cbl, or TC10 protein concentration, but high-fat feeding did decrease CAP protein concentration. APS, c-Cbl, CAP, and TC10 messenger RNA were present in the skeletal muscle and reflected the protein concentration of experimental groups. Despite insulin-stimulated plasma membrane IR- β tyrosine phosphorylation being unaffected by high-fat feeding, c-Cbl tyrosine phosphorylation, the kinase activity of IR- β toward APS, and glucose transporter 4 protein concentration were all significantly reduced in insulin-stimulated plasma membrane prepared from the skeletal muscle of high fat–fed animals. These findings suggest that the CAP/Cbl signaling cascade is present in skeletal muscle, activated by insulin, and impaired by high-fat feeding.

A novel isoform of Cbl-associated protein that binds protein kinase A

A novel isoform of Cbl-associated protein (CAP) was identified in a yeast two-hybrid screen for A-kinase anchoring proteins expressed in the heart. CAP is a scaffold protein implicated in insulin signaling and cytoskeleton regulation. The protein kinase A binding site is encoded by a previously unidentified, alternatively spliced exon.

Regulation of Cbl-associated protein/Cbl pathway in muscle and adipose tissues of two animal models of insulin resistance.

The phosphatidylinositol 3-kinase-independent pathway to induce glucose transport may involve the tyrosine phosphorylation of the protooncogene c-Cbl. In the present study, we examined whether acute exposure to insulin stimulates the tyrosine phosphorylation of Cbl and its association with Cbl-associated protein (CAP) in muscle and adipose tissue of rats in vivo. We report herein that insulin induces Cbl tyrosine phosphorylation and association with CAP in adipose tissue but not in muscle. We also examined the expression and tyrosyl-phosphorylation state of Cbl and CAP/Cbl association in adipose tissue of rats submitted to prolonged fasting and in monosodium glutamate (MSG)-insulin-resistant rats. An increase in Cbl phosphorylation is observed in the fat of MSG rats, parallel with an increase in association of CAP-Cbl as well as an augment in CAP and Cbl protein expression in the adipose tissue of these animals. These events are accompanied by a decrease in insulin-stimulated insulin receptor/ insulin receptor substrate (IRS)-1 tyrosine phosphorylation and an increase in the IRS-2/phosphatidylinositol 3-kinase/Akt/Foxo1 pathway. In adipocytes of fasted rats, there is a decrease in CAP and Cbl protein expression, insulin-induced Cbl phosphorylation, and the association with CAP. In parallel, there is also a decrease in the insulin receptor/IRSs/Akt/Foxo1 pathway. Thus, insulin is able to induce Cbl tyrosine phosphorylation and its association with CAP in the adipose tissue of normal rats. In addition, our data provide evidence that the CAP-Cbl pathway may have a role in the modulation of adiposity in fasting and in MSG-treated rats.