Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.Insulin rapidly stimulates glucose transport into fat and muscle cells by causing the insertion of additional glucose transporters of the GLUT4 isotype into the plasma membrane, in a process referred to as GLUT4 translocation. The overall process consists of generation of the specialized vesicles containing GLUT4 from the endosomal system, the movement of these vesicles from the perinuclear region to the plasma membrane, and the fusion of the vesicles with the plasma membrane (1). The steps in this process that insulin accelerates, and the complete signaling pathways from the insulin receptor that lead to their acceleration, have not yet been fully defined. One partial insulin signaling pathway that has been established to be required for GLUT4 translocation is the pathway that proceeds from the receptor through tyrosine phosphorylation of the insulin receptor substrates to activation of phosphatidylinositol 3-kinase and generation of phosphatidylinositol 3,4,5-trisphosphate. The latter leads to the activation of the protein kinase Akt and also protein kinase C /, and there is evidence that GLUT4 translocation requires the activation of one or both of these kinases (reviewed in Refs. 2 and 3). However, although a substrate linking either kinase to GLUT4 translocation has been sought for several years, hitherto none has been clearly identified.Recently, we reported the discovery of a new substrate for insulin-activated Akt in 3T3-L1 adipocytes, which was designated AS160 for Akt subtrate of 160 kDa (4). The most prominent feature of AS160 is the presence of a GTPase activating domain for a Rab. Since Rabs are small G proteins that play critical roles in vesicle formation, movement, and fusion (5), we investigated the role of AS160 in GLUT4 translocatio...

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

Insulin controls glucose flux into muscle and fat by regulating the trafficking of GLUT4 between the interior and surface of cells. Here, we show that the AS160 Rab GTPase activating protein (GAP) is a negative regulator of basal GLUT4 exocytosis. AS160 knockdown resulted in a partial redistribution of GLUT4 from intracellular compartments to the plasma membrane, a concomitant increase in basal glucose uptake, and a 3-fold increase in basal GLUT4 exocytosis. Reexpression of wild-type AS160 restored normal GLUT4 behavior to the knockdown adipocytes, whereas reexpression of a GAP domain mutant did not revert the phenotype, providing the first direct evidence that AS160 GAP activity is required for basal GLUT4 retention. AS160 is the first protein identified that is specially required for basal GLUT4 retention. Our findings that AS160 knockdown only partially releases basal GLUT4 retention provides evidence that insulin signals to GLUT4 exocytosis by both AS160-dependent and -independent mechanisms.

Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non^small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLCassociated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. Experimental Design:We tested mutation-specific antibodies byWestern blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. Results: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry^based DNA sequencing. Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.Lung cancer is a major cause of cancer-related mortality worldwide and is expected to remain a major health problem for the foreseeable future. Lung cancer is broadly divided into small-cell lung cancer (20% of lung cancers) and non -smallcell lung cancer (NSCLC; 80% of lung cancers). Somatic mutations in the epidermal growth factor receptor (EGFR) gene are found in a subset of NSCLC adenocarcinomas and are associated with sensitivity to the small-molecule EGFR tyrosine kinase inhibitors gefitinib (1, 2) and erlotinib (3). Different EGFR mutations have been reported, but the most common NSCLC-associated EGFR mutations are in-frame deletions in exon 19 (E746_A750del) and the point mutation replacing leucine with arginine at codon 858 in exon 21 (L858R; refs. 3 -5). These two mutations represent 85% to 90% of EGFR mutations in NSCLC patients. Data from clinical research have confirmed that patients with these mutations are highly responsive to EGFR inhibitors including gefitinib and erlotinib (5 -8).Based on these clinical findings, EGFR mutational analysis in lung adenocarcinoma may now be used to guide treatment decisions and to enroll patients in specific arms of clinical trials. Direct DNA sequencing of PCR-amplified genomic DNA has been developed to detect EGFR mutations i...

AS160 is a newly described substrate for the protein kinase Akt that links insulin signaling and GLUT4 trafficking. In this study, we determined the expression of and in vivo insulin action on AS160 in human skeletal muscle. In addition, we compared the effect of physiological hyperinsulinemia on AS160 phosphorylation in 10 lean؊to؊moderately obese type 2 diabetic and 9 healthy subjects. Insulin infusion increased the phosphorylation of several proteins reacting with a phosphoAkt substrate antibody. We focused on AS160, as this Akt substrate has been linked to glucose transport. A 160-kDa phosphorylated protein was identified as AS160 by immunoblot analysis with an AS160-specific antibody. Physiological hyperinsulinemia increased AS160 phosphorylation 2.9-fold in skeletal muscle of control subjects (P < 0.001). Insulin-stimulated AS160 phosphorylation was reduced 39% (P < 0.05) in type 2 diabetic patients. AS160 protein expression was similar in type 2 diabetic and control subjects. Impaired AS160 phosphorylation was related to aberrant Akt signaling; insulin action on Akt Ser 473 phosphorylation was not significantly reduced in type 2 diabetic compared with control subjects, whereas Thr 308 phosphorylation was impaired 51% (P < 0.05). In conclusion, physiological hyperinsulinemia increases AS160 phosphorylation in human skeletal muscle. Moreover, defects in insulin action on AS160 may impair GLUT4 trafficking in type 2 diabetes. Diabetes 54:1692-1697, 2005 P eripheral insulin resistance is a major clinical feature of type 2 diabetes. Insulin resistance in skeletal muscle from type 2 diabetic patients is associated with impaired signal transduction at the level of insulin receptor substrate 1 (IRS-1) and phosphatidylinositol (PI) 3-kinase (1-4) and glucose transport (1,2,5). Akt is a downstream effector of PI 3-kinase that is directly linked to the regulation of glucose transport (6). Although defects in IRS-1/PI 3-kinase and glucose transport have been observed in several cohorts of type 2 diabetic subjects, impairments at the level of the Ser/Thr kinase Akt have been less apparent (rev. in 7). The role of Akt in skeletal muscle insulin resistance has been a challenge to resolve due to the presence of multiple isoforms that offer compensatory regulation.Recently, a novel 160-kDa substrate of Akt has been identified in 3T3L1 adipocytes as AS160, a protein containing a Rab GTPase-activating protein (GAP) domain (8). Phosphorylation of AS160 is required for the insulininduced translocation of GLUT4 to the plasma membrane in 3T3L1 adipocytes (9). The expression of a dominant inhibitory mutant AS160 markedly reduces GLUT4 exocytosis, without altering endocytosis (10). We hypothesized that insulin action on AS160, the most proximal step identified thus far in the insulin-signaling cascade to glucose transport, is impaired in skeletal muscle from type 2 diabetic patients. Here we report that physiological hyperinsulinemia increases AS160 phosphorylation in human skeletal muscle. Moreover AS160 phosphorylation is impai...

This study describes a method for the identification of the substrates of specific serine kinases. An antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method was applied to the identification of substrates for the protein kinase Akt, which specifically phosphorylates the RXRXXS/T motif. 3T3-L1 adipocytes were treated with insulin to activate Akt, and the putative Akt substrate proteins were isolated by immunoprecipitation with an antibody against the phospho form of this motif. This led to the identification of a novel 160-kDa substrate for Akt. The 160-kDa substrate for Akt, which was designated AS160, has a Rab GAP domain. Recombinant AS160 was shown to be a substrate for Akt, and two sites of phosphorylation, both in RXRXXS/T motifs, were identified by mass spectrometry and mutation. Insulin treatment of adipocytes caused AS160 to redistribute from the low density microsomes to the cytosol.Protein phosphorylation is a key cellular regulatory mechanism. The human genome contains ϳ1000 kinases. A major issue is to identify the protein substrates for each kinase. A number of different approaches have been developed (reviewed in Ref. 1). These include: in vitro phosphorylation of cell homogenates with recombinant kinases, screening of expression libraries with recombinant kinases, searching for kinase interacting proteins by the yeast two-hybrid screen, and the generation of mutated kinases that can function only with an ATP derivative. Each of these methods has its advantages and limitations, and additional methods are needed.Each serine kinase typically phosphorylates Ser/Thr within a particular motif, and for many kinases the motif has been defined through identification of the sites of phosphorylation on substrate proteins and on peptide libraries (2). Phosphospecific antibodies against the phosphorylated form of these motifs are available or can be generated. Thus, immunoprecipitation with these antibodies, when combined with tandem mass spectrometry of tryptic peptides, offers an approach for the isolation and identification of substrates for specific serine kinases. In the past this approach has been used for the identification of substrates for tyrosine kinases through the use of antibodies against phosphotyrosine, but to our knowledge it has not previously been used with serine kinases.In the present study, we applied this method to find substrates for the protein kinase Akt (also known as protein kinase B), which specifically phosphorylates Ser or Thr in the motif RXRXXS/T (3). We have employed 3T3-L1 adipocytes, a cell type in which Akt is rapidly activated by insulin treatment (4). This method has led to the isolation of a novel target for Akt that contains a Rab GAP 1 domain and two PTB domains. EXPERIMENTAL PROCEDURESAntibodies-A key reagent for this study is the antibody for the Akt phosphomotif RXRXXpS/T, where X is any amino acid...

The insulin-signaling network regulates blood glucose levels, controls metabolism, and when dysregulated, may lead to the development of type 2 diabetes. Although the role of tyrosine phosphorylation in this network is clear, only a limited number of insulin-induced tyrosine phosphorylation sites have been identified. To address this issue and establish temporal response, we have, for the first time, carried out an extensive, quantitative, mass spectrometrybased analysis of tyrosine phosphorylation in response to insulin. The study was performed with 3T3-L1 adipocytes stimulated with insulin for 0, 5, 15, and 45 min. It has resulted in the identification and relative temporal quantification of 122 tyrosine phosphorylation sites on 89 proteins. Insulin treatment caused a change of at least 1.3-fold in tyrosine phosphorylation on 89 of these sites. Among the responsive sites, 20 were previously known to be tyrosine phosphorylated with insulin treatment, including sites on the insulin receptor and insulin receptor substrate-1. The remaining 69 responsive sites have not previously been shown to be altered by insulin treatment. They were on proteins with a wide variety of functions, including components of the trafficking machinery for the insulin-responsive glucose transporter GLUT4. These results show that insulin-elicited tyrosine phosphorylation is extensive and implicate a number of hitherto unrecognized proteins in insulin action. Diabetes 55:2171-2179, 2006 M etabolic control is primarily regulated by the insulin-signaling network. In healthy individuals, insulin stimulates glucose uptake from the bloodstream into adipose tissue and skeletal muscle while inhibiting glucose production in the liver. Dysregulation of this network associated with insulin resistance causes an increase in blood glucose and lipid levels, often initially associated with an increase in insulin levels and eventually culminating in type 2 diabetes (1). Understanding the signaling network activated by insulin stimulation is crucial for identifying the causes and effects of network dysregulation and insulin resistance.Insulin binds to the insulin receptor at the cell surface and activates its tyrosine kinase activity, leading to autophosphorylation and phosphorylation of several receptor substrates. Phosphorylation of selected tyrosine sites on receptor substrates is known to activate different pathways leading to increased glucose uptake, lipogenesis, and glycogen and protein synthesis, as well as to stimulation of cell growth (1,2). In addition to activation of these pathways by tyrosine phosphorylation, several mechanisms of downregulating the response to insulin stimulation have also been identified. For instance, serine phosphorylation on insulin receptor substrate (IRS)-1 induced by a variety of factors has been shown to interfere with the activating effects of tyrosine phosphorylation by decreasing binding to the insulin receptor or increasing degradation of IRS-1 (1,3,4). Ser/Thr phosphorylation of the insulin receptor has also bee...

Brentuximab vedotin (BV) is an antibody-drug conjugate that specifically delivers the potent cytotoxic drug MMAE to CD30-positive cells. BV is FDA-approved for treatment of relapsed/refractory Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL); however, many patients do not achieve complete remission and develop BV resistant disease. We selected for BV-resistant HL (L428) and ALCL (Karpas-299) cell lines using either constant (ALCL) or pulsatile (HL) exposure to BV. We confirmed drug resistance by MTS assay, and analyzed CD30 expression in resistant cells by flow cytometry, qRT-PCR, and Western blotting. We also measured drug exporter expression, MMAE resistance, and intracellular MMAE concentrations in BV-resistant cells. Additionally, tissue biopsy samples from 10 HL and 5 ALCL patients who had relapsed or progressed after BV treatment were analyzed by immunohistocytochemistry for CD30 expression. The resistant ALCL cell line, but not the HL cell line, demonstrated downregulated CD30 expression compared to the parental cell line. In contrast, the HL cell line, but not the ALCL cell line, exhibited MMAE resistance and increased expression of the MDR1 drug exporter compared to the parental line. For both HL and ALCL, samples from patients relapsed/resistant on BV persistently expressed CD30 by immunohistocytochemistry. One HL patient sample expressed MDR1 by immunohistocytochemistry. Although loss of CD30 expression is a possible mode of BV resistance in ALCL in vitro models, this has not been confirmed in patients. MMAE resistance and MDR1 expression are possible modes of BV resistance for HL both in vitro and in patients.