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Originally published In Press as doi:10.1074/jbc.C400180200 on July 16, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37431-37435, September 3, 2004
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RNAi-based Analysis of CAP, Cbl, and CrkII Function in the Regulation of GLUT4 by Insulin*

Prasenjit Mitra, Xuexiu Zheng, and Michael P. Czech{ddagger}

From the Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Received for publication, April 21, 2004 , and in revised form, July 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Stimulation of glucose transport by insulin in cultured adipocytes through translocation of intracellular GLUT4 glucose transporters to the plasma membrane has been suggested to require phosphatidylinositol (PI) 3-kinase-dependent and independent mechanisms. To test the involvement of a PI 3-kinase-independent pathway leading to activation of the TC10 GTPase, the putative intermediates CAP, c-Cbl, Cbl-b, and CrkII were selectively depleted in 3T3-L1 adipocytes using highly efficient small interfering (si) RNAs. Simultaneous depletion of the ubiquitination factors c-Cbl plus Cbl-b in cultured adipocytes had the expected effect of delaying dephosphorylation of EGF receptors upon removal of EGF. However, siRNA-mediated gene silencing of both Cbl isoforms or CAP or CrkII in these cells failed to attenuate insulin-stimulated deoxyglucose transport or Myc-tagged GLUT4-GFP translocation at either sub-maximal or maximal concentrations of insulin. The dose-response relationship for insulin stimulation of deoxyglucose transport in primary adipocytes derived from c-Cbl knock-out mice was also identical to insulin action on adipocytes from wild type mice. These data are consistent with the hypothesis that CAP, Cbl iso-forms, and CrkII are not required components of insulin signaling to GLUT4 transporters.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Glucose homeostasis is a tightly controlled process in mammals, requiring rapid disposal of ingested glucose into muscle and adipose tissue during high caloric intake and enhanced production of glucose by liver and kidney during starvation (1). Insulin is secreted during feeding and acts to stimulate the former process and inhibit the latter, thus maintaining appropriately low blood glucose concentrations (2). Insulin action to enhance glucose transport into muscle and fat cells reflects its ability to cause translocation of intracellular GLUT4 glucose transporter proteins to the plasma membrane (3, 4). The recycling of GLUT4 from intracellular compartments to the plasma membrane occurs in the basal state as well but at a much slower rate, and insulin action markedly enhances the exocytic pathway while inhibiting endocytosis (5). Insulin signaling to GLUT4 is dependent on the activation of the PI1 3-kinase signaling pathway whereby generation of D-3 polyphosphoinositides leads to the activation of downstream protein kinases such as Akt (2, 6–11). This cascade involves tyrosine phosphorylation of insulin receptor substrates and the recruitment of the p85 regulatory subunit of PI 3-kinase to these phosphotyrosines through binding of its SH2 domains (12–16). Disruption of PI 3-kinase activation by insulin through the action of inhibitors or dominant inhibitory constructs blocks GLUT4 translocation (17–19).

Despite the clear dependence of GLUT4 regulation by insulin on PI-3 kinase activity, several lines of evidence have indicated that an exclusive PI 3-kinase-dependent pathway might be insufficient for glucose uptake in adipocytes. Thus, a membrane-permeant analog of the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate failed to stimulate glucose up-take in cultured adipocytes in the absence of insulin (20). Similarly, activation of PI 3-kinase through interleukin 4 receptor or integrin receptors appeared not to be able to mimic insulin stimulation of glucose transport (21, 22). Moreover, two naturally occurring insulin receptor mutants that are unable to stimulate GLUT4 translocation appear to be fully capable of evoking PI-3 kinase activity (23). In addition, there may be cytoskeleton-based steps in the movements of GLUT4-containing vesicles toward the plasma membrane that do not require PI 3-kinase activity (24, 25). A definitive signaling pathway was recently described and hypothesized to act in tandem with the PI 3-kinase pathway to mediate insulin stimulation of GLUT4 translocation (26). This pathway was proposed to be initiated by association of CAP and APS adaptor proteins with the insulin receptor, which in turn recruit c-Cbl and/or Cbl-b proteins that are then tyrosine-phosphorylated. Phosphotyrosine on Cbl was found to associate with the CrkII adaptor protein bound to the GTPase exchange factor C3G, leading to GTP/GDP exchange and activation of the TC10 GTPase (27). Dominant negative constructs of CAP (26) and TC10 (28) have been shown to significantly inhibit GLUT4 recycling in response to insulin in cultured adipocytes.

Recent studies have questioned the role of the CAP/APS/Cbl/TC10 pathway in GLUT4 regulation in cultured adipocytes as well as in muscle (29, 30). Furthermore, Minami et al. (31) reported an increase in insulin sensitivity in APS knock-out mice rather than the expected decrease. Neither CAP, Cbl, CrkII, nor C3G in this TC10 pathway have been tested for their requirement in insulin signaling to GLUT4 by gene ablation in mice or by RNAi-based gene silencing in 3T3-L1 adipocytes, the model system in which they were studied. Our laboratory has developed a powerful method for selective depletion of proteins in adipocytes based on administration of small interfering RNAs (siRNA). Application of this approach has strongly implicated the involvement of the Akt2 protein kinase (11), the unconventional myosin Myo1c (24), and the adaptor protein EHBP1 (EH-domain binding protein 1)2 in the mechanism of insulin-stimulated glucose transport. In this present study, we generated functional siRNAs to efficiently suppress the expression of several members of the CAP/Cbl/TC10 pathway in 3T3-L1 adipocytes. We also analyzed primary adipocytes derived from c-Cbl knock-out mice to further validate our findings derived from RNAi-mediated gene silencing. Our results demonstrate that c-Cbl/Cbl-b, CAP, or CrkII can be depleted in cultured adipocytes without compromising stimulation of hexose transport or GLUT4 translocation by insulin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials—Rabbit polyclonal anti-CAP antibody and EGF was purchased from Upstate Biotechnology (Charlottesville, VA). Mouse monoclonal anti-Cbl-b (G-1), and rabbit polyclonal epidermal-growth factor receptor (EGFR) (1005) were from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal antibodies against phospho-EGF receptor (Tyr1068), p44/42 mitogen-activated protein kinase antibody and Akt antibody were from Cell Signaling Technology (Beverly, MA). c-Cbl, c-Cbl (pY700), and CrkII monoclonal antibody were from Pharmingen; fluorescein isothiocyanate-conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (Palo Alto, CA). Human insulin was obtained from Eli Lilly. Other reagents used were all of analytical reagent grade. siRNA Duplexes—The siRNA purchased from Dharmacon Inc. (Lafayette, CO) were designed to target the following cDNA sequences: scrambled, 5'-CAGTCGCGTTTGCGACTGG-3'; c-Cbl, 5'-GACACTTTCCGGATTACTA-3'; Cbl-b, 5'-GGACAGACGGAATCTCACA-3'; Crk733, 5'-GCGAGTCCCTAATGCCTAC-3'; Crk852, 5'-TCCCGATGAGGACTTCAGC-3'; CAP76, 5'-AGACATGGACCCTACCAAA-3'; CAP 1018, 5'-GATGAGCTCAGCAGTCAGC-3'.

All the siRNAs are the exact complement of their target DNA sequence except for Cbl-b where a di-nucleotide mismatch has been incorporated near the 5' end sense strand RNA (33).

Cell Culture and Electroporation—The 3T3-L1 and 3T3-F442A fibroblasts were grown to confluence and differentiated to mature adipocytes for up to 8 days (11, 34) prior to electroporation. Scrambled (20 nmol), c-Cbl (10 nmol), Cbl-b (10 nmol), c-Cbl+Cbl-b (10 nmol each), CAP76 (20 nmol), CAP1018 (20 nmol), Crk733 (20 nmol), and Crk852 (20 nmol) siRNAs were electroporated in mature cultured adipocytes (5 x 106cells/electroporation) at the setting of 0.18 kV and 960 microfarads using Bio-Rad gene pulser II. After electroporation, cells were immediately mixed with fresh medium before being reseeded onto multiple-well plates. Gene silencing efficiency and adipocyte function were assayed 48 h after electroporation (7–10 days after initiation of differentiation).

Immunofluorescence Microscopy—3T3-L1 and 3T3-F442A adipocytes were cotransfected with Myc-GLUT4-EGFP plasmid and c-Cbl+Cbl-b or scrambled siRNAs. 36 h after electroporation, adipocytes were serum-starved for another 12 h, stimulated with 100 nM insulin for 30 min, washed, and immunostained for Myc epitope using mouse anti-Myc antibody (clone 910) and rhodamine-labeled anti-mouse secondary antibody. Coverslips were mounted in Vectashield mounting medium (Vector Laboratories) and observed under IX70-inverted microscope (Olympus, Melville, NY).

Adipocytes Stimulation—Thirty-six hours after electroporation with siRNA, 3T3-L1 and 3T3-F442A adipocytes were serum-starved with Dulbecco's modified Eagle's medium containing 0.5% BSA for another 12 h before being stimulated with 100 nM insulin or 50 nM EGF for the indicated times. The cells were then lysed, resolved by SDS-PAGE, and blotted with the respective antibodies.

2-Deoxyglucose Uptake Assay—Deoxyglucose uptake was measured as described (11). In brief, siRNA-transfected cells reseeded on 12-well plates were cultured for 48 h prior to 3-h starvation with Krebs-Ringer Hepes buffer (130 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.3 mM MgSO4, 25 mM Hepes, pH 7.4) supplemented with 0.5% BSA and 2 mM sodium pyruvate. Cells were then stimulated with insulin for 25 min at 37 °C. Deoxyglucose uptake was initiated by addition of [1,2-3H]2-deoxy-D-glucose to a final assay concentration of 100 µM for 5 min at 37 °C. Assays were terminated by four washes with ice-cold Krebs-Ringer Hepes buffer, and the cells were lysed with 0.4 ml of 1% Triton X-100, and 3H was determined by scintillation counting. Nonspecific deoxyglucose uptake was measured in the presence of 20 µM cytochalasin B and subtracted from each determination to obtain specific uptake. The fold increase was calculated after normalizing the basal uptake in respective siRNA-transfected cells to one.

Glucose Transport in Primary Adipocytes—Primary adipocytes were isolated from control and c-Cbl knock-out mice, a kind gift from H. Gu (35), Laboratory of Immunology, NIAID, National Institutes of Health, Rockville. MD. Glucose uptake assay in primary mouse adipocytes was carried out following the method of Tozzo and Kahn (36). Briefly, adipose cells isolated from epididymal fat pads of four to five mice were incubated at 37 °C with constant shaking in a 4% suspension by volume (~40,000 cells/500 µl) in Krebs-Ringer phosphate buffer, pH 7.4, with Hepes (20 mM), 2.5% BSA (fraction V), and 200 nM adenosine. Cells were treated in the absence (basal) or presence of 1, 10, or 100 nM insulin for 30 min in presence of U-[14C]glucose, and the reaction was terminated by adding stop solution containing 1 mM phloretin and 0.05 mM cytochalsin B and separating cells from the medium by spinning the suspension through dinonyl phthalate oil (Eastman Kodak Co.).

Statistical Analysis—Comparison of data presented as mean ± S.E. was performed by Student's t test (unpaired), and differences at p < 0.05 were considered to be statistically significant. Percent reduction of individual protein expression was measured by densitometric analysis and is presented as a mean of several independent experiments.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
siRNA-mediated CAP Depletion Inhibits Cbl Phosphorylation on Tyr700 but Not GLUT4 Regulation in 3T3-L1 Adiocytes—We initiated our studies by silencing the expression of CAP, a bifunctional Cbl-binding adaptor protein with three SH3 domains and a sorbin homology motif (37). Previous studies indicated a CAP mutant lacking these SH3 domains expressed in 3T3-L1 adipocytes reduced insulin stimulated GLUT4 translocation by ~50% (38). One of two siRNAs we tested for inhibition of CAP expression (siRNA CAP76) caused a near total depletion of CAP, while another siRNA (CAP1018) and a scrambled control siRNA had no detectable effect on CAP protein expression (Fig. 1A). Using phospho-specific antibodies against Cbl, we next tested for the effect of CAP ablation on Cbl phosphorylation. Of the four principal tyrosine phosphorylation sites in Cbl, (Tyr371, Tyr731, Tyr700, Tyr774), phosphorylation of Tyr700 and Tyr774 have been shown to create docking sites for the SH2 domain of CrkII (39, 40). Since insulin reportedly did not induce tyrosine phosphorylation of Tyr774 in 3T3-L1 adipocytes (29), we analyzed the phosphorylation status of Tyr700 both in control and CAP-ablated cells. Both un-stimulated and insulin stimulated adipocytes showed comparable phosphorylation as determined by phospho-specific antibody Tyr700(P). Little or no effect of insulin on Cbl phosphorylation at tyrosine 700 was also observed after 5 min of insulin stimulation (data not shown). However, phosphorylation of tyrosine 700 was found to be significantly diminished by siRNA-mediated depletion of CAP in 3T3-L1 adipocytes, indicating a function for CAP in tyrosine phosphorylation of Cbl (Fig. 1B).



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  		FIG. 1.
siRNA-mediated CAP depletion blocks Cbl phosphorylation on Tyr700 but has no effect on insulin-stimulated deoxyglucose uptake. A, synthetic siRNA duplexes targeting different regions of CAP were electroporated in adipocytes at 20 nmol per 5 x 106 cells. 48 h after electroporation cells were harvested, equal amounts of lysates were resolved by SDS-PAGE and analyzed by Western blotting for expression of CAP protein. B, insulin-stimulated Cbl phosphorylation on tyrosine 700 residue in control (scrambled siRNA-treated) and CAP76 siRNA-treated adipocytes. Cells, 36 h after electroporation, were serum-starved for 12 h prior to assay and were harvested at indicated time points after insulin stimulation. Equal amounts of lysates for each time point were resolved in SDS-PAGE and blotted with phospho-Cbl antibody. C, dose-dependent insulin-stimulated deoxyglucose uptake in CAP76 or scrambled siRNA-treated adipocytes. Quantitative data are presented as mean ± S.E. of three independent experiments. Scr, scrambled siRNA.

 
We also analyzed the effect of CAP ablation on deoxyglucose uptake in response to insulin in 3T3-L1 adipocytes. As evidenced in Fig. 1C, both control and CAP-depleted adipocytes showed similar deoxyglucose transport rates in the presence of either 1 or 100 nM insulin. This result indicates that neither the reduction of CAP expression nor the inhibition of Cbl phosphorylation on Tyr700 has a significant effect on insulin-stimulated deoxyglucose uptake.

GLUT4 Translocation Is Normal in 3T3-L1 Adipocytes Depleted of c-Cbl plus Cbl-b—We next studied the consequences of selective attenuation of c-Cbl and Cbl-b in 3T3-L1 and 3T3-F442A adipocytes. Cbl proteins have been implicated in the regulation of endocytic trafficking of EGFR in a variety of cell types (41, 42). Binding of EGF to EGFR dimerizes and activates the intrinsic tyrosine kinase activities of the receptor resulting in multiple tyrosine phosphorylations at sites within its cytoplasmic domain that in turn engage specific cytoplasmic signaling proteins. Phosphorylation of EGFR at tyrosine 1068 serves as a binding site for GRB2 (43), while phosphorylation at tyrosine 1045 promotes binding of c-Cbl to the receptor (44). EGFR association with c-Cbl causes monoubiquitination of the receptor (45), which is then rapidly internalized through clathrin-coated pits, sorted through early endosomes, and ultimately degraded in lysosomes (46). Loss of EGFR tyrosine phosphorylation has been reported to precede such receptor internalization (47).

To assure that Cbl depletion by our siRNA-based procedure has some biological consequence in these present studies, we used phospho-specific antibody against EGFR Tyr1068(P) to determine EGFR phosphorylation in control and Cbl-depleted 3T3-F442A cells. We also developed conditions where the two isoforms of Cbl (c-Cbl plus Cbl-b) could be depleted simultaneously using siRNA species directed to both. As evidenced in Fig. 2E, EGF-induced EGFR phosphorylation at tyrosine 1068 persisted for 10 min in control cells, while upon attenuation of expression of c-Cbl plus Cbl-b proteins, EGFR Tyr1068 phosphorylation persisted even after 45 min of EGF stimulation. We then tested the effect of loss of both c-Cbl plus Cbl-b on insulin-stimulated deoxyglucose transport in 3T3-F442A adipocytes (data not shown) and 3T3-L1 adipocytes (Fig. 2). We achieved >95% attenuation of c-Cbl and Cbl-b expression (Fig. 2A); however, even this near total loss of c-Cbl and Cbl-b expression failed to modify insulin-stimulated deoxyglucose transport (Fig. 2D). In five independent experiments, Cbl-depleted 3T3-L1 adipocytes displayed comparable deoxyglucose transport rates in relation to adipocytes treated with scrambled siRNA when stimulated with submaximal (1 nM) or maximal concentrations (100 nM) of insulin.



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  		FIG. 2.
siRNA-mediated depletion of c-Cbl plus Cbl-b prolongs EGFR phosphorylation but has no effect on insulin-stimulated GLUT4 translocation and deoxyglucose uptake. A, synthetic siRNA duplexes targeting c-Cbl, Cbl-b, and scrambled control siRNA were electroporated in adipocytes as described in the legend to Fig. 1, and cells were assayed 48 h after electroporation. Representative Western blots for Cbl-b and c-Cbl protein expression upon treatment of 3T3-L1 adipocytes with siRNA directed against Cbl-b, c-Cbl, c-Cbl+Cbl-b, or scrambled siRNA. B, cells cotransfected with Myc-GLUT4-EGFP together with either scrambled siRNA or a mixture of c-Cbl and Cbl-b siRNAs were grown on coverslips for 48 h and stimulated with 100 nM insulin for 30 min at 37 °C. Representative images for GFP-positive cells and exofacial Myc staining are shown. All fluorescence intensity settings are identical for all images. C, percentage of transfected adipocytes showing Myc-GLUT4-GFP rim on the cell surface. Data are presented as the mean ± S.E. of three independent experiments with >100 cells counted in each experiment. D, dose-dependent insulin-stimulated deoxyglucose uptake in scrambled control and c-Cbl+Cbl-b siRNA-treated 3T3-L1 adipocytes. Quantitative data are presented as a mean ± S.E. of five independent experiments. Scr, scrambled siRNA. E, EGFR phosphorylation in 3T3-F442A cells treated with c-Cbl+Cbl-b siRNA or scrambled control siRNA. Synthetic siRNA duplexes targeting c-Cbl+Cbl-b and scrambled control siRNA were electroporated, and 36 h after electroporation cells were serum-starved for 12 h and harvested at the indicated time points after EGF stimulation. Equal amounts of lysates for each time point were resolved in SDS-PAGE and blotted with phospho-EGFR antibody (first panel), c-Cbl+Cbl-b antibody (second panel), and Akt antibody used as control (third panel).

 
The effect of combined depletion of c-Cbl and Cbl-b on the regulation of GLUT4 translocation was also analyzed (Fig. 2, B and C). Cotransfection of Myc-GLUT4-EGFP plasmid with the mixture of c-Cbl- and Cbl-b-directed siRNAs caused ~95% depletion of Cbl protein expression (see Fig. 2A) with no effect on insulin-stimulated Myc-GLUT4-GFP translocation to the plasma membrane (Fig. 2B). Quantifying the ratio of cell surface Myc rim signal over the total Myc-GLUT4-GFP signal in transfected cells revealed comparable Myc-GLUT4-GFP on the cell surface of both control and Cbl-depleted adipocytes (Fig. 2C). The data indicate that neither deoxyglucose transport nor GLUT4 recycling in response to insulin is perturbed following the loss of both Cbl isoforms in cultured adipocytes.

siRNA-mediated CrkII Depletion Has No Impact on Insulin-stimulated Deoxyglucose Transport in 3T3L1 Adipocytes—SH2 domain-containing adaptor protein CrkII is another important component in the CAP-Cbl-Tc10 pathway (48). Tyrosine-phosphorylated Cbl reportedly recruits CrkII to lipid rafts along with guanine nucleotide factor C3G, which in its turn activates the Rho family protein TC10 (49). Fig. 3 depicts the consequence of selective attenuation of CrkII expression on insulin regulation of glucose transport. Electroporation of siRNA species Crk733, but not Crk852, into 3T3-L1 adipocytes caused a 79% inhibition of CrkII protein expression as measured by densitometric analysis. However, contrary to the proposed role of CrkII in glucose transport stimulation, markedly decreased CrkII protein expression had no effect on insulin-stimulated glucose transport (Fig. 3C).



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  		FIG. 3.
siRNA-mediated CrkII depletion has no effect on insulin-stimulated deoxyglucose transport in 3T3-L1 adipocytes. Synthetic siRNA duplexes targeting different regions of CrkII were electroporated in 3T3-L1 adipocytes at 20 nmol per 5 x 106 cells. 48 h after electroporation, cells were harvested, resolved in SDS-PAGE, and analyzed for expression of CrkII (A) and p44/42 (B) used as control. C, dose-dependent insulin-stimulated deoxyglucose uptake in Crk733 and scrambled siRNA treated 3T3-L1 adipocytes. Quantitative data are presented as mean ± S.E. of five independent experiments. Scr, scrambled siRNA.

 
Primary Adipocytes from c-Cbl knock-out Mice Respond Normally to Insulin—During preparation of this manuscript, a report appeared that suggested some inhibition of insulin-stimulated glucose transport in cultured human adipocytes upon siRNA-based depletion of Cbl (32). Whether or not the levels of GLUT4 protein were reduced due to potential toxicity of the eight different siRNAs used in tandem in this human cell line was not shown in this study. To substantiate our data from 3T3-L1 cells, we analyzed insulin action on glucose uptake in primary adipocytes derived from c-Cbl knock-out mice (Fig. 4). As revealed in Fig. 4A, primary mouse adipocytes express c-Cbl but very little Cbl-b compared with 3T3-L1 adipocytes, and ablation of c-Cbl in the primary adipocytes of c-Cbl knock-out mice did not significantly enhance Cbl-b expression. Consistent with our data in cultured adipocytes, both control and c-Cbl-deficient primary adipocytes show similar glucose uptake increases when stimulated with 1, 10, or 100 nM insulin (Fig. 4B). Taken together, the data obtained in this study provide compelling evidence that Cbl proteins are not required for optimal insulin signaling and regulation of GLUT4 in either cultured 3T3-L1 or primary mouse adipocytes. Furthermore, CAP and CrkII are not apparently required for insulin regulation of hexose transport in 3T3-L1 adipocytes.



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  		FIG. 4.
Primary adipocytes from c-Cbl knock-out mice respond normally to insulin. Primary adipocytes were isolated from C57BL/6 and c-Cbl knock-out mice following the procedure described under "Experimental Procedures." A, epididymal fat was extracted from four male C-57BL/6 and four male c-Cbl knock-out mice, and equal amounts of fat cell lysates from both groups were resolved by SDS-PAGE and blotted with either c-Cbl or Cbl-b antibody. B, U-[14C]glucose (3 µM) was measured in the presence of 1, 10, and 100 nM insulin. Results were expressed as fold increase over basal glucose uptake values and presented as mean ± S.E. of three independent experiments.

 

   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK30898 (to M. P. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-2254; Fax: 508-856-1617; E-mail: michael.czech{at}umassmed.edu.

1 The abbreviations used are: PI, phosphatidylinositol; siRNA, short interfering RNA; RNAi, RNA interference; CAP, Cbl-activating protein; SH, Src homology; EGF, epidermal growth factor; EGFR, EGF receptor; BSA, bovine serum albumin; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; APS, adaptor protein with pleckstrim homology and Src homology 2 domains. Back

2 Guilherme, A., Soriano, N. A., Furcinitti, P. S., and Czech, M. P. (July 9, 2004) J. Biol. Chem. 10.1074/jbc.M401918200. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Shepherd, P. R., and Kahn, B. B. (1999) N. Engl. J. Med. 341, 248–257[Free Full Text]
  2. Saltiel, A. R., and Kahn, R. C. (2001) Nature 414, 799–806[CrossRef][Medline] [Order article via Infotrieve]
  3. Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758–4762[Free Full Text]
  4. Suzuki, K., and Kono, T. (1980) Proc. Natl. Acad. Sci. U. S. A. (1980) 77, 2542–2545[Abstract/Free Full Text]
  5. Bryant, N. J., Govers, R., and James, D. E. (2002) Nat. Rev. Mol. Cell. Biol. 3, 267–277[CrossRef][Medline] [Order article via Infotrieve]
  6. Kohn, A. D., Summers, S. A., Birnbaum, M. J., and Roth, R. A. (1996) J. Biol. Chem. 271, 31372–31378[Abstract/Free Full Text]
  7. Czech, M. P., and Corvera, S. (1999) J. Biol. Chem. 274, 1865–1868[Free Full Text]
  8. Bae, S. S., Cho, H., Mu, J., and Birnbaum, M. J. (2003) J. Biol. Chem. 278, 49530–49536[Abstract/Free Full Text]
  9. Cantley, L. C. (2002) Science 296, 1655–1657[Abstract/Free Full Text]
  10. Katome, T., Obata, T., Matsushima, R., Masuyama, N., Cantley, L. C., Gotoh, Y., Kishi, K., Shiota, H., and Ebina, Y. (2003) J. Biol. Chem. 278, 28312–28323[Abstract/Free Full Text]
  11. Jiang, Z., Zhou, Q. L., Coleman, K. A., Chounard, M., Boese, Q., and Czech, M. P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 7569–7574[Abstract/Free Full Text]
  12. Cheng, A., Dube, N., Gu, F., and Tremblay, M. L. (2002) Eur. J. Biochem. 269, 1050–1059[Medline] [Order article via Infotrieve]
  13. Ueki, K., Fruman, D. A., Yballe, C. M., Fasshauer, M., Klein, J., Asano, T., Cantley, L. C., and Kahn, R. C. (2003) J. Biol. Chem. 278, 48453–48466[Abstract/Free Full Text]
  14. Smith-Hall, J., Pons, S., Patti, M. E., Burks, D. J., Yenush, L., Sun, X. J., Kahn, C. R., and White, M. (1997) Biochemistry 36, 8304–8310[CrossRef][Medline] [Order article via Infotrieve]
  15. Lamphere, L., Carpenter, C. L., Sheng, Z. F., Kallen, R. G., and Lienhard, G. E. (1994) Am. J. Physiol. 266 E486–E494[Medline] [Order article via Infotrieve]
  16. Staubs, P. A., Reichart, D. R., Saltiel, A. R., Milarski, K. L., Maegawa, H., Berhanu, P., Olefsky, J. M., and Seely, B. L. (1994) J. Biol. Chem. 269, 27186–27192[Abstract/Free Full Text]
  17. Okada, T., Kawano, Y., Sakakibara, T., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3568–3573[Abstract/Free Full Text]
  18. Cheatham, B., Vlahos, C. J., Cheatham, L., Wang, L., Blenis, J., and Kahn, C. R. (1994) Mol. Cell. Biol. 14, 4902–4911[Abstract/Free Full Text]
  19. Sharma, P. M., Egawa, K., Huang, Y., Martin, J. L., Huvar, I., Boss, G. R., and Olefsky, J. M. (1998) J. Biol. Chem. 273, 18528–18537[Abstract/Free Full Text]
  20. Jiang, T., Sweeny, G., Rudolf, M. T., Klip, A., Traynor-Kaplan, A., and Tsien, R. Y. (1998) J. Biol. Chem. 273, 11017–11024[Abstract/Free Full Text]
  21. Isakoff, S. J., Taha, C., Rose, E., Marcusohn, J., Klip, A., and Skolnik, E. Y. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10247–10251[Abstract/Free Full Text]
  22. Guilherme, A., and Czech, M. P. (1998) J. Biol. Chem. 273, 33119–33122[Abstract/Free Full Text]
  23. Krook, A., Whitehead, J. P., Dobson, S. P., Griffith, M. R., Ouwens, M., Baker, C., Hayward, A. C., Sen, S. K., Maassen, J. A., Siddle, K., Tavare, J. M., and O'Rahilly, S. (1997) J. Biol. Chem. 272, 30208–30214[Abstract/Free Full Text]
  24. Bose, A., Guilherme, A., Robida, S. I., Nicoloro, S. M. C., Zhou, Q. L., Jiang, Z. Y., Pomerlau, D. P., and Czech, M. P. (2002) Nature 420, 821–824[CrossRef][Medline] [Order article via Infotrieve]
  25. Semiz, S., Park, J. G., Nicoloro, S. M. C., Furcinitti, P., Zhang, C., Chawla, A., Leazyk, J., and Czech, M. P. (2003) EMBO J. 22, 2387–2399[CrossRef][Medline] [Order article via Infotrieve]
  26. Baumann, C. A., Ribon, V., Kanzaki, M., Thurmond, D. C., Mora, S., Shigematsu, S., Bickel, P. E., Pessin, J. E., and Saltiel, A. R. (2000) Nature 407, 202–207[CrossRef][Medline] [Order article via Infotrieve]
  27. Watson, R. T., Shigematsu, S., Chiang, S. H., Mora, S., Kanzaki, M., Macara, I. G., Saltiel, A. R., and Pessin, J. E. (2001) J. Cell Biol. 154, 829–840[Abstract/Free Full Text]
  28. Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson, R. T., Neudauer, C. L., Macara, I. G., Pessin, J. E., and Saltiel, A. R. (2001) Nature 410, 944–948[CrossRef][Medline] [Order article via Infotrieve]
  29. Jebailey, L., Rudich, A., Huang, X., Ciano-Oliviera, C., Kapas, A., and Klip, A. (2003) Mol. Endocrinol. 18, 359–372[Medline] [Order article via Infotrieve]
  30. Thirone, A. C., Carvalheira, J. B., Hirata, A. E., Velloso, L. A., and Saad, M. J. (2004) Endocrinology 145, 281–293[Abstract/Free Full Text]
  31. Minami, A., Iseki M., Kishi, K., Wang, M., Ogura, M., Furukawa, N., Hayashi, S., Yamada, M., Obata, T., Takeshita, Y., Nakaya, Y., Bando, Y., Izumi, K., Moodie, S., Kajiura, F., Matsumoto, M., Takatsu, K., Takaki, S., and Ebina, Y. (2003) Diabetes 52 2657–2665[Abstract/Free Full Text]
  32. Ahn, M. Y., Katsanakis, K. D., Bheda, F., and Pillay, T. S. (2004) J. Biol. Chem. 279, 21526–21532[Abstract/Free Full Text]
  33. Doench, J. G., Petersen, C. P., and Sharp, P. A. (2003) Genes Dev. 17 438–442[Abstract/Free Full Text]
  34. Sakurai, K., Kawazuma, M., Adachi, T., Harigaya, T., Saito, Y., Hashimoto, N., and Mori, C. (2004) Br. J. Pharmacol 141, 209–214[CrossRef][Medline] [Order article via Infotrieve]
  35. Naramura, M., Kole, H. K., Hu, R. J., and Gu, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15547–15552[Abstract/Free Full Text]
  36. Tozzo, F., Gnudi, L., and Kahn, B. B. (1997) Endocrinology 138, 1604–1611[Abstract/Free Full Text]
  37. Ribon, V., Printen, J. A., Hoffman, N. G., Kay, B. K., and Saltiel, A. R. (1998) Mol. Cell. Biol. 18, 872–879[Abstract/Free Full Text]
  38. Kimura, A., Baumann, C. A., Chiang, S. H., and Saltiel, A. R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9098–9103[Abstract/Free Full Text]
  39. Reedquist, K. A., Fukazawa, T., Govindaswami, R., Langdon, W. Y., Shoelson, S. E., Drucker, B. J., and Band, H. (1996) J. Biol. Chem. 271, 8435–8442[Abstract/Free Full Text]
  40. Dikic, I., Szymkiewicz, I., and Soubeyran, P. (2003) Cell. Mol. Life Sci. 60, 1805–1827[CrossRef][Medline] [Order article via Infotrieve]
  41. Lill, N. L., Douillard, P., Awwad, R. A., Ota, S., Lupher, M. L., Jr., Miyake, S., Meissner-Lula, N., Hsu, V. W., and Band, H. (2000) J. Biol. Chem. 275, 367–377[Abstract/Free Full Text]
  42. Huang, Y., Kim, S. O., Jiang J., and Frank, S. J. (2003) J. Biol. Chem. 278, 18902–18913[Abstract/Free Full Text]
  43. Rojas, M., Yao, S. Y., and Lin, Y. (1996) J. Biol. Chem. 271, 27456–27461[Abstract/Free Full Text]
  44. Levkowitz, G., Waterman, H., Ettenberg, S. A., Katz, M., Tsygankov, A. Y., Alroy, I. Lavi, S., Iwai, K., Reiss, Y., Ciechanover, A., Lipkowitz, S., and Yarden, Y. (1999) Mol. Cell 4, 1029–1040[CrossRef][Medline] [Order article via Infotrieve]
  45. Haglund, K., Sigismund, S., Polo, S., Szymkiewicz, I., Di Fore, P. P., and Dikic, I. (2003) Nat. Cell Biol. 5, 461–466[CrossRef][Medline] [Order article via Infotrieve]
  46. Jiang, X., and Sorkin, A. (2003) Traffic 4, 529–543[Medline] [Order article via Infotrieve]
  47. Burke, P., Schooler, K., and Wiley, H. S. (2001) Mol. Biol. Cell 12, 1897–1910[Abstract/Free Full Text]
  48. Ribon, V., and Alan, R., Saltiel, A. R. (1997) Biochem. J. 324 839–845[Medline] [Order article via Infotrieve]
  49. Saltiel, A. R., and Pessin, J. E. (2002) Trends Cell Biol. 12, 65–71[CrossRef][Medline] [Order article via Infotrieve]

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