In NIH-3T3 Fibroblasts, Insulin Receptor Interaction with Specific Protein Kinase C Isoforms Controls Receptor Intracellular Routing*

Pietro Formisano, Francesco Oriente, Claudia Miele, Matilde CarusoDagger , Renata Auricchio, Giovanni Vigliotta, Gerolama Condorelli, and Francesco Beguinot§

From the Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano" and Centro di Endocrinologia ed Oncolgia Sperimentale del Consiglio Nazionale delle Ricerche (CNR), "Federico II" University of Naples Medical School, Naples, Italy

    ABSTRACT
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Insulin increased protein kinase C (PKC) activity by 2-fold in both membrane preparations and insulin receptor (IR) antibody precipitates from NIH-3T3 cells expressing human IRs (3T3hIR). PKC-alpha , -delta , and -zeta were barely detectable in IR antibody precipitates of unstimulated cells, while increasing by 7-, 3.5-, and 3-fold, respectively, after insulin addition. Preexposure of 3T3hIR cells to staurosporine reduced insulin-induced receptor coprecipitation with PKC-alpha , -delta , and -zeta by 3-, 4-, and 10-fold, respectively, accompanied by a 1.5-fold decrease in insulin degradation and a similar increase in insulin retroendocytosis. Selective depletion of cellular PKC-alpha and -delta , by 24 h of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure, reduced insulin degradation by 3-fold and similarly increased insulin retroendocytosis, with no change in PKC-zeta . In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs (3T3Mut), insulin-induced coprecipitation of PKC-alpha , -delta , and -zeta with the IR was reduced by 10-, 7-, and 3-fold, respectively. Similar to the 3T3hIR cells chronically exposed to TPA, untreated 3T3Mut featured a 3-fold decrease in insulin degradation, with a 3-fold increase in intact insulin retroendocytosis. Thus, in NIH-3T3 cells, insulin elicits receptor interaction with multiple PKC isoforms. Interaction of PKC-alpha and/or -delta with the IR appears to control its intracellular routing.

    INTRODUCTION
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The turn on of the insulin signaling mechanism by the insulin receptor (IR)1 involves a complex network of protein-protein interactions (1). Insulin-bound receptors phosphorylate a variety of docking proteins which include the IRS and Shc systems. Once phosphorylated, the docking proteins recruit and activate multiple insulin effectors (1). By employing the IRSs to engage Src homology 2 domain proteins, the IR avoids the stoichiometric constraints encountered by receptors which directly recruit these signaling molecules to their autophosphorylation sites (1, 2). The IRSs widen the connection and the tuning opportunities of the insulin signaling (3). There is evidence, however, that certain insulin bioeffects also follow the direct interaction of the IR with major insulin effectors. These effectors include phosphatidylinositol 3-kinase (4, 5) and, possibly, protein kinase C (PKC) (6, 7).

PKCs represent a family of structurally and functionally related serine/threonine kinases derived from multiple genes as well as from alternative splicing of single mRNA transcripts (8, 9). The individual isoforms differ in their regulatory domains and in their dependence on Ca2+, as well as in their tissue distribution and intracellular localization (10, 11). PKCs appear to play a dual role in the insulin signaling network. First, PKCs control insulin-dependent receptor kinase activation (12-15) and may regulate IRS-1 signaling as well (12, 13). Second, at least in certain cells and tissues, insulin activation of PKCs is required to evoke insulin effects on glucose transport and its intracellular metabolism (16, 17). Current evidence (6) indicates that chimeric receptors consisting of the EGF receptor extracellular domain fused to the cytoplasmic domain of the IR form stable complexes with PKC-alpha following EGF binding. It has also been reported that insulin increases PKC activity in Tyr(P) Ab precipitates from KB cells (22). However, the molecular mechanisms of PKC activation in response to insulin as well as the role of each individual PKC isoform in insulin signal transduction is still unsettled.

In previous reports (23) we demonstrated that Arg1152 and Lys1153 in the regulatory domain of the IR kinase are crucial for enabling IR phosphorylation by PKC. A peptide encoding the receptor sequence surrounding these residues inhibited phosphorylation of IR by PKC. In contrast, a mutant peptide in which the Arg and Lys were substituted by neutral amino acids exhibited no inhibitory effects, suggesting that IR phosphorylation by PKC follows direct IR-PKC interaction (23). In the present work we have shown that insulin controls IR association with PKC-alpha , -delta , and -zeta . In turn, in the NIH-3T3 cells, PKC-alpha and/or -delta association with IR appears crucial for enabling proper intracellular sorting of the receptor to the insulin degradative route.

    EXPERIMENTAL PROCEDURES
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Materials-- Rabbit polyclonal antibodies toward specific PKC isoforms were purchased from Life Technologies, Inc. Ab50 and B10 receptor antibodies were a generous gift from Drs. D. Accili and P. Gorden (National Institutes of Health, Bethesda, MD). Protein electrophoresis reagents were from Bio-Rad. Western blotting and ECL reagents were from Amersham Corp. Media and sera for cell culture were from Life Technologies Inc. All other reagents were from Sigma.

Mutant Cell Clones, Extract Preparations, and PKC Assays-- The NIH-3T3 cell clones expressing the QA mutant insulin receptors have been previously reported (23). For determination of PKC activity, the cells were solubilized with 20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 mg/ml aprotinin, and 25 mg/ml leupeptin (extraction buffer) and then clarified by centrifugation at 10,000 × g for 15 min at 4 °C. Upon protein quantitation, equal aliquots of the extract were added to the lipid activators (10 mM phorbol 12-myristate 13-acetate, 0.28 mg/ml phosphatidyl serine, and 4 mg/ml dioleine, final concentrations), and the 32P/substrate solution (50 mM Ac-MBP(4-14), 20 mM ATP, 1 mM CaCl2, 20 mM MgCl2, 4 mM Tris, pH 7.5, and 10 mCi/ml (3,000 Ci/mM) [gamma -32P]ATP), in the presence or the absence of 25 µM PKC pseudosubstrate inhibitor peptide PKC(19-36) (24). The samples were incubated for 20 min at room temperature and rapidly cooled on ice, and 20-µl aliquots were spotted onto phosphocellulose disc papers (Life Technologies, Inc.). Discs were washed twice with 1% H3PO4, followed by two more washes with water, and disc-bound radioactivity was quantitated by liquid scintillation counting.

Co-precipitation Studies-- Cell extracts were prepared as described above. Samples were precipitated with protein A-Sepharose-bound Ab50 or B10 insulin receptor antibodies, and the immunocomplexes were resuspended either in extraction buffer, for quantitation of PKC activity, or in Laemmli buffer (25), for SDS-polyacrylamide gel electrophoresis protein separation. These proteins were then blotted on nitrocellulose filters, probed with isoform-specific PKC antibodies, and detected by the ECL method as described by the manufacturer. Alternatively, the immunoprecipitations were performed using isoform-specific PKC Abs, followed by immunoblotting and detection with insulin receptor antibodies. Quantitation of the autoradiographs was obtained by laser densitometry.

Insulin Binding, Internalization, and Intracellular Processing-- 125I-Labeled insulin binding and internalization were analyzed as described previously (26). Internalization rates were calculated according to Lund et al. (27). Degraded and intact 125I-insulin in the incubation media or the cell lysates were determined by trichloroacetic acid precipitation as described in Formisano et al. (26).

    RESULTS
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PKC Co-precipitation with the Insulin Receptor-- NIH-3T3 cells expressing human wild-type insulin receptors (3T3hIR cells) were exposed for 30 min to 100 nM insulin or 1 µM TPA. This treatment increased the ability of plasma membrane preparations from the cells to phosphorylate the Ac-MBP(4-14) substrate for PKC by 2- and 10-fold, respectively (Fig. 1, top panel). Insulin effect was accompanied by a similarly sized increase in phosphorylation of the PKC substrate by specific IR Ab precipitates from these same cells (Fig. 1, bottom panel). At variance, substrate phosphorylation by the IR Ab precipitates from TPA-stimulated cells was increased by only 35 ± 3% (value ± S.D.) above the basal levels. Simultaneous preincubation of the cells with both insulin and TPA did not significantly enhanced the effect of insulin alone. Ac-MBP(4-14) phosphorylation by the immunoprecipitates from both basal and insulin and/or TPA-stimulated cells were blocked by the specific PKC inhibitory peptide PKC(19-36), indicating co-precipitation of PKC with the insulin receptor.


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Fig. 1.   PKC activity in membrane preparations and IR Ab precipitates from 3T3hIR cells. Top panel, PKC activity was assayed by incubating the Ac-MBP(4-14) substrate with membrane preparations from basal and insulin- or TPA-stimulated cells, as described under "Experimental Procedures." Bottom panel, cell lysates were immunoprecipitated with Sepharose-bound IR Ab, and PKC activity was assayed in the absence or in the presence of excess PKC(19-36) inhibitory peptide. Data represent the means ± S.D. of four triplicate experiments.

To further investigate the PKC-IR co-precipitation, we immunoblotted the IR Ab precipitates with isoform-specific PKC Abs. In precipitates from insulin-unstimulated cells, PKC-alpha and -delta were detectable at low levels at the expected Mr 80,000 (Fig. 2). A faint band was also revealed by PKC-zeta Abs, corresponding to the predicted Mr 65,000 (Fig. 2C). PKC-epsilon was undetectable in these IR Ab precipitates although clearly recognizable in total lysates of the cells. In contrast PKC-gamma , -beta 1, and -beta 2 could not be demonstrated in either the cell lysates or the IR precipitates (not shown). Upon stimulation of the cells with 100 nM insulin for 30 min, the levels of PKC-alpha , -delta , and -zeta detected on blots increased by 7-, 3.5-, and 3-fold, respectively, while PKC-epsilon remained undetectable. Almost identical results were obtained by precipitating the cell lysates with isoform-specific PKC Abs followed by blotting and detection with IR Abs (data not shown). As shown in Fig. 3, the effect of insulin on PKC-alpha , -delta , and -zeta co-precipitation with the IR exhibited very similar dose responses. Insulin effects were well detectable at 10 -10 M, half-maximal at 2-5 × 10 -9, and reached a plateau at 10 -7 M. Differing from dose responses, time courses of the insulin effect on IR-PKC co-precipitation were specific for each PKC isoform. In the case of PKC-alpha , the insulin effect was maximal after 30 min of exposure, remaining unchanged for up to 2 h, and then vanishing in 16 h (Fig. 4, top panel), while, in the case of PKC-delta , the effect reached a maximum after 15 min and disappeared by 60 min (Fig. 4, middle panel). For PKC-zeta , the insulin effect was diphasic, with an early spike (maximum in 5 min), which returned to basal levels in 15 min and was followed by a more sustained increase lasting for up to 16 h (Fig. 4, bottom panel).


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Fig. 2.   Co-precipitation of IR with individual PKC isoforms. 3T3hIR cells were exposed to 100 nM insulin for 30 min as indicated. Lysates from the cells were then precipitated with Sepharose-bound IR Ab (alpha -IR precip.) separated by SDS-polyacrylamide gel electrophoresis, and immunoblotted with specific PKC-alpha , -delta , -epsilon , and -zeta Abs. Aliquots of the cell lysates were directly immunoblotted with no previous precipitation (total lysates). The experiment shown is representative of four independent experiments.


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Fig. 3.   Insulin dose-response curve of IR-PKCs co-precipitation. 3T3hIR cells were incubated with the indicated concentrations of insulin. Cell extracts were then precipitated with Sepharose-bound IR Ab and blotted with PKC-alpha (filled circles), PKC-delta (open triangles), and PKC-zeta Abs (open circles) as described under "Experimental Procedures." Detection was achieved by ECL and autoradiography, and the intensity of the bands was quantitated by laser densitometry. Data represent the means of three independent experiments with each individual PKC isoform.


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Fig. 4.   Time course of IR-PKCs co-precipitation. 3T3hIR cells were exposed to 100 nM insulin for the indicated times. Cell extracts were then precipitated with Sepharose-bound IR Ab and blotted with PKC-alpha (top panel), PKC-delta (middle panel), and PKC-zeta Abs (bottom panel). After ECL and autoradiography (see "Experimental Procedures"), the intensity of the bands was quantitated by laser densitometry. Each data point represents the mean ± S.D. of three independent experiments.

Effects of PKC Inhibition and Cellular Depletion on IR Routing-- Evidence is available that PKC has an important role in regulating the internalization and degradation of several tyrosine kinase receptors following binding with their specific ligand (6). We therefore addressed the questions whether this is also the case for the IR kinase, and whether specific PKC isoforms are involved in this regulation. To this end, we analyzed insulin-induced IR internalization and intracellular routing following either simultaneous inhibition of PKC-alpha , -delta , and -zeta activities with staurosporine or cell depletion of TPA-sensitive PKCs (PKC-alpha and -delta ) by a 24-h preincubation with 1 µM TPA. Preincubation of the cells with 6 µM staurosporine before insulin stimulation inhibited PKC activity in total cell lysates by almost 55%. Concomitantly, as revealed by immunoblot studies, the levels of PKC-alpha , -delta , and -zeta in IR Ab precipitates were reduced by 3-, 4-, and 10-fold, respectively (Fig. 5, top panel), suggesting that PKC activation is necessary for its association with the insulin receptor. Chronic treatment of the cells with TPA decreased PKC activity by >80% and also reduced recovery of PKC-alpha and -delta in the IR Ab precipitates by 6- and 5-fold, respectively. At variance from the staurosporine however, similar amounts of the PKC-zeta isoform were evidenced in the immunoprecipitates from TPA-treated cells and in the precipitates from untreated cells. Upon entering cells, normal insulin-bound IRs return to the plasma membrane mainly through an intracellular compartment where insulin is detached from the receptor and degraded (26, 28). Small amounts of internalized receptors are also rapidly recycled through a distinct retroendocytotic mechanism (26, 29). Following TPA and staurosporine preincubation, however, the amount of trichloroacetic acid-soluble (degraded) 125I-insulin released by the cells decreased by 66 ± 7 and 32 ± 3%, respectively (Fig. 5, middle panel). These changes were accompanied by 60 ± 5 and 22 ± 4% respective increases in the amount of trichloroacetic acid-precipitable (intact) 125I-insulin released into the medium by TPA- and staurosporine-treated cells, respectively (Fig. 5, bottom panel). No significant change on insulin-induced IR internalization by the cells occurred in either the TPA- or the staurosporine-treated cells (data not shown). Thus, preserved association of the IR with PKC-alpha and -delta but not PKC-zeta correlated with normal receptor intracellular sorting.


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Fig. 5.   Effect of PKC inhibition on IR-PKC coprecipitation and insulin degradation and retroendocytosis. Top panel, 3T3hIR cells were preincubated for either 30 min with 6 µM staurosporine or 24 h with 1 µM TPA and further stimulated with insulin, as indicated. Cell extracts were then precipitated with IR Ab, blotted with PKC-alpha , -delta or -zeta Abs and revealed by ECL. A representative autoradiograph is shown. Middle and bottom panels, cells treated with either staurosporine for 30 min or TPA for 24 h were incubated with 125I-insulin, acid-washed to remove extracellular insulin, and warmed at 37 °C for 30 further min. Aliquots of media were then precipitated with trichloroacetic acid. trichloroacetic acid-soluble radioactivity represents insulin degradation (middle panel), while trichloroacetic acid-precipitable radioactivity represents the intact insulin (bottom panel). Bars represent the means ± S.D. of four triplicate experiments.

IRMut-PKC Interaction and Intracellular Routing-- To further explore the potential for PKC-IR interaction to regulate IR intracellular routing, we have studied the internalization and intracellular sorting of the R1152Q/K1153A double mutant insulin receptor (IRMut). In previous studies, we have shown that this receptor is not phosphorylated by PKC, but responds to insulin with auto- and substrate phosphorylation, and transduction of metabolic and mitogenic responses (23). At variance with the 3T3hIR cells, insulin exposure did not increase PKC activity in plasma membranes from cells expressing the mutant receptors (3T3Mut; Fig. 6, top panel). Based on IR-PKC co-precipitation, the amounts of PKC-alpha , -delta , and -zeta that associated with the insulin-activated IRs were, respectively, 10-, 7-, and 3-fold less in NIH-3T3 cells expressing IRMut than in the 3T3hIR cells (Fig. 6, bottom left). The levels of the each individual PKC isoform in total cell lysates were identical in the two cell types, however (Fig. 6, bottom right), indicating that IRMut was unable to properly associate with and activate PKC following insulin binding, despite normal levels of these kinases in the cells. Based on the appearance of trypsin-resistant insulin binding (intracellular receptors), the IRMut underwent rapid and time-dependent internalization in response to insulin, superimposable to that of the wild-type IR (data not shown). Preincubation with 100 nM insulin for 0.5, 1, and 16 h also reduced subsequent 125I-insulin binding by 20, 28, and 31% in the 3T3Mut cells and by 19, 25, and 28% in the 3T3hIR cells, indicating that the mutant receptor undergoes normal insulin-dependent down-regulation as well (Fig. 7, top panel). However, insulin degradation levels were reduced by 3-fold in the 3T3Mut cells as compared with 3T3hIR cells (Fig. 7, middle panel). Conversely, intact 125I-insulin release into the medium was 3-fold greater in the 3T3Mut than in the 3T3HIR cells (Fig. 7, bottom panel). Thus, similar to wild-type IR in PKC-depleted cells with IRMut, lack of interaction with PKCs is accompanied by a preferential receptor convey to the cell surface through the retroendocytotic rather than the insulin-degradative route.


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Fig. 6.   Membrane PKC activity and insulin-induced IR-PKC co-precipitation in 3T3Mut cells. Top, PKC activity was assayed in membrane preparations from basal and insulin-stimulated 3T3Mut cells as described in the legend to Fig. 1. Bottom, 3T3hIR and 3T3Mut cells were exposed to 100 nM insulin as described under "Experimental Procedures." Cell extracts were either precipitated with IR Ab and blotted with PKC-alpha , -delta and -zeta Abs (alpha -IR precip.) or directly blotted with the specific PKC Abs (total lysates) as described in Fig. 2. A representative experiment is shown.


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Fig. 7.   Receptor down-regulation, insulin degradation, and retroendocytosis in 3T3Mut cells. Top panel, 3T3hIR and 3T3Mut cells were preincubated with 100 nM insulin for the indicated times and then thoroughly washed to remove all free and cell-surface bound insulin. 125I-Insulin binding was subsequently determined as described under "Experimental Procedures." Data represent the means ± S.D. of at least three triplicate experiments. Insulin degradation (middle panel) and retroendocytosis (bottom panel) were measured in 3T3hIR and 3T3Mut cells, as described in Fig. 5. Bars represent the means ± S.D. of four triplicate experiments.

    DISCUSSION
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PKC activation in response to insulin has been reported in several tissues and cell types (18, 30). However, neither the molecular mechanisms by which the activation occurs nor the functional role of PKC in the insulin signaling system have been completely elucidated. In the present work, we have shown that, in NIH-3T3 cells expressing human IRs, insulin induces membrane recruitment of PKC-alpha , -delta , and -zeta , but not of the -epsilon isoforms. PKC-alpha , -delta , and -zeta recruitment in response to insulin linearly correlated with their appearance in the IR Ab precipitates. This observation is consistent with a previous report by Liu and Roth (22) in which the recovery of an insulin-stimulated PKC activity in phosphotyrosine Ab precipitates from insulin-stimulated cells was described. While eliciting a 10-fold greater PKC translocation to the plasma membrane, TPA was less effective than insulin in promoting co-precipitation of PKC-alpha and -delta with the IR and was unable to promote that of PKC-zeta . Neither EGF or platelet-derived growth factor elicited any significant IR-PKC co-precipitation (not shown). It appears therefore that, in NIH-3T3 cells, a specific association of PKC-alpha , -delta , and -zeta with IR occurs in response to insulin.

We have further observed that, in NIH-3T3 cells, inhibition of PKC-alpha , -delta , and -zeta activity by staurosporine also inhibited insulin-induced PKC-IR association, suggesting that the insulin-induced activation of these PKC isoforms is necessary to allow their subsequent association with the receptor. Staurosporine pretreatment of the cells also increased the routing of the insulin-receptor complexes through the retroendocytotic pathway, thus shifting the internalized receptors from the degradative to the retroendocytotic compartment. This change in receptor sorting was not accompanied by alterations in insulin-induced receptor internalization or down-regulation, indicating that PKC-IR association is crucial in specifically controlling the intracellular sorting of the receptor following insulin-dependent internalization. Consistent with these data and with the relevance of a direct PKC-IR interaction for proper sorting of the receptor, the IRQA mutant, which is unable to interact with PKC-alpha , -delta , and -zeta , but is normally responsive to insulin in term of auto- and substrate phosphorylation, kinase activation, and signaling (23), exhibited identical abnormalities in intracellular cell sorting as the wild-type human IR in staurosporine-treated cells. It is possible that the abnormal routing of IRQA is caused by decreased phosphorylation by one or more PKC isoforms because the QA double mutation impairs receptor phosphorylation by these kinases both in vitro and in intact cells (23). The altered intracellular routing of IRQA might also be directly caused by its decreased ability to activate PKC. This is less likely, however, because TPA activation of PKC in IRQA-expressing cells does not restore normal receptor routing (data not shown). A different mutant (IRQK) also exhibits similar abnormalities in the intracellular sorting as the IRQA (26). Similar to IRQA, IRQK is unable to interact with PKC-alpha , -delta , and -zeta upon insulin exposure (not shown). At variance with the IRQA, however, IRQK does not respond to insulin in terms of autophosphorylation and kinase activation (31), indicating that receptor phosphorylation and kinase activation are not sufficient for enabling receptor-PKC association.

Pretreatment of NIH-3T3 cells with TPA before insulin stimulation depleted the cells of PKC-alpha and -delta , but had no effect on PKC-zeta levels. Accordingly, the levels of PKC-zeta co-precipitating with IRs following insulin stimulation were identical in cells treated with TPA or not, while the levels of PKC-alpha and -delta were greatly reduced. Nevertheless, in TPA-pretreated cells, we observed a shift of the internalized insulin-IR complexes from the degradative toward the retroendocytotic route that was almost 2-fold more pronounced than that caused by staurosporine. We suggest, therefore, that PKC-alpha and/or -delta but not PKC-zeta are responsible for PKC control of IR routing in these cells. Based on the findings reported in the present work, PKC-alpha appears the most likely candidate for this regulatory role. In fact, (i) the time course of insulin-induced IR engagement in the degradative route better correlates with the time course of insulin-dependent PKC-alpha -IR co-precipitation than with that of PKC-delta -IR co-precipitation, and (ii) chronic exposure of the cells to TPA, to which the IR sorting mechanism is extremely sensitive, determines a 3-fold greater depletion of PKC-alpha than -delta from the cells. In addition, consistent with this possibility, Seedorf et al. (6) have recently shown that chimeric receptors engineered with the EGF receptor extracellular domain fused to the cytoplasmic domain of several different tyrosine kinase receptors, including IR, form stable complexes with PKC-alpha upon EGF binding and promote receptor tyrosine kinase internalization and degradation. TPA-sensitive PKCs are known to bind and phosphorylate cytoskeletal proteins such as F-actin (32, 33), talin (34), and other cytoskeleton-associated proteins (35, 36), as well as proteins regulating vesicle formation and trafficking such as dynamin I (37). It is tempting therefore to speculate that, once bound to the IR, PKC-alpha might control the IR intracellular routing by interacting with specific cytoskeletal elements.

Evidence is available that PKCs are required for insulin-induced regulation of gene expression (18, 38), protein synthesis (30), glucose uptake (18, 20, 21), and pyruvate dehydrogenase activity (19). Previous studies have also demonstrated an important role of the PKC system in controlling the IR kinase and signaling in several physiological (39) and pathological (40) conditions. By describing the role of PKC in controlling the IR intracellular sorting, the findings in the present work indicate the existence of an additional step where PKCs may control the function of the insulin receptor and thus insulin action.

    ACKNOWLEDGEMENTS

We are grateful to Drs. E. Consiglio and G. Vecchio for their continuous support and advice during the course of this work, to Drs. P. Gorden and D. Accili (National Institutes of Health, Bethesda, MD) for generously donating anti-insulin receptor antibodies. We also thank Dr. M. Bifulco for helpful discussions and Dr. D. Liguoro for the technical help.

    FOOTNOTES

* This work was supported in part by the Biomed2 Program of the European Community, Grant BMH4-CT-0751 (to F. B.), Telethon Grant E.554 (to F. B.), a grant of the Associazione Italiana per la Ricerca sul Cancro (AIRC) (to P. F.), the Ministero dell' Università e della Ricerca Scientifica, and the Progetto Finalizzato Biotecnologie del CNR.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The authors dedicate this paper to the late Professor Gaetano Salvatore.

Dagger Recipient of a fellowship of the Federazione Italiana per la Ricerca sul Cancro (FIRC).

§ To whom correspondence should be addressed: Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano" Federico II University of Naples Medical School, Via Pansini, 5, 80131 Naples, Italy. Tel.: 39 81 7463248; Fax: 39 81 7701016; E-mail: beguino{at}unina.it.

1 The abbreviations used are: IR, insulin receptor; IRS, insulin receptor substrate; Ab, antibody; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; EGF, epidermal growth factor.

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Abstract
Introduction
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Discussion
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