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Address correspondence to Jeffrey E. Pessin, Dept. of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651. Tel.: (631) 444-3083. Fax: (631) 444-3022. email: pessin{at}pharm.sunysb.edu
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Abstract |
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Key Words: insulin; signal transduction; adipocyte; lipid raft; compartmentalization
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Introduction |
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In vitro binding assays have indicated that active GTP-bound TC10 can directly interact with a number of potential effectors that were originally identified as binding proteins for Cdc42 and/or Rac (Neudauer et al., 1998; Murphy et al., 1999; Joberty et al., 2000; Lin et al., 2000). In mammalian cells, over 20 potential effector-binding proteins have been identified; however, the functional role of these in specific cellular events appears to be highly dependent upon cell context. For example, although expression of Cdc42 in fibroblasts has marked effects on actin organization, there is no significant change in the adipocyte actin cytoskeleton. In contrast, TC10 expression results in a marked disruption of adipocyte cortical actin organization, leading to an inhibition of insulin-stimulated GLUT4 translocation (Chiang et al., 2001; Kanzaki and Pessin, 2002; Kanzaki et al., 2002). Part of this difference can be accounted for by the distinct spatial compartmentalization of TC10 compared with Cdc42 and other Rho family members. Unlike other Rho family proteins that undergo carboxyl-terminal geranylgeranylation, TC10 contains a CAAX sequence that specifies farnesylation and dual palmitoylation responsible for targeting to plasma membrane lipid raft microdomains (Watson et al., 2001). Thus, the compartmentalization of these proteins implies that functionally relevant downstream effectors must also be spatially restricted to their appropriate sites of action.
In this regard, several reports have implicated atypical PKCs (PKC/
) as direct substrates for the phosphoinositide-dependent protein kinase 1 (PDK1). Insulin activates PDK1 through the generation of phosphatidylinositol (PI)-3,4,5P3 by the stimulation of the type 1A PI 3-kinase (Standaert et al., 1997; Kotani et al., 1998; Bandyopadhyay et al., 1999a). On the other hand, PKC
/
has also been reported to form a quaternary complex with Par6, Par3/ASIP, and activated Cdc42 in various cell types (Joberty et al., 2000; Lin et al., 2000; Noda et al., 2001). The Par proteins were originally identified as proteins involved in asymmetric cell division and polarized growth in the Caenorhabditis elegans development (Etemad-Moghadam et al., 1995; Watts et al., 1996). Par6 is composed of a PDZ (PSD-95/Dlg/ZO-1) domain downstream of a motif that is similar to a Cdc42/Rac-interacting binding (CRIB) domain, and both are apparently required for the association of Par6 with Cdc42. In addition, Par6 and atypical PKCs both contain PB1 (Phox and Bem1) domains that are required for forming heterodimeric complexes (Ponting et al., 2002). Par3, also termed ASIP, contains three PDZ domains and specifically binds to both Par6 and atypical PKCs at cellcell contact sites in fibroblasts and epithelial cells (Izumi et al., 1998; Suzuki et al., 2001). Thus, Par6 and Par3 proteins appear to provide scaffolding functions, linking atypical PKCs and the Rho family small GTPases Cdc42 and Rac. Although it is not known whether TC10 can form a similar signaling complex in vivo, GTP-bound active TC10 has been reported to bind the CRIB domain of Par6 using in vitro binding assays (Joberty et al., 2000). Furthermore, it has been reported that overexpression of Par3 in adipocytes inhibits insulin-induced glucose uptake and GLUT4 translocation (Kotani et al., 2000).
To reconcile the apparent role of PI 3-kinase signaling with the scaffolding function of Par6Par3, we have examined the intracellular compartmentalization, Par6Par3 interaction, and phosphorylation of PKC/
in adipocytes. Our data demonstrate that TC10 stimulates PKC
/
phosphorylation and recruitment to plasma membrane lipid raft microdomains in adipocytes through the Par6Par3 complex. In contrast, activation of PI 3-kinase signaling results in PKC
/
phosphorylation without detectable recruitment to the plasma membrane. Importantly, insulin stimulation of adipocytes results in an identical PKC
/
localization as TC10 activation that is completely distinct from PI 3-kinase activation. Furthermore, insulin-induced phosphorylation of GSK-3ß appears to be mediated not only by the PI 3-kinasePKB pathway, but also by the TC10Par6atypical PKC signaling pathway. Thus, PKC
/
appears to function as a convergent downstream target that can differentiate these two pathways through restricted spatial compartmentalization.
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Results |
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To confirm this observation in vivo, we coexpressed PKC-EGFP with either empty vector, TC10/T31N, or TC10/Q75L in 3T3L1 adipocytes (Fig. 1 C). The expressed PKC
-EGFP was primarily localized to the cytoplasm with no evidence for any membrane association (Fig. 1 C, ac). As previously reported (Kanzaki and Pessin, 2001), the expressed TC10/T31N protein was predominantly localized to the plasma membrane (Fig. 1 C, df). However, there was no significant redistribution of PKC
-EGFP, which remained predominantly cytosolic. TC10/Q75L was also primarily concentrated at the plasma membrane, but in this case there was a marked colocalization and recruitment of PKC
-EGFP to the plasma membrane (Fig. 1 C, gi). In parallel, expression of Par6 resulted in a diffuse cytosolic distribution that was not significantly different when coexpressed with TC10/T31N (Fig. 1 D, af). In contrast, Par6 was recruited to the plasma membrane when coexpressed with TC10/Q75L (Fig. 1 D, gi).
In adipocytes, expression of the guanine nucleotide exchange factor C3G activates TC10 and potentiates the insulin stimulation of GLUT4 translocation (Chiang et al., 2001). Therefore, we activated the endogenous TC10 protein by C3G overexpression and assessed the subsequent recruitment of PKC/
(Fig. 2 A). In intact cells, the expressed PKC
-EGFP protein was primarily cytosolic (Fig. 2 A, ac; arrowhead). However, upon coexpression with C3G there was a marked redistribution of PKC
-EGFP to the plasma membrane (Fig. 2 A, ac; arrow). In addition, pretreatment of the transfected adipocytes with the Rho-specific toxin Clostridium difficile toxin B completely prevented the C3G-stimulated recruitment of PKC
-EGFP (unpublished data).
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Insulin stimulates plasma membrane recruitment of PKC/
and the Par6Par3 complex through TC10 activation
Previous works have demonstrated that insulin stimulation results in the activation of TC10 (Chiang et al., 2001; Watson et al., 2001). Therefore, to determine whether a physiological agonist can also induce the plasma membrane recruitment of the endogenous Par3 and PKC/
proteins, immunofluorescent localization was performed in basal and insulin-stimulated adipocytes (Fig. 3). As previously observed, PKC
/
was distributed throughout the cells with no indication of compartmentalized localization at either low or high magnification (Fig. 3 A, a and b). As expected, insulin stimulation resulted in a distinct PKC
/
translocation to the plasma membrane (Fig. 3 A, c and d). In parallel, Par3 was also distributed throughout the cytosol in the basal state and underwent insulin-stimulated translocation to the plasma membrane (Fig. 3 B, ad).
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To confirm the functional role of Par6 in the insulin-induced plasma membrane translocation of endogenous PKC/
, we next examined the effect of Par6-
CRIB and Par6-DD/AA by confocal fluorescent microscopy (Fig. 9). As previously observed, insulin stimulation resulted in the translocation of PKC
/
in control nontransfected cells (Fig. 9, b, c, e, and f; arrowheads). In contrast, cells expressing either Par6-
CRIB or Par6-DD/AA failed to undergo an insulin-induced translocation of endogenous PKC
/
(Fig. 9, af; arrows). Similarly, endogenous PKC
/
failed to undergo insulin-stimulated plasma membrane translocation in cells expressing TC10/T31N (Fig. 9, gi).
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Finally, we also examined the effect of dominant-interfering Par6 mutants and TC10/T31N on GSK-3ß phosphorylation (Fig. 10 C). The insulin stimulation of GSK-3ß phosphorylation was inhibited by expression of Par6-CRIB and Par6-DD/AA (Fig. 10 C, lanes 16), but not by expression of wild-type Par6 (Fig. 10 C, lanes 7 and 8). Similar to the dominant-interfering Par6 mutants, expression of TC10/T31N also inhibited insulin-stimulated GSK-3ß phosphorylation (Fig. 10 C, lanes 9 and 10). Together, these data demonstrate that at least one pool of GSK-3ß is phosphorylated by PKC
/
through a mechanism requiring TC10 and interactions with the Par6 protein complex.
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Discussion |
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In this regard, recent evidence has demonstrated the presence of two distinct insulin signaling pathways that function in concert to mediate GLUT4 translocation and glucose uptake (Baumann et al., 2000; Chiang et al., 2001; Saltiel and Kahn, 2001). One pathway occurs through the insulin stimulation of IRS protein tyrosine phosphorylation, leading to the association and activation of the type 1A PI 3-kinase (Cheatham et al., 1994; Okada et al., 1994; Corvera and Czech, 1998). The subsequent formation of PI3,4,5P3 recruits other downstream signaling molecules such as PKB and PDK1 to nonlipid raft regions of the plasma membrane through their PH domains (Alessi et al., 1997; Stokoe et al., 1997; Stephens et al., 1998). Although PKC/
does not have a PH domain, PKC
/
can associate with PDK1 and accounts for the subsequent phosphorylation on the activation loop T410/T402 residues (Chou et al., 1998; Le Good et al., 1998). More recently, a second pathway has been proposed that results from the tyrosine phosphorylation of Cbl and its recruitment to lipid raft microdomains through the adaptor proteins APS and CAP (Ribon and Saltiel, 1997; Baumann et al., 2000; Liu et al., 2002). In turn, tyrosine-phosphorylated Cbl engages the CrkIIC3G complex that recruits the C3G guanylnucleotide exchange activity to the lipid raft microdomain regions where TC10 is also compartmentalized (Chiang et al., 2001; Watson et al., 2001, 2003).
The data presented in this report provide an important connection between these pathways by demonstrating that PKC/
is a convergent downstream target of both the IRS PI 3-kinase and Cbl-TC10 signaling cascades. Because insulin activates PDK1 and induces T410/T402 phosphorylation, it has been assumed that PKC
/
is recruited to the plasma membrane by PDK1 (Le Good et al., 1998; Balendran et al., 2000). However, our data demonstrate that TC10-dependent (and not PI 3-kinasedependent) signals are responsible for PKC
/
plasma membrane localization, at least in adipocytes. More precisely, the TC10-dependent recruitment spatially restricts PKC
/
to the large caveolin-positive rosette structures in the plasma membrane of adipocytes. However, this interaction directly results from the association of the Par6Par3PKC
/
complex with activated TC10. This is consistent with the ability of TC10 to bind Par6 in vitro. Similiarly, the highly homologous Rho family member Cdc42 can also form a quaternary complex with Par6, Par3, and atypical PKCs (Joberty et al., 2000). This conclusion is further strengthened by the observation that overexpression of Par3 inhibits insulin-stimulated PKC
/
activation and GLUT4 translocation, presumably by disrupting PKC
/
phosphorylation and/or localization (Kotani et al., 2000).
In addition to PKC/
, PKB is also a downstream target of the PI 3-kinase pathway and phosphorylates substrates with an RXRXXS consensus motif (Lawlor and Alessi, 2001). Although the substrate sites for PKC
/
-dependent phosphorylation are more degenerate (RXS, RXXS, or RXXSXR), they share strong similarity to the PKB substrate recognition motif (Nishikawa et al., 1997). Although serine 9 of GSK-3ß is a consensus PKB phosphorylation site, several reports directly demonstrate that this site can also be phosphorylated by atypical PKCs (Ballou et al., 2001; Oriente et al., 2001). More recently, scratch-induced migration in astrocytes resulted in GSK-3ß phosphorylation through a Cdc42Par6PKC
signaling cascade independent of PKB (Etienne-Manneville and Hall, 2003). Although the physiological significance of TC10Par proteinPKC
regulation of GSK-3ß remains to be determined, our data demonstrate that in adipocytes, GSK-3ß phosphorylation is controlled not only by PKB, but also by PKC
/
through both TC10 and PI 3-kinase signals. Importantly, only the TC10 pathway results in the recruitment of PKC
/
to plasma membrane lipid raft microdomains.
Consistent with this idea, it is becoming increasingly apparent that lipid raft microdomains play a central importance in insulin action, including insulin-stimulated GLUT4 translocation. For example, multiple papers have demonstrated that disruption of these structures using various pharmacological agents or a dominant-interfering caveolin mutant all perturb insulin-stimulated GLUT4 translocation (Nystrom et al., 1999; Watson et al., 2001). More recently, we have reported that TC10 regulates a unique cortical actin structure (caveolin-associated F-actin) in fully differentiated 3T3L1 adipocytes consisting of F-actin spikes emanating from inside of the clustered caveolin-enriched rosette structures (Kanzaki and Pessin, 2002). Together, the data presented in this paper suggest an intriguing hypothesis that the caveolin-enriched lipid raft microdomains might function as important signaling platforms that orchestrate insulin signaling molecules including PKC/
. This hypothesis is also consistent with several reports showing a functional role of atypical PKCs in actin cytoskeleton regulation in other cell types (Gomez et al., 1995; Coghlan et al., 2000).
In summary, the data presented in this paper demonstrate that PKC/
serves as a convergent downstream target for both the PI 3-kinase and TC10 signals, and can be phosphorylated once either of these pathways is activated. Nevertheless, the spatial compartmentalization of PKC
/
is markedly different after activation of these pathways. Moreover, in fully differentiated adipocytes, insulin primarily recruits PKC
/
to the lipid raft microdomains and not to ruffling/lamellipodia regions of the plasma membrane despite coactivation of the PI 3-kinase pathway.
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Materials and methods |
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Cell culture and transient transfection of 3T3L1 adipocytes
Murine 3T3L1 preadipocytes were maintained, differentiated into adipocytes, and transfected by electroporation as described previously (Thurmond et al., 1998). After electroporation, cells were plated on glass coverslips and allowed to recover in complete medium.
Immunofluorescence and image analysis
Transfected and intact adipocytes were washed in PBS and fixed for 20 min in 4% PFA/PBS. The cells were washed briefly in PBS, permeabilized in PBS containing 0.1% saponin and 0.4% BSA for 10 min, and were then blocked in 5% donkey serum (Sigma-Aldrich) for 1 h at RT. Primary and secondary antibodies were used at 1:100 dilutions (unless otherwise indicated) in 0.4% BSA/PBS, and samples were mounted on glass slides with Vectashield® (Vector Laboratories). Cells were imaged using a confocal fluorescence microscope (model LSM510; Carl Zeiss MicroImaging, Inc.). Images were then imported into Adobe Photoshop® (Adobe Systems, Inc.) for processing, and composite files were generated.
Preparation and processing of plasma membrane sheets
Adipocyte plasma membrane sheets were prepared as described previously (Kanzaki et al., 2000). In brief, cells were incubation with 0.5 mg/ml poly-D-lysine for 1 min and then swollen in a hypotonic buffer (23 mM KCl, 10 mM Hepes, 2 mM MgCl2, and 1 mM EDTA, pH 7.5) by three successive rinses. The swollen cells were sonicated, and the bound plasma membrane sheets were fixed with 2% PFA and blocked with 5% donkey serum. The membrane sheets were then incubated with primary antibodies for 90 min at RT. The primary antibodies were detected with Texas redconjugated donkey antimouse antibody and Alexa® 488conjugated donkey antirabbit antibody for 2 h at RT.
Immunoprecipitation and immunoblotting
After experimental treatments, the cells were solubilized in 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.5% saponin, 150 mM NaCl, 2% glycerol, 5 mM sodium fluoride, 1 mM sodium vanadate, 1 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin A. The extracts were centrifuged at 13,000 g for 20 min to remove insoluble material, and 50 µg total protein was resolved by SDS-PAGE followed by immunoblotting and was visualized with the SuperSignal® Chemiluminescence Detection kit (Pierce Chemical Co.). For immunoprecipitation, whole-cell extracts were incubated for 2 h at 4°C with 5 µg monoclonal myc antibody. The samples were then precipitated with protein G PLUS-Sepharose (Santa Cruz Biotechnology, Inc.) and immunoblotted as described above.
Nondetergent sucrose gradient fractionation
3T3L1 adipocytes were either left untreated or were treated with 100 nM insulin for 3, 5, or 10 min. Cells were then washed with ice-cold PBS, rapidly scraped in 0.5 M sodium carbonate buffer (pH 11.0), and homogenized on ice. The homogenates were then sonicated four times for 20 s and combined with a buffer containing 25 mM MES (pH 6.5), 150 mM NaCl, and 250 mM sodium carbonate plus 35% (wt/vol) sucrose. The sample was then loaded on a 535% continuous sucrose gradient and centrifuged at 39,000 rpm in a rotor (model SW41; Beckman Coulter) at 4°C for 19 h.
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Acknowledgments |
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This work was supported by National Institutes of Health research grants DK33823 and DK59291 (to J.E. Pessin), DK60591 and DK61618 (to A.R. Saltiel), and DK61188 (to J.B. Hwang), and by American Diabetes Association grant 1-03-JF-14 (to M. Kanzaki).
Submitted: 27 June 2003
Accepted: 5 December 2003
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