INTRODUCTION

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Phosphoinositide (PI) 3-kinase, a lipid kinase composed of an SRC homology 2 (SH2) domain-containing regulatory subunit and a 110-kDa catalytic subunit, catalyzes phosphorylation of the D3 position of PIs (46, 48). This enzyme was first identified complexed with SRC kinase and the middle T antigen of polyomavirus and was later found to associate with various tyrosine-phosphorylated proteins in response to stimulation of cells with growth factors or cytokines (46, 48). Activation of PI 3-kinase, either by targeting of the enzyme to the plasma membrane (27) or as a consequence of direct interaction between the SH2 domain of the regulatory subunit and phosphorylated tyrosine residues present within specific motifs (5), results in the triggering of various important biological actions. Thus, with the use of either a dominant negative protein that blocks the interaction between PI 3-kinase and tyrosine-phosphorylated proteins (21, 33, 41) or pharmacological inhibitors of the enzyme, such as wortmannin or LY294002 (12, 47), PI 3-kinase has been shown to participate in intracellular trafficking, organization of the cytoskeleton, cell growth and transformation, prevention of apoptosis, cell differentiation, and several metabolic actions of insulin (11, 17, 23, 24, 33, 41, 46, 48). However, relatively little is known about the downstream effectors of PI 3-kinase that mediate each of these biological effects.

Recently, two types of serine-threonine kinase have been shown to act downstream of PI 3-kinase. One of these kinases is Akt (also known as protein kinase B), which contains a pleckstrin homology domain. Several growth factors induce rapid activation of Akt in intact cells (10, 16, 28). However, the mechanism of Akt activation in cells is not fully understood. Phosphatidylinositol 3,4-bisphosphate, one of the products of PI 3-kinase activity in vivo, binds directly to the pleckstrin homology domain of Akt and stimulates kinase activity in vitro (18, 28), suggesting that Akt may be directly activated by this lipid in intact cells. On the other hand, activation of Akt was shown to parallel its phosphorylation status, and replacement of serine and threonine residues that are sites of ligand-induced phosphorylation in the enzyme abolished its activation (3, 31). These data, together with the identification of a kinase that phosphorylates and activates Akt (4, 44), suggest that Akt activity is regulated mainly by phosphorylation. Whichever mechanism is primarily responsible for regulation of Akt, the observation that pharmacological or molecular biological inhibitors of PI 3-kinase prevent Akt activation in intact cells (10, 16, 26, 29) indicates that Akt acts downstream of PI 3-kinase.

The protein kinase C (PKC) family of serine-threonine kinases comprises at least 11 members (37). One class of PKC isozymes, termed atypical PKC, is distinct from other members of this family in several respects. For example, the atypical PKC enzymes, consisting of PKC and PKC, are not activated by diacylglycerol or phorbol ester, whereas members of the other two classes, conventional PKC and novel PKC, are activated by these reagents both in vitro and in vivo (1, 2, 36-38, 40, 49). PKC, also identified as an atypical PKC isozyme, is the human counterpart of mouse PKC (42). Evidence suggests that atypical PKC is the second type of serine-threonine kinase that acts downstream of PI 3-kinase. PKC is activated in vitro by phosphatidylinositol 3,4,5-trisphosphate (36), another product of PI 3-kinase activity (46). When expressed in 3Y1 fibroblasts or HepG2 hepatoma cells expressing various mutant platelet-derived growth factor receptors, PKC contributed to trans activation of the tetradecanoyl phorbol acetate-responsive element in response to platelet-derived growth factor or epidermal growth factor in a PI 3-kinase-dependent manner (2). Furthermore, the activity of atypical PKC stimulated by either insulin or bacterial lipopolysaccharide was shown to be inhibited by either a pharmacological inhibitor or a dominant negative mutant of PI 3-kinase (22, 34, 43). All of these observations indicate that atypical PKC isozymes are downstream effectors of PI 3-kinase.

Stimulation of glucose uptake into skeletal muscle and adipocytes is one of the most important actions of insulin. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, an essential step for glucose uptake in muscle and adipocytes, were markedly attenuated by pharmacological inhibitors or a dominant negative mutant of PI 3-kinase (13, 39, 41). Furthermore, a constitutively active mutant of PI 3-kinase promoted glucose uptake and translocation of GLUT4 in quiescent adipocytes (20). These observations suggest a central role for PI 3-kinase in regulation of glucose transport. However, a downstream effector of PI 3-kinase that mediates stimulation of glucose uptake has not been identified.

Although Akt and atypical PKC act downstream of PI 3-kinase, it remains unclear which actions of PI 3-kinase are mediated by which protein kinase. It is also not known whether Akt and atypical PKC act in the same signaling pathway or whether they transmit signals through different pathways. To address these important questions, we have investigated the roles of these protein kinases in insulin-stimulated glucose uptake. We recently showed that a mutant Akt in which the sites of ligand-induced phosphorylation were replaced by alanine acts in a dominant negative manner (26). Because this mutant did not affect insulin-induced glucose uptake (26), we have now examined the possible role of atypical PKC as a downstream effector of PI 3-kinase in this process. We have investigated the effects of dominant negative mutants and a constitutively active mutant of PKC in order to determine whether this enzyme participates in the regulation of glucose uptake by insulin in 3T3-L1 adipocytes. We have also investigated whether Akt and PKC function in the same or different signaling pathways.

	MATERIALS AND METHODS

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Cells and antibodies. 3T3-L1 preadipocytes were maintained and induced to differentiate into adipocytes as described previously (41). To establish CHO-IR cells that express (in addition to human insulin receptors) tagged PKC (CHO-IR/PKC cells), we transfected CHO-IR cells with both pSV40-high (which confers resistance to hygromycin) and an SRD vector encoding T7 epitope-tagged mouse PKC (2). Transfected cells were selected and cloned as described previously (25). CHO cells that express FLAG epitope-tagged Akt (CHO-Akt cells) have been described previously (26). To prepare a construct encoding mouse PKC tagged with the T7 epitope, we performed PCR with a sense primer (5'-AAG GCC ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CCC AGC AGG ACC GAC), an antisense primer (5'-GAG GTC GAA GTC TTG CAG CCC), and a full-length mouse PKC cDNA as the template. The resulting PCR product, containing nucleotides 4 to 762 of PKC cDNA fused with a DNA sequence encoding the T7 epitope, was digested with PstI and ligated to the PstI site of cDNA encoding mouse PKC. The resulting cDNA construct encoded T7 epitope-tagged mouse PKC and was subcloned into an SR expression vector.

RT-PCR. Transcripts encoding PKC or PKC were detected by reverse transcription (RT)-PCR analysis. Complementary DNA was synthesized, with the use of a FastTrack 2.0 kit and a cDNA Cycle kit (Invitrogen), from ~300 µg of polyadenylated RNA extracted from 3T3-L1 adipocytes or total RNA extracted from mouse brain. PCR was then performed with 1/10 of the resulting cDNA as the template and with primers that correspond to nucleotides 247 to 265 and 576 to 595 of mouse PKC cDNA or to nucleotides 145 to 164 and 700 to 719 of mouse PKC cDNA. The amplification protocol comprised 30 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min.

Construction of and infection with adenovirus vectors. Various PKC mutant constructs are shown in Fig. 1. Complementary DNAs encoding wild-type mouse PKC (WT) and a kinase-deficient mutant in which glutamate is substituted for Lys²⁷³ in the kinase domain (KD) were as described previously (2). To prepare constructs encoding PD and PDKD, hemagglutinin (HA) epitope-tagged, NH₂-terminal-deletion mutants of PKC, we performed PCR with sense (5'-TTA GGT ACC ATG TAC CCA TAC GAT GTT CCG GAT TAC GCT AGC CTC GCC AAA CGT TTC AAT AGG CGC) and antisense (5'-GAT ACC ACT CTC CCT GGT) primers and wild-type mouse PKC cDNA as a template. The resulting PCR product, containing nucleotides 442 to 735 of PKC cDNA fused with a DNA sequence encoding the HA epitope, was digested with EcoT22I and ligated to the EcoT22I site of cDNA encoding either WT or KD. To construct NKD, we performed PCR with sense (5'-TTA GGT ACC ATG TAC CCA TAC GAT GTT CCG GAT TAC GCT AGC CTC TCG TCC AGT CTA GGT CTG CAG) and antisense (5'-GAT AGA ATG CAG CCC GAC) primers and KD cDNA as a template. The resulting product, containing nucleotides 742 to 966 of PKC cDNA fused with a DNA sequence encoding the HA epitope, was digested with ClaI and fused with the ClaI site of cDNA encoding WT. Complementary DNA encoding the wild-type or various mutant proteins was subcloned into pAxCAwt (35), and adenovirus vectors containing these cDNAs were generated by transfecting 293 cells with the corresponding pAxCAwt plasmid together with DNA-terminal protein complex (35), as described previously (41). The resulting vectors were termed AxCAWT, AxCAKD, AxCAPD, AxCAPDKD, and AxCANKD, respectively.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Structures of various mutants of PKC, KD, NKD, and PDKD contain a Lys273-to-Glu mutation (K273E). PD, PDKD, and NKD contain the HA epitope at their truncated NH2 termini. The first and last amino acids of each protein are numbered. PD, CR, and CD, pseudosubstrate, cysteine-rich, and catalytic domains, respectively.

Kinase assays. CHO cells or 3T3-L1 adipocytes were deprived of serum for 16 to 20 h, incubated in the absence or presence of insulin, and then immediately frozen with liquid nitrogen. For assay of the kinase activity of endogenous or T7 epitope-tagged PKC, the frozen cells were lysed as described previously (2) and the lysate was centrifuged (15,000 × g for 20 min). The protein concentration in the resulting supernatants was determined with the use of the bicinchoninic acid protein assay reagent (Pierce), and equal amounts of protein were subjected to immunoprecipitation with polyclonal antibodies or MAbs to PKC or with antibodies to the T7 epitope tag. After washing twice with buffer A (50 mM MOPS [morpholinepropanesulfonic acid]-HCl [pH 7.5], 0.5% Triton X-100, 10% glycerol, 0.1% bovine serum albumin, 5 mM EDTA, 5 mM EGTA, 20 mM NaF, 50 mM -glycerophosphate, 2 mM sodium orthovanadate, 2 mM dithiothreitol, 1 µg of leupeptin per ml, and 2 mM phenylmethylsulfonyl fluoride), once with buffer A containing 1 M NaCl, and then once with a solution containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, and 1 mM EGTA, the immunoprecipitates were incubated for 14 min at 30°C with 0.4 µCi of [-³²P]ATP in a reaction mixture (25 µl) containing 35 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 0.5 mM EGTA, 0.1 mM CaCl₂, 40 µM unlabeled ATP, and 30 µM myelin basic protein (MBP) as a substrate. When indicated, phosphatidylserine (PS) (100 µg/ml) was also present in the reaction mixture.

Glucose uptake and translocation of GLUT4. Glucose uptake was assayed as described previously (41). In brief, 3T3-L1 adipocytes were incubated for 16 h in Dulbecco's modified Eagle's medium containing 5.6 mM glucose and 0.5% fetal bovine serum. The cells were washed twice with DB buffer (140 mM NaCl, 2.7 mM KCl, 1 mM CaCl₂, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄ [pH 7.4], 0.5 mM MgCl₂) and then incubated with 100 nM insulin for 15 min, 0.5 µg of growth hormone (GH) per ml for 10 min, or 300 mM sorbitol for 60 min. DB buffer (1 ml) containing bovine serum albumin (1 mg/ml) and 0.1 mM 2-deoxy-D-[1,2-³H]glucose (1 µCi) was added to each well, and after 5 min, the cells were washed and then solublized with 0.1% SDS. The radioactivity incorporated into the cells was measured with a liquid scintillation counter.

	RESULTS

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Expression of PKC in 3T3-L1 adipocytes. Atypical PKC isozymes comprise PKC and PKC. To examine the relative abundances of these two isoforms in 3T3-L1 adipocytes, we prepared antibodies specific for each. T7 epitope-tagged PKC or PKC was transiently expressed in COS7 cells, and cell lysates were subjected to immunoprecipitation with antibodies either to PKC (190), to PKC (170), or to the T7 epitope or with control serum. The immunoprecipitates were then subjected to immunoblot analysis with antibodies to T7 (Fig. 2A). Proteins of ~80 kDa reactive with antibodies to T7 were detected in the immunoprecipitates prepared from the lysates expressing T7-tagged PKC with 190 or with antibodies to T7 and in the immunoprecipitates prepared from the lysates expressing T7-tagged PKC with 170 or with antibodies to T7. These results indicate that 190 and 170 specifically recognize PKC and PKC, respectively. In contrast, polyclonal antibodies generated against a peptide corresponding to the COOH terminus of rat PKC (CT) detected both T7-tagged PKC and PKC (Fig. 2B), indicating that these antibodies recognize both PKC and PKC.

View larger version (49K): [in this window] [in a new window]   FIG. 2.   Expression of PKC, but not PKC, in 3T3-L1 adipocytes. (A) Specificity of antibodies to PKC and PKC. COS7 cells cultured in 6-cm-diameter dishes were transiently transfected, with the use of Lipofectamine (Gibco), with 6 µg of SRD or SR vectors encoding T7 epitope-tagged PKC or PKC. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to T7, to PKC (190), or to PKC (170) or with control serum (Cont), and the resulting immunoprecipitates were subjected to immunoblot analysis with antibodies to T7. (B) Recognition of both PKC and PKC by antibodies generated in response to a peptide corresponding to the COOH terminus of rat PKC (CT). COS7 cells transiently transfected as described for panel A were lysed and subjected to immunoprecipitation with either antibodies to T7 or normal mouse globulin (NMG). The resulting immunoprecipitates were then subjected to immunoblot analysis with either antibodies to T7 (lower panel) or CT (upper panel). (C) Analysis of PKC and PKC protein expression in mouse brain and 3T3-L1 adipocytes. Lysates prepared from mouse brain or 3T3-L1 adipocytes were subjected to immunoprecipitation with 190, 170, or control rabbit serum, and the immunoprecipitates were subjected to immunoblot analysis with CT. (D) Analysis of PKC and PKC transcripts in mouse brain and 3T3-L1 adipocytes. PCR was performed with primers specific for mouse PKC or PKC cDNA and with either the indicated concentration of full-length PKC or PKC cDNA subcloned into the SRD vector (left panel) or cDNA synthesized from RNA extracted from either mouse brain or 3T3-L1 adipocytes (right panel). Amplification products were analyzed by agarose gel electrophoresis and ethidium bromide staining. Lane M, molecular size standards. Data are representative of those from three experiments.

Activation of PKC by insulin in a PI 3-kinase-dependent manner. 3T3-L1 adipocytes were incubated for various times in the absence or presence of 100 nM insulin, lysed, and subjected to immunoprecipitation with control serum or polyclonal antibodies to PKC generated against a peptide corresponding to amino acids 197 to 213 of mouse PKC (197). These antibodies were previously shown not to cross-react with PKC (1). Kinase activity in the immunoprecipitates was then assayed with MBP as a substrate. The kinase activity precipitated by 197 was markedly greater than that precipitated by control serum (Fig. 3B). Activation of PKC was evident within 3 min of exposure of cells to insulin; the activity was maximal (about three times that of the basal value) at 5 min and remained increased at 10 min (Fig. 3A). Prior treatment of the cells with 100 nM wortmannin or infection of the cells with AxCAp85, an adenovirus vector that encodes a dominant negative mutant of PI 3-kinase (41), prevented insulin-induced activation of PKC (Fig. 3B).

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Stimulation of PKC activity by insulin in 3T3-L1 and CHO cells. (A) Time course of insulin stimulation of PKC activity in 3T3-L1 adipocytes. 3T3-L1 cells cultured in 6-cm-diameter plates were incubated in the presence of 100 nM insulin for the indicated times, after which the cells were lysed and subjected to immunoprecipitation with polyclonal antibodies to PKC (197). The resulting immunoprecipitates were then assayed for kinase activity with MBP as a substrate. Data are expressed relative to the activity at zero time. (B) Effects of inhibitors of PI 3-kinase on insulin stimulation of PKC activity in 3T3-L1 adipocytes. 3T3-L1 cells were incubated in the absence or presence of 100 nM wortmannin for 20 min or were infected at the indicated MOI (PFU per cell) with an adenovirus vector encoding p85 (AxCAp85), and they were then incubated in the absence or presence of 100 nM insulin for 5 min. Cells were then lysed and subjected to immunoprecipitation (IP) with either 197 or normal rabbit serum (NRS). Immunoprecipitates were assayed for PKC activity as described for panel A. Data are expressed relative to the activity of control 197 precipitates. (C) Insulin stimulation of PKC activity in transiently transfected CHO-IR cells. CHO-IR cells were transiently transfected with constructs encoding T7-tagged wild-type PKC or KD. After 48 h, the cells were deprived of serum, incubated in the absence or presence of 100 nM insulin for 3 min, and then lysed. The lysates were subjected to immunoprecipitation with antibodies to T7, and the immunoprecipitates were subjected either to immunoblot analysis with antibodies to T7 (upper panel) or to the PKC kinase assay (lower two panels). (D) Effect of p85 on insulin-stimulated PKC activity in CHO-IR/PKC cells. CHO-IR/PKC cells were infected with AxCAp85 at the indicated MOI (PFU per cell). Cells were then incubated in the absence or presence of 100 nM insulin for 3 min, after which cell lysates were subjected to immunoprecipitation with antibodies to the T7 epitope and PKC activity was assayed in the resulting precipitates. Data are expressed relative to the activity of precipitates from uninfected cells not exposed to insulin. Quantitative data in panels A, B, and D are means ± standard errors from three experiments, and those in panel C are means from two experiments.

Dominant negative effects of kinase-defective mutants of PKC on insulin-induced activation of PKC. To investigate the roles of PKC in cells by specifically inhibiting the activity of the endogenous enzyme, we have constructed adenovirus vectors that encode dominant negative mutants of PKC. Infection of cells with an adenovirus (AxCAKD) encoding KD revealed that the mutant enzyme was not activated either by insulin treatment in intact cells (Fig. 4A) or by addition of PS to the kinase assay mixture (Fig. 4B); PS stimulated the activity of the wild-type enzyme ~2.5-fold. Infection of CHO-IR/PKC cells with AxCAKD resulted in inhibition of insulin-induced activation of PKC (measured in immunoprecipitates prepared with antibodies to the T7 epitope) in an MOI-dependent manner (Fig. 5A), without an effect on the amount of T7 epitope-tagged PKC in the cells (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Effects of insulin in vivo and PS in vitro on the kinase activities of various PKC mutants. (A) 3T3-L1 adipocytes cultured in six-well plates were infected (or not) with adenovirus vectors encoding PD (PD), KD (KD), or NKD (NKD) at an MOI of 150, 30, or 50 PFU/cell, respectively. The cells were then incubated in the absence or presence of 100 nM insulin for 5 min, lysed, and subjected to immunoprecipitation with a MAb to PKC. The immunoprecipitates were assayed for PKC activity. (B) KB cells cultured in six-well plates were infected (or not) with adenovirus vectors encoding WT (WT), KD (KD), PD (PD), or NKD (NKD) at an MOI of 1, 3, 15, or 5 PFU/cell, respectively; these MOIs resulted in the expression of similar amounts of PKC proteins. The cells were lysed, the total lysates were subjected to immunoprecipitation with a MAb to PKC, and the immunoprecipitates were assayed for PKC activity in the absence or presence of PS (100 µg/ml). Data are means ± standard errors from three experiments.

View larger version (42K): [in this window] [in a new window]   FIG. 5.   Effects of KD and NKD on insulin-induced activation of PKC. (A and B) CHO-IR/PKC cells were infected with AxCAKD (A) or AxCANKD (B) at the indicated MOI (PFU per cell), incubated in the absence or presence of 100 nM insulin for 5 min, and lysed. The total lysates were subjected to immunoprecipitation with a MAb to the T7 epitope, and the immunoprecipitates were then assayed for PKC activity. (C) 3T3-L1 adipocytes were infected with AxCANKD at the indicated MOI (PFU per cell), incubated in the absence or presence of 100 nM insulin for 5 min, and lysed. The total lysates were subjected to immunoblot analysis with a MAb to PKC (lower panel) or to immunoprecipitation with polyclonal antibodies to PKC (197); the immunoprecipitates were then assayed for PKC activity (upper panel). Quantitative data are expressed as fold stimulation relative to uninfected cells not exposed to insulin and are means ± standard errors from three experiments.

Inhibition of insulin-stimulated glucose uptake and GLUT4 translocation by dominant negative mutants of PKC. We next examined the effects of the dominant negative mutants of PKC on insulin-stimulated glucose uptake and translocation of GLUT4 in 3T3-L1 adipocytes. Cells were infected with either AxCAKD or AxCANKD and then assayed for insulin-induced glucose uptake. Overexpression of either KD or NKD resulted in a dose-dependent inhibition of insulin-stimulated glucose uptake (Fig. 6A and B). Basal glucose transport was not affected by infection of cells with the viruses encoding either of the dominant negative mutants of PKC (Fig. 6A and B). The amounts of GLUT4 protein, assessed by immunoblot analysis, in infected and noninfected cells were also similar (Fig. 7). Glucose uptake stimulated by either GH or hyperosmolarity (300 mM sorbitol) was not affected by AxCAKD (Fig. 6C).

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Effects of KD and NKD on glucose uptake in 3T3-L1 adipocytes. (A and B) 3T3-L1 adipocytes were infected with AxCAKD (A) or AxCANKD (B) at the indicated MOI (PFU per cell) and then incubated in the absence or presence of 100 nM insulin for 15 min. Cells were then assayed for glucose uptake. (C) 3T3-L1 adipocytes were infected (or not) with AxCAKD at an MOI of 150 PFU/cell, incubated in the absence or presence of GH (0.5 µg/ml) for 10 min or 300 mM sorbitol for 60 min, and then assayed for glucose uptake. Data are means ± standard errors from three experiments.

View larger version (30K): [in this window] [in a new window]   FIG. 7.   Effects of various PKC mutants on the amount of GLUT4 protein in 3T3-L1 adipocytes. Cells were infected (or not) with AxCAKD, AxCAPD, AxCANKD, or AxCAWT at an MOI of 150 PFU/cell, and total cell lysates were subjected to immunoprecipitation with a MAb to GLUT4. The immunoprecipitates were then subjected to immunoblot analysis with polyclonal antibodies to GLUT4.

View larger version (36K): [in this window] [in a new window]   FIG. 8.   Effect of overexpression of wild-type PKC on the inhibition of insulin-stimulated glucose uptake by NKD. 3T3-L1 adipocytes were infected (or not) with AxCANKD at an MOI of 100 PFU/cell and, after 12 h, with AxCAWT at the indicated MOI. After an additional 36 h, the cells were assayed for glucose uptake. Data are means ± standard errors from three experiments.

View larger version (74K): [in this window] [in a new window]   FIG. 9.   Effects of PKC mutants on translocation of GLUT4 to the plasma membrane of 3T3-L1 adipocytes. Uninfected cells (A and B) or cells that were infected with AxCAKD (C) or AxCAPD (D through F) at an MOI of 150 PFU/cell were incubated in the absence (A through E) or presence (F) of 100 nM wortmannin for 20 min and then in the absence (A, D, and F) or presence (B, C, and E) of 100 nM insulin for 5 min. Plasma membrane fragments were then prepared for immunofluorescence microscopy with antibodies to GLUT4 and tetramethyl rhodamine isothiocyanate-labeled secondary antibodies. Data are representative of those from at least three independent experiments.

Enhancement of glucose transport and GLUT4 translocation by a constitutively active mutant of PKC. To investigate whether activation of PKC is capable of stimulating glucose uptake in 3T3-L1 adipocytes, we prepared an adenovirus vector that encodes a constitutively active mutant of PKC. Because the activity of PKC enzymes is negatively regulated by the pseudosubstrate domain, we constructed a mutant PKC that lacks this domain (PD). As expected, the kinase activity of PD was markedly greater than that of the wild-type enzyme, and this activity was little affected by insulin treatment in vivo or PS in vitro (Fig. 4). Infection of 3T3-L1 adipocytes with a virus (AxCAPD) that encodes this mutant resulted in stimulation of glucose uptake in an MOI-dependent manner (Fig. 10). The extent of the stimulation paralleled the extent of expression of the mutant protein (data not shown). At an MOI of 150 PFU/cell, the extent of the stimulatory effect was similar to that achieved by 100 nM insulin (Fig. 10). PDKD, a mutant PKC that lacks both kinase activity and the pseudosubstrate domain, did not stimulate glucose uptake (data not shown), suggesting that the activation of sugar transport by PD was due to its kinase activity and not to nonspecific effects of viral infection. Furthermore, the amounts of GLUT4 (Fig. 7) and of insulin-stimulated PI 3-kinase activity precipitated with antibodies to phosphotyrosine (data not shown) were not increased by expression of PD. Insulin did not increase glucose uptake further in cells that had been infected with AxCAPD at an MOI of 150 PFU/cell, and treatment of such cells with wortmannin had little effect on glucose uptake (Fig. 10). Moreover, GLUT4 translocation, as assessed by the plasma membrane lawn assay, was stimulated by PD, and this stimulation was not affected by insulin or wortmannin (Fig. 9).

View larger version (36K): [in this window] [in a new window]   FIG. 10.   Effect of PD on glucose uptake in 3T3-L1 adipocytes. Cells were infected with AxCAPD at the indicated MOI (PFU per cell) and incubated in the absence or presence of 100 nM wortmannin for 20 min and then with or without 100 nM insulin for 10 min. Glucose uptake was then assayed. Data are means ± standard errors from three experiments.

Localization of PKC and Akt to different signaling pathways. Finally, we examined whether PKC and Akt, both of which are downstream effectors of PI 3-kinase, act in the same or different signaling pathways. Insulin induced a fivefold increase in Akt activity in 3T3-L1 adipocytes (Fig. 11A). Infection of cells with AxCAKD did not affect insulin-induced activation of Akt, even at an MOI of 150 PFU/cell, a dose sufficient to inhibit insulin-induced glucose uptake by ~50% (Fig. 6A). Infection with AxCAPD did not result in the activation of Akt, even at an MOI of 150 PFU/cell (Fig. 11B), a virus dose sufficient to activate glucose uptake to an extent similar to that achieved by insulin (Fig. 10). Essentially similar results were obtained with CHO cells (data not shown). These observations indicate that Akt does not function downstream of PKC.

View larger version (27K): [in this window] [in a new window]   View larger version (29K): [in this window] [in a new window]   View larger version (26K): [in this window] [in a new window]   FIG. 11.   Localization of PKC and Akt to different signaling pathways. (A and B) Effects of KD (A) and PD (B) on Akt activity in 3T3-L1 adipocytes. Cells were infected with AxCAKD (A) or AxCAPD (B) at the indicated MOI (PFU per cell) and then incubated in the absence or presence of 100 nM insulin for 10 min. Cell lysates were subjected to immunoprecipitation with antibodies to Akt, and the resulting precipitates were assayed for Akt kinase activity. (C and D) Effects of a dominant negative mutant of Akt on insulin-induced activation of Akt (C) and PKC (D) in CHO-Akt cells. Cells were infected at the indicated MOI (PFU per cell) with an adenovirus vector (AxCAAkt-AA) encoding Akt-AA, incubated in the absence or presence of 100 nM insulin for 10 min, and lysed. Lysates were subjected to immunoprecipitation with antibodies to either the FLAG epitope (C) or PKC (D), and the resulting immunoprecipitates were assayed for Akt or PKC activity, respectively. (E and F) Effects of Akt-AA on insulin-induced activation of Akt2 (E) and PKC (F) in 3T3-L1 adipocytes. Cells were infected with AxCAAkt-AA at the indicated MOI (PFU/cell), incubated in the absence or presence of 100 nM insulin for 3 min, and lysed. Lysates were subjected to immunoprecipitation with antibodies to Akt2 (E) or to PKC (F), and the precipitates were assayed for Akt and PKC activity, respectively. Data are means ± standard errors from three experiments.

View larger version (36K): [in this window] [in a new window]   FIG. 12.   Combined effects of Akt-AA and KD on insulin-induced glucose uptake. 3T3-L1 adipocytes were infected with AxCAKD at the indicated MOI, and after 8 h, they were infected (or not) with AxCAAkt-AA at an MOI of 200 PFU/cell. After 40 h, the cells were assayed for insulin-induced glucose uptake. Data are expressed as a percentage of maximal insulin-induced glucose uptake and are means ± standard errors from three experiments.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Evidence suggests that stimulation of glucose transport by insulin is mediated mainly by a pathway triggered by PI 3-kinase (12, 20, 21, 39, 41). Because both Akt and atypical PKC isozymes act downstream of PI 3-kinase, it has been of interest to determine whether insulin-stimulated glucose uptake is mediated through one of these protein kinases or through an as-yet-unknown effector of the lipid kinase. To address this question, we have now investigated the role of an atypical PKC isoform, PKC, in intact cells by specifically inhibiting the activity of the endogenous enzyme. Overexpression of kinase-deficient mutants of atypical PKC has been shown to inhibit various biological actions, including activation of mitogen-activated protein kinase, DNA synthesis, nuclear factor-B-dependent trans activation, and v-ras-induced transformation (7-9, 15), although it is not clear whether such mutants inhibit the activity of the endogenous enzymes.

We have now shown that expression of kinase-defective mutants of PKC (KD and NKD) inhibited insulin-induced activation of both transfected and endogenous PKC. Because the insulin-induced increase in PI 3-kinase activity, measured in immunoprecipitates prepared with antibodies to phosphotyrosine, was not affected by either of these mutant proteins, it appears that they do not inhibit the insulin receptor kinase or subsequent phosphorylation of IRS1; rather, they prevent specific signaling downstream of PI 3-kinase. Normal activation of Akt by insulin in cells expressing KD also supports this conclusion. These observations thus demonstrated that KD and NKD act in a dominant negative manner.

Overexpression of either KD or NKD inhibited insulin stimulation of glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. Basal glucose transport in cells expressing these mutants was similar to that in uninfected cells. Furthermore, glucose uptake induced by either GH or hyperosmolarity attributable to sorbitol, both of which promote glucose transport through a PI 3-kinase-independent mechanism (41), was not affected by KD, suggesting that KD inhibited insulin-induced glucose uptake not by an effect on a component of the transport machinery but by blocking specific signals mediated through PKC. Indeed, the amount of GLUT4 protein in cells infected with AxCAKD or AxCANKD was unchanged.

To investigate further the role of PKC in glucose uptake and translocation of GLUT4, we examined the effects of a constitutively active mutant of PKC. The kinase activity of