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Molecular Endocrinology, doi:10.1210/me.2003-0253
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Molecular Endocrinology 18 (5): 1287-1300
Copyright © 2004 by The Endocrine Society

Partitioning-Defective Protein 6 Regulates Insulin-Dependent Glycogen Synthesis via Atypical Protein Kinase C

Peter Weyrich, Katja Kapp, Gerhard Niederfellner, Martina Melzer, Rainer Lehmann, Hans-Ulrich Häring and Reiner Lammers

Medical Clinic IV, University of Tübingen, Tübingen 72076, Germany

Address all correspondence and requests for reprints to: Reiner Lammers, Medical Clinic IV, Otfried-Müller-Strasse 10, 72076 Tübingen, Germany. E-mail: reiner.lammers{at}med uni-tuebingen.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The atypical isoforms of protein kinase C (aPKCs) play an important role in insulin signaling and are involved in insulin-stimulated glucose uptake in different cell systems. On the other hand, aPKCs also are able to negatively regulate important proteins for insulin signaling, like phosphatidylinositol 3-kinase and protein kinase B/Akt. To find aPKC-interacting proteins that may promote positive or negative activities of aPKCs, a yeast two-hybrid screen was performed. Partitioning-defective protein 6 (Par6) was detected in human cDNA libraries of different adult insulin-sensitive tissues. Although Par6 is known as an aPKC-interacting protein during development, no role for Par6 in insulin signaling has been reported so far. We therefore studied the effects of Par6 overexpression in C2C12 murine myoblasts. In these cells, Par6 associated constitutively with endogenous aPKCs, and the expression level as well as the activity of aPKCs were increased. Insulin-dependent association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate 1 was hampered and the phosphorylation of Akt/glycogen synthase kinase-3{alpha}/ß was significantly impaired after stimulation with insulin or with platelet-derived growth factor. Consequently, insulin-dependent glycogen synthesis was down-regulated (1.44 vs. 2.24 fold, P < 0.01). We therefore suggest that Par6 acts as a negative regulator of the insulin signal.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOSE UPTAKE AND translocation of the glucose transporter Glut4 are tightly regulated by insulin in skeletal muscle and adipose tissue of mammals. This is essential for maintenance of glucose homeostasis, especially in the postprandial state. In type 2 diabetes mellitus, this regulation is disturbed due to insulin resistance of muscle and adipose tissues, a pathological state with a still unknown molecular basis. A large family of serine-threonine-kinases that is involved in insulin signaling and possibly in insulin resistance states are protein kinases C (PKC). The family of PKC comprises classical PKCs (PKC{alpha}, ßI, ßII, {gamma}), novel PKC (PKC{delta}, {epsilon}, {eta}, {theta}), and the atypical isoforms PKC{zeta} and PKC{iota}/{lambda} (aPKCs). aPKCs are ubiquitously expressed and evolutionarily conserved to a remarkable extent throughout many different species. They are involved in a wide variety of biological processes like cellular proliferation (1), differentiation (2, 3), survival (4), regulation of cytoskeleton (5, 6), and cell polarity (7, 8, 9, 10). Recent data suggest an important role of especially aPKCs in insulin signaling (see Ref.11 for review). After insulin stimulation, autophosphorylation of the cytosolic ß-subunit of the insulin receptor permits the interaction with the adapter proteins insulin receptor substrates 1–4 (IRS), which become tyrosine-phosphorylated and subsequently allow the recruitment of the p85-subunit of phosphatidylinositol 3-kinase (PI3K) via its src-homology-2 domains. The catalytical p110-subunit of PI3K then generates 3-phosphatidylinositol derivatives in the plasma membrane, which finally activate aPKCs either directly or via the membrane-associated 3-phosphoinositide-dependent protein kinase 1 (PDK1), which phosphorylates Thr410/403 in the activation loop of PKC{zeta}/{iota}, respectively. The functional relevance of aPKCs for insulin signaling and glucose transport was shown both by studies with specific inhibitors for aPKCs (12, 13, 14) or by overexpression of wild-type, constitutively active, or dominant-negative mutants of aPKCs in different cell systems (13, 15, 16, 17, 18, 19). Recently, insulin-induced association of PKC{zeta} with a Glut4 positive vesicular compartment has been described in a rat skeletal muscle model, where PKC{zeta} phosphorylates the vesicle-associated membrane protein 2 located on Glut4 containing vesicles (19). However, the exact mechanism linking aPKC activation to subsequent Glut4 translocation remains still elusive.

A negative regulatory function of aPKCs in the insulin-signaling cascade also has been reported. aPKCs are able to phosphorylate IRS1 on still unknown Ser/Thr residues. Ser/Thr phosphorylation of IRS1 plays a dual role as it either enhances or terminates the insulin signal. It was shown that aPKCmediated phosphorylation of IRS1 accelerates the rate of Tyr dephosphorylation of IRS1, and thereby down-regulates both the IRS1·p85 association and the activity of PI3K in rat hepatoma Fao cells (20), rat adipose tissue cells, and NIH-3T3 fibroblasts (21). Thus, aPKCs themselves provide an autoregulatory negative feedback mechanism for PI3K-induced aPKC-activation and insulin signaling.

Another molecule involved in insulin signaling and negatively regulated by aPKCs is protein kinase B (Akt). There is considerable evidence suggesting an important role of Akt for insulin-induced glucose uptake as well as glycogen synthesis (see Refs.22 and 23 for review). Similar to aPKCs, Akt becomes activated after insulin stimulation through 3-phosphoinositide-mediated recruitment to the plasma membrane via its amino-terminal pleckstrin homology (PH) domain and subsequent phosphorylation by PDK1 (24). PKC{zeta} is able to associate with its regulatory domain to the domain of Akt (25). Different studies revealed that through this interaction, PKC{zeta} confers an inhibitory effect on Akt after stimulation with platelet-derived growth factor (PDGF) (26), epidermal growth factor or lysophosphatidic acid (27). The exact mechanism of this inhibition remains unclear, but it was shown by Doornbos and co-workers (26) that a kinase-inactive mutant of PKC{zeta} fails to inhibit Akt, although this mutant still interacts with Akt to the same extent as wild-type PKC{zeta}. By contrast, wild-type PKC{zeta} was able to even inhibit a constitutively active mutant of Akt (Thr308Asp/Ser473Asp) in quiescent cells. This led to the conclusion that PKC{zeta} exerts its inhibitory function on Akt most likely by phosphorylation of Thr308/Ser473-independent sites. A possible relevance of this PKC{zeta}-mediated Akt inhibition in diabetes emerged from studies performed on the role of the lipid second messenger ceramide. Intracellular ceramide is generated upon TNF{alpha}-mediated activation of sphingomyelinase (28) and upon exposure to free fatty acids by de novo synthesis (29). One mechanism by which ceramide is believed to cause insulin resistance is down-regulation of insulin-induced Akt kinase activity (30, 31, 32). This down-regulation depends on the interaction of Akt with PKC{zeta} (33) and activation of aPKCs, which also is mediated by ceramide (34, 35). In conclusion, current experimental evidence supports both positive and negative regulatory effects of aPKCs within the insulin-signaling cascade.

In the present study, we performed a yeast two-hybrid screen with different regions of PKC{zeta} or PKC{iota} as baits to find new interacting proteins which may modulate the function of aPKCs in insulin-sensitive tissues. Among others, we found partitioning-defective protein 6 (Par6), which is already known as an aPKC-interacting protein. Hitherto, functions of Par6 mainly have been ascribed to developmental processes in various metazoans. Par6 is involved in asymmetric cell division in the nematode Caenorhabditis elegans during development (36) and formation of cell polarity in neuroblasts/oocytes of Drosophila melanogaster (37, 38). For the human ortholog, it was shown that Par6 regulates the assembly of epithelial tight junctions in human Madin-Darby canine kidney cells (39, 40), and that Par6 plays a role in cellular transformation of NIH-3T3 fibroblasts (41). A possible function of Par6 in insulin signal transduction has not been described until now, although Par6 is strongly expressed in adult insulin-sensitive tissues like liver, skeletal muscle, and pancreas (40, 41, 42, 43). To investigate such a role of Par6, we employed C2C12 murine myoblasts and stably overexpressed Par6. Here, we provide evidence that Par6 negatively regulates both Akt and PI3K activity, leading to a reduced phosphorylation of the Akt substrate glycogen synthase kinase (GSK) 3{alpha}/ß and to a reduced insulin-dependent glycogen synthesis in C2C12 cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Par6 Is a PKC{zeta}/{iota}-Interacting Protein in Insulin-Sensitive Tissues
To identify proteins that interact with aPKCs, a yeast two-hybrid screen was performed and identified Par6 in cDNA libraries of insulin-sensitive tissues. Par6-containing clones were present in liver, pancreas, muscle, and brain cDNA libraries, independent of which aPKC-bait was used. Coimmunoprecipitation of Par6 with aPKCs was possible in human embryonic kidney (HEK) 293 cells transiently co-overexpressing PKC{iota} or PKC{zeta}. Thus, the in vivo interaction of Par6 with aPKCs was verified for both isoforms (data not shown). Par6 interaction with PKC{zeta} has been described earlier (40, 41). Because strong expression of Par6 also was reported for insulin-sensitive tissues and various cell lines by other groups (40, 41, 42, 43), we further explored whether Par6 may play a role within the insulin-signaling cascade. For this purpose, we established cell lines of C2C12 murine myoblasts overexpressing Par6 (C2C12/Par6+). C2C12/Par6+ were not morphologically altered and did not differ in proliferation rates (data not shown). Endogenous Par6 was not detectable in parental C2C12 cells, neither by RT-PCR nor by Western blotting with a polyclonal rabbit antibody, which is able to detect both human and murine Par6 (data not shown). For this reason, native C2C12 could serve as a negative control in the ensuing cell culture experiments.

Par6 Constitutively Associates with Endogenous aPKCs and Increases aPKC Expression/ Phosphorylation Levels in C2C12 Cells
To verify the aPKC-Par6 interaction in our C2C12/Par6+ model, we examined whether Par6 associated with endogenous aPKCs in these cells. Indeed, endogenous aPKCs were coimmunoprecipitated with Par6 in C2C12/Par6+ cells, but not in native C2C12 cells. Stimulation of serum starved C2C12/Par6+ cells with 100 nM insulin for 15 min did not affect the Par6-aPKC association (Fig. 1AGo). As the antibody used for Western blotting of aPKCs was directed against the carboxyl terminus, we could not distinguish between murine isoforms of aPKC (PKC{zeta} and PKC{lambda}). We next examined eight independent C2C12/Par6+ lines that overexpressed different levels of Par6. In these cell lines, phosphorylation of aPKCs on Thr410/403, an indicator for kinase activity of PKC{zeta}/{lambda}, increased proportionally with Par6 expression levels under basal conditions. Of note, levels of aPKC protein also were increased in C2C12/Par6+, but to a similar extent in all lines (see Fig. 1BGo for three representative C2C12/Par6+ lines). For all other signaling proteins (Akt, GSK3{alpha}/ß, p85, IRS1, ERK1/2, Shc) examined in this work no change of expression in C2C12/Par6+ was detectable (see Figs. 3AGo, 4Go, 5Go, A and B, and 6A). An increase of aPKC activity in C2C12/Par6+ was verified by an in vitro immunokinase assay with aPKC-dependent incorporation of [{gamma}-32P]ATP into a peptide substrate. aPKC activity was increased in C2C12/Par6+ compared with native C2C12 under basal conditions (3736 ± 331 vs. 3143 ± 467 cpm; n = 4; P = 0.052) and after insulin stimulation (5113 ± 1078 vs. 4682 ± 863 cpm; n = 3; P = 0.13), although this increase in aPKC activity did not reach levels of statistical significance. We therefore analyzed whether Par6 expression affects the PKC{zeta}-dependent serine phosphorylation of IRS1 in intact cells. For this experiment, we employed a phospho-specific antibody raised against Ser318 of rat-IRS1, which recently was characterized as a PKC{zeta}-dependent serine phosphorylation site of IRS1 in an in vitro assay (44). We used baby hamster kidney (BHK) cells, as phosphorylation levels of IRS1 under basal conditions are lower than in other cell systems. Cotransfection of Par6/PKC{zeta} markedly increased Ser318-phosphorylation of transfected IRS1, whereas coexpression of PKC{zeta} alone had no influence on Ser318 phosphorylation of IRS1 (Fig. 1DGo). Upon coexpression of the other proteins, IRS1 expression was reduced, which is a transient expression specific effect that underscores the increase in Ser318 phosphorylation. The difference between the in vitro and in vivo results indicates that the Par6-activated form of the aPKC population may not be Triton soluble. Because Par6 itself is partially insoluble in Triton, it could sequester aPKCs to the insoluble fraction, and only then aPKCs would be activated. In summary, overexpressed Par6 associated constitutively with aPKCs in C2C12/Par6+ and caused a dose-dependent increase in phosphorylation and activity of endogenous aPKCs.



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  		Fig. 1. Effects of Par6 Overexpression on Endogenous aPKCs in C2C12 Cells

A, Parental (C2C12) and Par6 overexpressing (C2C12/Par6+) C2C12 cells were grown to confluency, serum starved overnight, and stimulated with insulin (15 min, 100 nM). Cell lysates were subjected to immunoprecipitation (IP) with Par6 antibody, resolved by SDS-PAGE and immunoblotted with an antibody to aPKCs. IgG indicates the heavy chain of rabbit IgG. B, Cell lysates of nontransfected C2C12 and three independent C2C12/Par6+ cell clones (Par6/5, Par6/13 and Par6/14) with different Par6 expression levels were analyzed by Western blotting with antibodies for aPKCs and a phospho-specific antibody directed against p-Thr410/Thr403 of PKC{zeta}/{lambda}. C, aPKCs were immunoprecipitated from lysates of untreated or insulin-stimulated (15 nM, 15 min) C2C12 and C2C12/Par6+ cells and assayed for kinase activity as described in Material and Methods. Data from four experiments are shown as mean + SEM. D, BHK cells were transiently transfected with constructs of the indicated proteins. Lysates of unstimulated BHK cells were immunoblotted with a phospho-specific antibody raised against Serine318 of IRS1 and reprobed for IRS1 expression.

 


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  		Fig. 3. Insulin-Stimulated Akt1 Activation Is Significantly Inhibited in C2C12/Par6+ Cells via an aPKC-Dependent Mechanism

A, C2C12/Par6+ were treated with different insulin concentrations and analyzed for phosphorylation/expression of endogenous Akt1 (upper panel) and GSK3{alpha}/ß (lower panel). Blots shown are representative for three independent C2C12/Par6+ cell lines with comparable Par6 overexpression. B, To quantify Akt1/GSK3{alpha} phosphorylation, densitometric data acquired of each phospho-specific Western blot signal were related to the corresponding signal from the protein expression blots. Normalized mean values + SEM obtained are plotted in arbitrary units; paired columns with significant differences (P < 0.05) are marked with an asterisk. The densitometric data also represent three independent C2C12/Par6+ clones with comparable expression levels of Par6. C, Lysates of untreated and insulin-stimulated (15 nM, 15 min) C2C12 and C2C12/Par6+ cells were assayed for Akt1 activity as described in Materials and Methods. Data from four experiments are shown as mean + SEM, paired values which differed significantly (P < 0.05) are marked with an asterisk. D, C2C12 and C2C12/Par6+ myoblasts were incubated in the absence or presence of 25 µM cell-permeable aPKC-specific pseudosubstrate inhibitor (aPKC-PS) for 1 h before subsequent insulin (Ins) stimulation (15 nM, 15 min). Cell lysates were analyzed for phosphoThr308-Akt1 and Akt1 protein. Densitometric data obtained from phospho-specific bands corrected by corresponding protein expression signal intensities from three independent experiments are shown as mean + SEM in arbitrary units; paired values that differed significantly (P < 0.05) are marked with an asterisk.

 


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  		Fig. 4. Effects of Par6 Overexpression on Akt1 and ERK1/2 Phosphorylation under Chronic Insulin Stimulation

Serum-starved C2C12 and C2C12/Par6+ cells were incubated with or without 100 nM insulin for 12 h as indicated. After a 2-h period of serum starvation, cells were stimulated again with insulin (15 nM, 15 min). Cell lysates were analyzed with phospho-specific antibodies for Akt1 (Thr308, upper panel) and ERK1/2 (Thr202/204, lower panel), or for Akt1 and ERK1/2 protein.

 


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  		Fig. 5. Effects of Par6 Overexpression on Insulin Induced p85·IRS1 Association

A, C2C12 and C2C12/Par6+ cells were serum starved and then stimulated with different concentrations of insulin for 15 min. After lysis of cells, equal amounts of protein were subjected to Western blot (WB) analysis with p85 antibody either directly or after immunoprecipitation (IP) of IRS1 (IgG indicates the heavy chain of rabbit IgG). B, The same experiment as in panel A was performed with the p85-antibody for IP and the IRS1 antibody for immunoblotting.

 
Insulin-Stimulated Synthesis of Glycogen Is Significantly Inhibited in C2C12/Par6+ Cells Compared with Native C2C12
To test whether increased aPKC activity in C2C12/Par6+ acts positively or negatively on insulin signaling, we next examined insulin-stimulated glycogen synthesis. Confluent and serum-starved native C2C12 and C2C12/Par6+ cells did not exhibit statistically significant differences in glycogen synthesis (56.1 ± 10.1 vs. 47.44 ± 8.25 cpm/10 µg protein; P <= 0.21; n = 3) under basal conditions. After a 60-min stimulation with various concentrations of insulin, the relative increase of glycogen synthesis was significantly reduced in C2C12/Par6+. The difference between parental and Par6 transfected C2C12 cells peaked under the maximal insulin stimulation of 100 nM (2.24- vs. 1.44-fold of mean basal values; P <= 0.001; n = 3; Fig. 2AGo). Expression of Par6 protein itself was constant in these experiments performed with C2C12/Par6+, and neither serum starvation nor insulin stimulation affected its expression (Fig. 2BGo). To summarize, Par6 overexpression led to a decreased insulin-dependent glycogen synthesis in C2C12, and this inhibition already was significant at insulin concentrations present under physiological conditions in blood plasma.



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  		Fig. 2. Insulin-Stimulated Glycogen Synthesis Is Significantly Inhibited in Par6 Overexpressing Compared with Native C2C12 Cells

A, Native C2C12 (filled squares) and C2C12/Par6+ (open squares) myoblasts were stimulated with different insulin concentrations for 60 min, and glycogen synthesis was measured. Data from three independent experiments are shown as means ± SEM, paired values that differed significantly (P < 0.05) are marked with an asterisk. B, Representative cell lysates of insulin-stimulated C2C12 and C2C12/Par6+ were immunoblotted with a Par6-antibody.

 
Par6 Overexpression Inhibits Insulin-Dependent Activation of Akt1
To understand the mechanism of decreased insulin-dependent glycogen synthesis in C2C12/Par6+, we next analyzed signaling molecules located upstream in cellular glycogen synthesis, namely Akt and GSK3{alpha}/ß. C2C12 and C2C12/Par6+ cells were stimulated with different concentrations of insulin and lysates subjected to Western blot analysis with an antibody raised against phospho-Thr308, a site localized in the activation loop of Akt1. Insulin-dependent phosphorylation of endogenous Akt1 was inhibited in all examined C2C12/Par6+ clones compared with parental cells, including those with low Par6 overexpression, and despite of identical Akt1 expression (Fig. 3AGo, upper panel). We next examined whether this Akt1 inhibition was reflected by a parallel decrease in phosphorylation of GSK3{alpha}/ß, which is an established substrate of Akt1. For this purpose, an antibody directed against the Akt phosphorylation site of GSK3{alpha}/ß (Ser21/9) was used. Indeed, insulin-dependent Ser21/9 phosphorylation of GSK3{alpha}/ß was compromised in C2C12/Par6+ cells, indicating the functional relevance of Akt1 inhibition for downstream signaling (Fig. 3AGo, lower panel). Densitometric quantification of Akt1/GSK3{alpha}/ß phosphorylation normalized to expression levels revealed a statistically significant inhibition of both insulin-induced Akt1 and GSK3{alpha} phosphorylation at higher insulin concentrations (15 nM insulin; Akt1, 48.6 ± 15.4%; GSK3{alpha}, 57.8 ± 20.8% of parental C2C12 cells; see Fig. 3BGo). Although we could detect only weak phosphorylation signals of GSK3ß in C2C12 cells after insulin stimulation, GSK3ß phosphosignals were also diminished in C2C12/Par6+ (data not shown). To verify whether reduced Akt1 phosphorylation also reflects Akt1 activity in our C2C12/Par6+ model, we quantified Akt1 activity by use of an immunokinase assay. In this assay, the insulin-dependent increase in Akt activity was significantly lower in C2C12/Par6+ cells (1.4-fold increase vs. 2.1-fold in C2C12; P = 0.02; n = 4; Fig. 3CGo). To further confirm whether this reduced insulin-dependent Akt activity was the consequence of an increased aPKC activity in C2C12/Par6+ cells, we treated the cells with a myristoylated, cell-permeable inhibitory pseudosubstrate of aPKCs before insulin stimulation. The oligopeptide chosen for this experiment comprised part of the pseudosubstrate domain of aPKCs, which is identical in both murine PKC{zeta} and PKC{lambda}. Although inhibition of aPKCs had no influence on insulin-induced phospho-Thr308 of Akt1 in native C2C12, treatment of C2C12/Par6+ with the inhibitory peptide resulted in increased Akt1 phosphorylation levels, now equivalent to control values of C2C12 (Fig. 3DGo).

Impact of Chronic Insulin Stimulation
To see whether inhibition of Akt1 in C2C12/Par6+ is also apparent under chronically elevated insulin concentrations, we stimulated C2C12/Par6+ for 12 h with 100 nM insulin before cells were stimulated with 15 nM insulin again. This long-term exposure to insulin led to decreased Akt1 phosphorylation in both C2C12 and C2C12/Par6+ compared with short exposure times. Nevertheless, lowered Akt1 phosphorylation levels in C2C12/Par6+ also were evident after a 12-h incubation period with insulin compared with nontransfected cells (Fig. 4Go, upper panel). We next analyzed whether other insulin signaling pathways were altered in C2C12/Par6+. As mentioned above, from the endogenous proteins tested, only aPKCs had a different expression level in C2C12/Par6+. Regarding insulin-dependent activation, we compared phosphorylation of Shc and ERK1/2 (p42/p44-MAPK) between native and transfected cells. Although tyrosine phosphorylation of Shc did not differ (data not shown), phosphorylation of Thr202/204 of ERK1/2 was increased in C2C12/Par6+ cells after short and prolonged stimulation with insulin (Fig. 4Go, lower panel). This indication for an increased ERK activity may be explained by an aPKC-dependent rescue of the ERK signaling cascade by phosphorylation of the Raf kinase inhibitory protein (45). Alternatively, Akt1 itself is able to phosphorylate Raf-1 at Ser259 and thereby promotes a negative regulation of the Ras/Raf-1/ERK1/2 signaling cascade (46). Therefore, increased activity of endogenous aPKCs and decreased insulin-stimulated Akt1 activity both may account for the increase in insulin-dependent phosphorylation of ERK1/2 observed in C2C12/Par6+.

The Association of the PI3-Kinase p85 Subunit with IRS1 Is Decreased in C2C12/Par6+ Cells
PKC{zeta} is known to act as a negative feedback regulator on PI3K activity by phosphorylating IRS1 at still unknown Ser/Thr residues (20). This mechanism impairs the ability of IRS1 to associate with the p85 subunit of PI3K and thereby lowers PI3K activity (21). As our C2C12/Par6+ cells showed an increased aPKC activity, it was interesting to test if the insulin-responsive association of p85 with IRS1 was affected in these cells. Densitometric analysis of both p85 and IRS1 signals did not reveal significant differences between transfected and native cell lines (data not shown). Nevertheless, coimmunoprecipitation of p85 with IRS1 (Fig. 5AGo) and of IRS1 with p85 (Fig. 5BGo) was markedly lowered in C2C12/Par6+ cells after insulin stimulation. This decreased p85·IRS1 interaction implies an inhibition of PI3K activity, as the access of PI3K to phospholipids becomes compromised under these conditions. The role of IRS1-Ser318 phosphorylation in this process is currently under investigation.

Akt1/GSK3{alpha}/ß Phosphorylation Is Also Reduced after Stimulation with PDGF in C2C12/Par6+ Cells
PKC{zeta}-dependent down-regulation of Akt activity by direct interaction of PKC{zeta} with the PH domain of Akt has been described by several authors (26, 27). To address the question whether PKC{zeta}-dependent direct Akt inhibition is instrumental in C2C12/Par6+ cells, we next examined the response of Akt1 to stimulation with PDGF. Phosphoinositide generation after PDGF stimulation does not depend on IRS proteins but on a direct association of the p85 subunit with the receptor (47). Therefore, an impaired Akt1 activity after PDGF stimulation would indicate the relevance of other mechanisms beyond PKC{zeta} dependent down-regulation of PI3K via IRS1 phosphorylation. Thus, C2C12/Par6+ cells were stimulated with PDGF B/B (1 nM, 15 min), and Akt1/GSK3{alpha}/ß phosphorylation was analyzed as described above. Indeed, PDGF-induced Akt1 phosphorylation on Thr308 was significantly inhibited (52.1 ± 9.9%; P < 0.01; n = 5) in C2C12/Par6+ cells compared with controls (Fig. 6Go, A and B). As before, the functional relevance of Akt1 inhibition for GSK3{alpha}/ß in PDGF-treated C2C12/Par6+ was confirmed by phospho-specific Western blotting of GSK3{alpha}/ß (GSK3{alpha}, 39.6 ± 14.3%; P < 0.01; n = 5). aPKC expression and phosphorylation on Thr410/403 in these experiments was only dependent on Par6 expression (Fig. 6AGo, bottom panel) but not sensitive to PDGF stimulation. To exclude that inhibition of PDGF-induced Akt1 phosphorylation in C2C12/Par6+ is a consequence of potentially altered PDGF-dependent PI3K activity, we tried to analyze PDGF receptor (PDGF-R)-associated PI3K activity with an immunokinase assay. Unfortunately, all antibodies tested for immunoprecipitation of the murine isoform of the PDGF-R were not able to precipitate the receptor. We therefore studied PDGF-dependent association of the PI3K with the PDGF-R by immunoprecipitation of the p85 subunit. One antibody detected a very weak signal for the PDGF-R that increased with PDGF stimulation and did not differ between native C2C12 and C2C12/Par6+ cells. Reblotting with phosphotyrosine antibody clearly showed a similar association of the PI3K with the PDGF-R in both cell lines (Fig. 6CGo). This finding therefore suggests that the compromised Akt activation in response to insulin stimulation does not only depend on Par6/aPKC-dependent effects on IRS1, but also on a second mechanism directly involving Akt.



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  		Fig. 6. Akt/GSK3{alpha}/ß-Phosphorylation Is Reduced in C2C12/Par6+ Cells after PDGF Stimulation

C2C12 and C2C12/Par6+ cells were serum starved and stimulated with 1 nM PDGF B/B for 15 min. Phosphorylation of Akt1, GSK3{alpha}/ß, and PKC{zeta} was detected (A) and quantified (B) by scanning densitometry as described in Fig. 3BGo. Mean values + SEM are represented in arbitrary units after normalization to expression levels. Differences with statistical significance (P < 0.05) are marked with an asterisk. The immunoblots shown are representative for five independent experiments. C, C2C12 and C2C12/Par6+ cells were stimulated with PDGF as indicated, lysed and subjected to immunoprecipitation with p85 antibody and analyzed for tyrosine phosphorylation of associated PDGF-R.

 
Par6 Constitutively Interacts with Akt1 and Akt2 in Transiently Transfected HEK293 Cells
Par6 has been described as a scaffold protein that is essential for formation of various multimeric protein complexes (40, 42, 48, 49). It therefore was an attractive hypothesis that Par6 may act as a scaffold that strengthens the PKC{zeta}·Akt1 association that is known to promote PKC{zeta}-dependent down-regulation of Akt1 (26). We generated Akt1 and Akt2 constructs tagged with a myc-epitope at the carboxyl terminus and tested Par6 for interaction with Akt1/2myc in transiently overexpressing HEK293 cells. Both Akt isoforms coimmunoprecipitated with Par6 by immunoprecipitation with Par6-antibody (Fig. 7AGo), as well as with an antibody directed against the myc-epitope (Fig. 7BGo). Next, we tested the stability of the Par6·Akt1/2myc complex in HEK293 cells when co-overexpressed with either PKC{zeta} or PKC{iota}. Despite of high expression levels in transfected HEK293 cells, a coimmunoprecipitation of Akt1/2myc with Par6 (Fig. 7CGo, lanes 2 and 6) was not possible anymore when PKC{zeta}/{iota} was concomitantly overexpressed (Fig. 7CGo, lanes 3 and 4 and 7 and 8). Thus, the affinity of PKC{zeta}/{iota} for Par6 apparently exceeds that of Akt1/2myc, and a triple complex is either not stable or is not formed. Similarly, although we were able to coimmunoprecipitate endogenous aPKCs with Par6 in C2C12/Par6+ cells (Fig. 1AGo), immunoprecipitation of endogenous Akt with Par6 was not possible in C2C12/Par6+ (data not shown). This finding also confirms a rather weak interaction of Akt1/2 with Par6. Furthermore, in NIH-3T3 cells stably overexpressing the Akt2myc construct, endogenous aPKCs were coimmunoprecipitated with Akt2myc, although we could not detect endogenous Par6 in our NIH-3T3 line (data not shown). These results support our conclusion that Par6, although able to interact with both aPKCs or Akt1/2, is not forming a scaffold for the PKC{zeta}·Akt1 complex.



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  		Fig. 7. Experiments on the Role of Par6 as a Potential Scaffold for Akt1·aPKC Interaction

A, HEK293 cells were transiently transfected with expression plasmids encoding Par6, Akt1myc, or Akt2myc. After 24 h of serum starvation, cells were harvested and lysates subjected to immunoprecipitation (IP) with a Par6 antibody. Immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose membranes which were probed with a myc antibody. Expression controls of corresponding cell lysates are shown in the lower panel. B, Akt1/2myc and Par6 overexpressing HEK293 cells were treated as in panel A, but the immunoprecipitation was performed with a myc-antibody and immunoblotting with the Par6 antibody. C, HEK293 cells overexpressing Par6, aPKC ({zeta} or {iota}) and myc-epitope-tagged Akt1/2 isoforms as indicated were harvested and proteins immunoprecipitated with a Par6-antibody. Immunoprecipitates were analyzed for aPKCs and subsequently for Akt1myc and Akt2myc. IgG indicates the heavy chain of the Par6-antibody used for IP. D, Serum-starved C2C12 and C2C12/Par6+ cells were treated for 2 h with the indicated concentrations of C6-ceramide coupled to BSA, stimulated with 10 nM insulin for 15 min and harvested. Akt1 phosphorylation was measured with a phospho-specific antibody directed against the ceramide-sensitive Ser473 site (upper panel). Densitometric data obtained from three independent experiments were normalized to expression levels and mean values + SEM are shown in arbitrary units (lower panel).

 
Ceramide-Induced Inhibition of Akt1 Is Not Significantly Increased in C2C12/Par6+
The signaling molecule ceramide is generated by de novo synthesis as well as from hydrolysis of sphingomyelin by sphingomyelinase. There is considerable evidence suggesting a role of this lipid second messenger in the pathogenesis of insulin resistance (31, 50, 51). Ceramide exerts its inhibitory effect on insulin signaling by inactivation of Akt1 (30, 52). To investigate whether Par6 overexpression interferes with this mechanism of Akt1 inhibition, we tested the effects of ceramide on insulin-dependent Akt1-phosphorylation in C2C12/Par6+ cells. Serum starved myoblasts were incubated for 2 h with different concentrations of BSA-coupled C6-ceramide. During the last 15 min, 10 nM of insulin was added to maximally stimulate Akt1 phosphorylation, and Western blotting with an antibody specific for the ceramide-sensitive phosphorylation site in the hydrophobic domain of Akt1 (Ser473) was performed. Ceramide preincubation down-regulated insulin-dependent Akt phosphorylation in Par6 overexpressing C2C12 cells more than in untransfected cells, though not to a statistically significant extent (Fig. 7DGo), whereas incubation with the inactive analogon dihydro-C6-ceramide did not affect Akt1 phosphorylation (data not shown). Because ceramide works in the parental as well as in the Par6 overexpressing C2C12, we conclude that Par6 is not required for nor does it interfere with ceramide action on Akt1 activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present manuscript, we show that insulin signaling was significantly impaired in C2C12 cells that overexpress Par6. Less p85/PI3K was associated with IRS1, whereas Akt kinase activity and GSK3{alpha}/ß phosphorylation were inhibited. Consequently, glycogen synthesis was significantly reduced in C2C12/Par6+ after stimulation with insulin.

In our yeast two-hybrid study, Par6 was found as an aPKC-interacting protein in human cDNA libraries derived from brain, pancreas, and skeletal muscle, the latter tissues indicating that Par6 is expressed in insulin-sensitive tissues. Although Par6 is expressed in several human and murine cell lines (39, 40, 41), we could not detect endogenous Par6 expression in the C2C12 murine myoblasts used for this study. We therefore consider this cell line suitable for investigating the effects of Par6 on insulin signaling, with untransfected cells serving as a valid negative control.

In the C2C12/Par6+ cell lines examined in this work, expression levels of endogenous aPKCs were increased markedly. Par6 also increased Thr 410/403 phosphorylation of aPKCs in their activation loop proportionally to its expression level, and this increase in phosphorylation was reflected by an enhanced aPKC kinase activity. Such a Par6-dependent aPKC activation has also been reported for Par6 overexpressing COS and Madin-Darby canine kidney cells (41, 43). It was therefore surprising that the increase in aPKC in vitro immunokinase activity was not as pronounced as the increase in aPKC Thr410/403 phosphorylation in C2C12/Par6+ cells. This may indicate a limited usefulness of the in vitro assay for aPKC protein complexes, especially when proteins like Par6 are only partially Triton insoluble. By contrast, the marked increase in Ser318 phosphorylation of the PKC{zeta} substrate IRS1 in PKC{zeta}/Par6 overexpressing BHK cells clearly indicated an increased activity in intact cells. Additional evidence for the increased aPKC kinase activity in C2C12/Par6+ cells is provided by our finding that ERK1/2 phosphorylation was enhanced after insulin stimulation. PKC{zeta} phosphorylates Raf-kinase-inhibitor protein, which subsequently dissociates from its interaction partner Raf-1 and thereby relieves Raf-1 kinase activity in cells (45). Assuming that this mechanism is also present in C2C12 cells, increased aPKC activity could account for the enhanced ERK1/2 phosphorylation observed in C2C12/Par6+ cells. As Par6 itself does not contain any kinase like domain, one may speculate that Par6 recruits aPKCs to specific subcellular compartments where they may get better access to their physiological substrates. The activation of aPKCs by Par6 then likely is insulin independent and constitutive. In summary, our data show that the increase in aPKC activity accounts for the inhibitory effects on insulin signaling in C2C12/Par6+, as inhibition of endogenous aPKCs rescued insulin-induced Akt phosphorylation in these cells.

The increase in aPKC activity also suggests a mechanism for the inhibition of insulin-dependent IRS1·p85 association observed in our C2C12/Par6+ cells. Insulin stimulation increases the association of PKC{zeta} with IRS1 and facilitates subsequent Ser/Thr phosphorylation of IRS1 (21). This phosphorylation impairs the IRS1 function by 1) causing its dissociation from the insulin receptor (20, 53, 54), 2) decreasing the phosphotyrosine content of IRS1 (20, 21, 54, 55, 56, 57), and 3) impairing the association of IRS1 with the p85 subunit of PI3K (20, 58). These events lead to a down-regulation of PI3K activity and provide a negative feedback mechanism in insulin signaling (20, 21, 56).

In PDGF-treated C2C12/Par6+ cells, both Thr308-phosphorylation of Akt1 and Ser21/9-phosphorylation of GSK3{alpha}/ß were lower than in native C2C12. PDGF-stimulated activation of PI3K does not depend on IRS proteins (59). Therefore, this finding cannot be explained by impairment of insulin-dependent IRS1·p85 association. As both tyrosine-phosphorylation of the PDGF-R and p85·PDGF-R association increased to a comparable extent in native and transfected C2C12 cells, the interference with Akt1 phosphorylation must occur downstream of the PI3K in C2C12/Par6+ cells. For this reason, we analyzed whether the second inhibitory mechanism of aPKCs described for insulin signal transduction is influenced by Par6 overexpression.

PKC{zeta} can interact directly with the PH domain of Akt1 and thereby down-regulates Akt1 activity (25, 27). This is further supported by COS-1 cells overexpressing PKC{zeta}, in which Akt1 activity was shown to be reduced after PDGF treatment (26). While this manuscript was under review, Powell et al. (60) described that a ceramide-induced increase in PKC{zeta} activity led to an enhanced phosphorylation of Akt1 on Thr34 in its PH domain, which hinders the recruitment of Akt1 to the plasma membrane and thereby down-regulates Akt activation in insulin signaling. Our finding that Par6 associated with Akt1 and Akt2 in transiently transfected HEK293 cells suggested that Par6 could act as a scaffold for the interaction of PKC{zeta} and Akt1. However, this function was not supported by other data obtained from HEK293 cells, as co-overexpression of PKC{zeta}/{iota} destroyed the Par6·Akt1/2 complex (see Fig. 7Go). Similarly, we could not coimmunoprecipitate endogenous Akt with overexpressed Par6 in C2C12 cells. To finally exclude a role for the interaction of Par6 with Akt, the region of Par6 that binds to Akt has to be identified and the effect of the corresponding mutant investigated.

Free fatty acids, which are typically elevated in patients suffering from type 2 diabetes, mediate an intracellular de novo synthesis of the lipid second messenger ceramide (29) and may be responsible for the phenomenon that ceramide levels are increased in insulin-responsive tissues of diabetic animals (51). Ceramide down-regulates insulin-dependent Akt1 kinase activity and glucose transport and is therefore discussed as an important mediator of insulin resistance (30). We therefore measured Akt1 phosphorylation after ceramide stimulation in C2C12/Par6+ cells. Par6 overexpression did not additionally enhance Akt1 inhibition in C2C12/Par6+ compared with parental cells, at least not to a significant level. This result suggests that Par6 overexpression does not interfere with this mechanism and indirectly supports the hypothesis that Par6 has no function as a scaffold for the PKC{zeta}·Akt1 interaction. On the other hand, published data on C2C12 cells provided evidence that in these cells ceramide mediated down-regulation of Akt1 can mainly be attributed to an enhanced activity of the PP2A phosphatase, which in turn dephosphorylates Akt1 at Ser473 (61). Therefore, a function of Par6 for the PKC{zeta}-dependent mechanism of ceramide-mediated down-regulation of Akt1 still cannot be excluded, and future studies in other cell systems have to address this issue.

Another Par6-interacting molecule that recently has been described as an important player in insulin signal transduction is the small Rho-GTPase Cdc42. Microinjection of anti-Cdc42 antibody or small interfering RNA for Cdc42 resulted in impaired insulin-induced glucose uptake/Glut4-translocation of 3T3-L1 adipocytes (62). This work also showed that insulin stimulation increased Cdc42 activity. As Par6 only associates with the GTP-bound, active Cdc42 via its semi-CRIB (Cdc42/Rac interactive binding) and part of its PSD-95/Discs large/Zo-1 (PDZ) domain (40, 63), one can speculate whether Par6 acts as a regulator for this Cdc42-dependent Glut4 translocation mechanism. Furthermore, Cdc42 associates with the p85 subunit of PI3K (64, 65). By this way, Par6 may be recruited to PI3K, thereby possibly facilitating the access for (Par6-bound) PKC{zeta} to its substrate IRS1 and promoting inhibition of IRS1 by the mechanism described above. Cdc42 also accounts for the regulation of GSK3ß activity at the leading edge of primary rat astrocytes during migration (49). In this scratch-induced astrocyte migration model, GSK3ß forms a complex with both Par6 and PKC{zeta}, and GSK3ß phosphorylation possibly depends on the Cdc42-Par6-PKC{zeta} pathway. Of note, the same paper reported that transient overexpression of Par6 apparently activated PKC{zeta} directly and led to an increased phosphorylation of GSK3ß in COS1 cells. This is in contrast to our model system where GSK3{alpha}/ß phosphorylation was mediated by Akt and reduced in permanently Par6 overexpressing cell lines. Cdc42 also has been described to colocalize with endogenous Akt at the leading edge of migrating fibroblasts (66). Hence, it is possible that the interaction of Par6 with Akt, as seen in transiently overexpressing HEK293 cells, may be of relevance for this colocalization or for other pathways beyond insulin signaling.

Yet another member of the partitioning-defective protein family protein has been reported to interfere with insulin signal transduction (67). In this study, overexpression of ASIP (aPKC isotype-specific-interacting protein) in 3T3-L1 adipocytes resulted in impaired glucose-uptake and translocation of the glucose-transporter Glut4 after insulin stimulation. ASIP is the human ortholog of Par3 (C. elegans) and Bazooka (D. melanogaster), respectively. It contains three consecutive PDZ domains, and therefore acts like Par6 as a scaffold protein. The first PDZ domain of ASIP/Par3 is able to interact with the carboxyl-terminal PDZ domain of Par6, independently of whether Par6 is in complex with aPKCs or not (40). In this way, an aPKC·Par6·Par3 heterotrimeric complex can be formed. This complex has been shown to localize to the anterior periphery of asymmetric dividing cells in the C. elegans zygote (68) and to play a role in formation of epithelial tight junctions both in C. elegans (9) and mammalian cells (10, 69). Taken together, our data obtained from Par6 overexpressing C2C12 cells and the work from Kotani et al. (67) overexpressing ASIP/Par3 in 3T3-L1 adipocytes both imply a potential role of this heterotrimeric aPKC·Par6·Par3 complex for insulin signal transduction.

Our results suggest that overexpression of Par6 in insulin-sensitive tissues could contribute to insulin resistance states and thus identifies a new candidate gene responsible for the development of type 2 diabetes. The effects of Par6 on insulin signaling in C2C12 cells are summarized in a model shown in Fig. 8Go. Future detailed studies on expression of human Par6 and on the influence of hyperglycemia or hyperinsulinemia on its expression should help to clarify the relevance of Par6 for insulin-signaling in the in vivo situation.



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  		Fig. 8. Model of Par6 Effects on Insulin Signaling

A, Insulin binding to the cell initiates the generation of phosphatidylinositolphosphates and activates PDK, which subsequently activates Akt. Activated Akt inhibits GSK3, which then cannot inhibit glycogen synthase (GS) any more. aPKCs are also activated by PDK after insulin stimulation. Beyond their positive effects on downstream events, aPKCs provide negative feedback signals in insulin signaling by phosphorylation of IRS1, which leads to down-regulation of IRS1 associated PI3K activity. B, With overexpression of Par6, activity of aPKCs increases. Activated aPKCs reduce IRS1·p85/PI3K association and negatively regulate Akt independently of the IRS1 feedback loop. IR, Insulin receptor; PM, plasma membrane.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Yeast Two-Hybrid Screen
Different regions of human aPKCs [PKC{iota}, amino acids (aa) 1–200 and aa 1–253; PKC{zeta}, aa 1–200; and full-length PKC{zeta}, aa 1–593] were cloned in frame to the DNA-binding domain of the LexA fusion protein vector pBTM116 (kindly provided by S. Hollenberg and J. Cooper, Fred Hutchinson Cancer Research Center, Seattle, WA). The bait plasmids were transformed into the Saccharomyces cerevisiae strain L40 (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ) and transformants used to screen human cDNA libraries of four different tissues (skeletal muscle, liver, pancreas, brain) subcloned into the GAL4 transcription activation domain expression vector pACT2 (CLONTECH, Palo Alto, CA). Yeast plasmid DNA of colonies growing on HIS-LEU-LYS-TRP-URA drop-out media (BIO 101, Inc., Carlsbad, CA) was isolated, rescued into the Escherichia coli strain HB101, retransformed into the aPKC bait strains, and assayed for ß-galactosidase activity. The specificity of the interaction between aPKCs and potential candidates was proven by transforming the candidate plasmid also into the L40 LexA and the L40 LexA-laminin strain.

Antibodies and Antisera
Rabbit polyclonal antisera were raised against a glutathione-S-transferase fusion protein with the carboxyl terminal 98 aa (aa 247–345) of human Par6{alpha} (GenBank accession no. NM_016948), the carboxyl-terminal 16 aa of human PKC{zeta}, the carboxyl-terminal 203 aa of SHC, a phosphoserine 318 peptide of rat IRS1, and against a peptide corresponding to the carboxyl terminus of p85. For detection of IRS1, a mouse monoclonal antibody was generated that is directed against the peptide 781HQHLRLSSSSGRLRYTA797. The Akt1 mouse antibody was from BD Transduction Laboratories (San Jose, CA), rabbit antisera to phospho-Akt1 (Thr308/Ser473), phospho-GSK3{alpha}/ß (Ser21/9), phospho-p42/44-ERK (Thr202/204), and to phospho-PKC{zeta}/{lambda} (Thr410/403) from Cell Signaling Technology (Beverly, MA), rabbit antibodies to PKC{zeta}, p42/44-ERK and PDGF-R from Upstate Biotechnology Inc. (Lake Placid, NY), and mouse antibody to GSK3{alpha}/ß from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-coupled secondary antibodies for Western blotting were purchased from Sigma (St. Louis, MO). The monoclonal antibody 9E10 directed against the myc-epitope was purified from murine hybridoma cell supernatants.

Construction of Expression Plasmids
cDNAs of human Par6{alpha}, Akt1, and Akt2 were cloned by standard procedures into the cytomegalovirus immediate early promoter-based expression plasmid pRK5 (70). Myc-epitope tags were added to the carboxyl terminus of Akt1/2 by PCR. The cDNAs encoding human PKC{zeta}/{iota} were a generous gift from H. Mischak (Hannover, Germany).

Cell Culture and Treatment
HEK293 cells were cultivated in F12/DMEM supplemented with 10% fetal calf serum and 2 mM glutamine. C2C12 murine myoblasts were grown in DMEM supplemented with 10% fetal calf serum and 2 mM glutamine. For experiments involving stimulation of C2C12 cells, the following protocol was used: cells were seeded at a density of 1 x 104 cells/cm2 in six-well plates, grown until approximately 90% confluence, maintained for further 24 h under low serum conditions (0.5% fetal calf serum) and then stimulated for 15 min with insulin or PDGF B/B. For treatment with C6-ceramide (C6, Sigma) or its inactive analogon dihydro-C6-ceramide (DH-C6), C6 first was dissolved in Me2SO (96% EtOH for DH-C6) to produce a stock solution of 25 mM. After a 25-fold dilution in DMEM containing 5% BSA and constant stirring for 45 min at room temperature, a 1 mM working solution with BSA-coupled C6/DH-C6 was obtained. This working solution was added for 2 h to the serum-starved C2C12 cells to final concentrations of 20 or 100 µM. During the last 10 min of this stimulation protocol, insulin was added at a concentration of 10 nM before cells were harvested and analyzed. The myristoylated aPKC inhibitor peptide [N-myristoyl-SIYRRGARRWRKL] was obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA).

Transfection and Analysis of Cells
For transfection, 50,000 C2C12 cells/well were seeded in six-well plates, cultivated for 24 h, and transfected according to the method of Chen and Okayama (71) with the expression vector pRK5-Par6{alpha} and pSV2neo in a 9:1 ratio, the latter providing a neomycin resistance gene for subsequent G418 selection. C2C12 colonies were analyzed for expression and positive clones were expanded. Stimulated C2C12 cells were washed twice with PBS and lysed in HEPES buffer [50 mM (pH 7.5), 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 100 mM NaF, 10 mM Na4P2O7, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml aprotinin]. Equal amounts of protein (60 µg per lane) were mixed with Laemmli buffer, boiled for 5 min and subjected to 10% SDS-PAGE. Proteins were transferred to nitrocellulose filters, decorated with antibodies and analyzed with the ECL system (Amersham Biosciences, Buckinghamshire, UK). For immunoprecipitation, lysates were adjusted for equal protein concentration with HNTG [20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM Na3VO4 (pH 7.5)] and placed for 5 h at 4 C on a rotating wheel in the presence of 20 µl of a 1:1 slurry of protein A- or G-Sepharose and antibody. The Sepharose was washed three times with HNTG, boiled for 5 min with Laemmli buffer, and analyzed by blotting as described above. Transient transfection of HEK293 cells was performed as described earlier (70).

Glycogen Synthesis Assay
Glycogen synthesis in C2C12 cells was measured by a method adapted from Berti et al. (72). In brief, C2C12 cells were washed three times with HEPES-buffered saline [20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2, 0.1% BSA (pH 7.4)] and then stimulated with different concentrations of insulin for 60 min at 37 C in the same solution. A mixture of D-glucose/D-[14C]-glucose (5 mM final concentration, 0.3 µCi/well, Dupont-NEN, Boston, MA) was added for further 60 min at 37 C. After discarding supernatants and three washes with ice-cold PBS, cell lysis was performed in 30% (wt/vol) KOH (30 min, room temperature). Lysates were heated for 30 min at 96 C, cooled on ice, and aliquots were removed for protein determination (Bradford assay). The glycogen was precipitated by adding 900 µl of ice-cold ethanol. After centrifugation for 4.5 min at 9000 x g, pellets were washed with 1 ml of ethanol and resuspended in 500 µl H2O. Radioactivity was determined by liquid scintillation counting. Glycogen synthesis was expressed as cpm/10 µg protein of insulin-stimulated cells divided by nonstimulated controls. For each concentration of insulin, triplicates were assayed.

Immunokinase Assays
Activation of aPKCs was assessed as described elsewhere (15, 73). In brief, cells were lysed with a buffer containing 20 mM Tris (pH 7.5), 0.25 M sucrose, 1.2 mM EGTA, 1 mM Na4P2O7, 1 mM NaF, 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM Na3VO4, 20 mM ß-mercaptoethanol, 1 mM PMSF, and 10 µg/ml aprotinin. Lysates were adjusted to equal protein amounts, and aPKCs were immunoprecipitated (4 C, 4 h) with an aPKC rabbit antibody. Immunoprecipitates were collected on protein A-Sepharose beads, washed, and incubated for 8 min at 30 C in 50 µl of 50 mM Tris (pH 7.5), 1 mM EGTA, 5 mM MgCl2, 1 mM NaF, 100 µM Na4P2O7, 100 µM PMSF, 40 µM of the substrate peptide PKC{epsilon} serine 159 analog (Biosource Technologies Inc., Camarillo, CA), 2 µg phosphatidylserine, and 66 µM [{gamma}-32P]-ATP (10 mCi/ml, Amersham Biosciences). 32P-labeled substrate was trapped on p81 phosphocellulose filters and radioactivity measured after three washes with phosphoric acid (0.75%) and acetone.

Activity of Akt was measured according to a protocol published by the manufacturer of the Akt specific substrate peptide (Upstate) with a sequence (RPRAATF) surrounding the phosphorylation site of GSK-3. In brief, cells were lysed with 50 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 5 mM Na4P2O7, 10 mM ß-glycerolphosphate, 1 mM Na3VO4, 0.1% ß-mercaptoethanol. After determination of protein concentration, Akt1 was immunoprecipitated with Protein G-Sepharose-bound sheep antibody directed against aa 466–480 of rat/mouse Akt1 (Upstate). Beads were washed three times with 50 mM Tris (pH 7.5), 0.03% Brij-35, 0.1 mM EGTA and preincubated with assay buffer (10 mM 3[N-morpholino]propanesulfonic acid, 12.5 mM ß-glycerolphosphate, 2.5 mM EGTA, 0.5 mM Na3VO4, 1 mM dithiothreitol) on ice. After addition of substrate peptide (final concentration 100 µM) and [{gamma}-32P]-ATP (80 µM), the reaction mixture was incubated for 10 min at 30 C. Finally, 32P-labeled substrate peptide was measured on p81 filters as described above.

Densitometry and Statistical Analysis
For quantification of signal intensity of phospho-specific immunoblots, the EasyWin32 Herolab Software was used. Densitometric data obtained from phospho-specific signals were divided by the densitometric values obtained from the corresponding immunoblots detecting protein expression. At least three independent experiments were performed to obtain mean values ± SEM. For statistical analysis of densitometry and glycogen synthesis, (two-tailed) Student’s t test was used. P <= 0.05 was considered to be statistically significant.


   ACKNOWLEDGMENTS
 
PKC{zeta} and {iota} cDNAs were kindly provided by H. Mischak (Hannover, Germany).


   FOOTNOTES
 
This work was supported by a grant from the European Community (QLRT-1999-00674).

Abbreviations: aa, Amino acids; aPKC, atypical isoforms of PKC; ASIP, aPKC isotype-specific-interacting protein; BHK, baby hamster kidney; DH-C6, dihydro-C6-ceramide; GSK, glycogen synthase kinase; HEK, human embryonic kidney; IRS, insulin receptor substrate; Par6, partitioning-defective protein 6; PDGF, platelet-derived growth factor; PDGF-R, PDGF receptor; PDK-1, 3-phosphoinositide-dependent protein kinase 1; PKC, protein kinase C; PDZ, PSD-95/Discs large/Zo-1; PH, pleckstrin homology; PI3K, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride.

Received for publication June 26, 2003. Accepted for publication February 10, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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