Protein Kinase Cζ Is a Negative Regulator of Protein Kinase B Activity*

Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCζ but not PKCα and PKCδ can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCζ was also found in COS-1 cells transiently expressing PKB and PKCζ, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCζ interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCζ was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCζ. In contrast, co-expression of a kinase-dead PKCζ mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCζ. A clear inhibitory effect of PKCζ was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCζ in vivoand that PKCζ acts as a negative regulator of PKB.

Protein kinase B (PKB), 1 also referred to as c-Akt or RAC-PK is a 60-kDa serine/threonine kinase which is the cellular homologue of the viral oncogene v-Akt (1)(2)(3). So far, three isoforms of PKB have been isolated: PKB␣, PKB␤, and PKB␥ (1,2,4,5). Overexpression of PKB family members has been correlated with different cancers such as breast cancer and some pancreatic and ovarian cancers (2,6,7). Recently, PKB has been found to yield an anti-apoptotic signal, which is crucial for cell survival in both fibroblasts and neuronal cells (8,9). Other reports have indicated a role for PKB in the regulation of glycogen synthesis by inhibition of glycogen synthase kinase-3 (GSK-3) (10,11). In addition, glucose uptake and metabolism in 3T3-L1 adipocytes have been shown to be regulated by PKB by mediating the translocation of the glucose transporter GLUT4 to the plasma membrane (12,13). Moreover, a role for PKB has been described in the regulation of protein synthesis through indirect activation of the p70 ribosomal S6 kinase (p70 S6K ) (14).
PKB comprises a NH 2 -terminal Akt homology (AH) domain of 148 amino acids, a catalytic domain of 264 amino acids showing high homology with cyclic AMP-dependent protein kinase A (PKA) and protein kinase C (PKC) and a short COOHterminal tail of 68 amino acids. A pleckstrin homology (PH) domain of 106 amino acids is present within the AH domain. Treatment of cells with different growth factors, insulin, or phosphatase inhibitors results in rapid activation of PKB (10,14,15). Also heat shock, hyperosmolarity stress, and intracellular cAMP elevation were shown to activate PKB in vivo (16,17). Growth factor and insulin-induced activation is almost completely prevented by overexpression of a dominant negative form of phosphatidylinositol (PI) 3-kinase (⌬p85) or by pretreatment of cells with the PI 3-kinase inhibitors wortmannin and LY294002 (14). Furthermore, a PDGF receptor mutant that is not able to stimulate PI 3-kinase activity also fails to activate PKB (14,18). These data demonstrate that insulin and growth factor-induced signals leading to PKB activation are transduced via the PI 3-kinase pathway. In contrast, stress, okadaic acid, and cAMP induced activation of PKB is PI 3-kinase independent since wortmannin is unable to block this pathway of PKB activation (15)(16)(17). Thus, in vivo, PKB can be activated via at least two pathways: a PI 3-kinase dependent and a PI 3-kinase independent pathway.
Initial studies by Konishi and co-workers (25,26) showed that the ␣, ␦, and isoforms of PKC are able to interact with PKB in vitro. In this paper we show that PKB can only be co-immunoprecipiatated with PKC and in addition we found that this interaction is under control of PKB activity. To understand the possible function of the PKB-PKC association, we investigated whether the interacting kinases regulate the activity of the respective kinases. Although no effect was found of PKB on PKC activity, both PDGF-and p110-CAAX-induced activation of PKB is abolished by co-expression of PKC. The activity of GSK-3, a downstream target of PKB is also affected by PKC co-expression. Finally, we found that the constitutive active PKB mutant (T308D/S473D) is inhibited by PKC in a PDGF-independent fashion. The results obtained establish PKC as a negative regulator of PKB activity.

MATERIALS AND METHODS
Expression Constructs-The pSG5 (Stratagene, La Jolla, CA) constructs containing HA-tagged wild-type bovine PKB␣, PKB␣ "kinase dead" (K179A), PKB DD and PKB AA were a gift from Dr. Paul Coffer (Department of Pulmonary Diseases, University Hospital Utrecht, The Netherlands). The DNA fragments encoding the AH domain of PKB (PKBAH) and PKB lacking the AH domain (PKB⌬AH) were amplified by polymerase chain reaction and subcloned as a BamHI/KpnI fragment into the eukaryotic expression vector pBK-CMV (Stratagene, La Jolla, CA) containing a HA epitope tag (pBK-HA). p110-CAAX, p110-R916P-CAAX (PLAP-CAAX), and the pMT2SM constructs containing Myc-tagged wild-type mouse PKC and Myc-tagged kinase-dead PKC have been described earlier (27,30).
Cell Culture, Transfections, and Immunoprecipitations-COS-1 and CHO cells were grown in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal calf serum (Life Technologies, Inc.) at 37°C in a humidified atmosphere with 7% CO 2 . Transient transfections in COS-1 cells were performed at 40% confluency by a DEAE-dextran method. In short, DNA was diluted in 500 g/ml DEAE-dextran (Sigma) in phosphate-buffered saline and added to the cells. Following a 30-min incubation at 37°C, medium containing 80 M chloroquine (Sigma) was added and the cells were incubated for 2.5-3 h at 37°C and subsequently shocked with 10% dimethyl sulfoxide (Sigma) for 2.5 min. Twenty-four hours after transfection cells were serum starved for 16 h. Stimulated and unstimulated cells were washed once with ice-cold phosphate-buffered saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 40 mM ␤-glycerophosphate, 1 mM sodium vanadate, 50 mM sodium fluoride, and 10 g/ml aprotinin) and incubated on ice for 5 min. Lysates were centrifuged and supernatants were precleared with protein A-Sepharose beads (Pharmacia, Uppsala, Sweden) for 1 h at 4°C. HA-PKB was immunoprecipitated from aliquots (200 g of protein) of the precleared extracts using 6 g of the monoclonal anti-HA antibody (12CA5) coupled to protein G-Sepharose beads (Sigma), whereas Myc-PKC was immunoprecipitated from precleared lysates by 1 g of the monoclonal anti-Myc antibody (9E10) (Boehringer, Mannheim, Germany) coupled to protein A-Sepharose beads. Endogenous PKC was immunoprecipitated from CHO cells using a polyclonal PKC antibody (27) coupled to protein G-Sepharose beads, whereas PKC␣ and PKC␦ were immunoprecipitated by monoclonal PKC␣ and PKC␦ antibodies (Transduction Laboratories, Lexington, KY), respectively. Normal rabbit serum was used as control antiserum for the co-immunoprecipitation studies. Immunoprecipitations were washed twice with lysis buffer and twice with low salt buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 ) prior to Western blot analysis or twice with high salt buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , and 0.5 M LiCl) and twice with low salt buffer prior to activity measurements.
Western Blotting-Cell extracts and immunoprecipitations were separated on an 8% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Boehringer, Mannheim, Germany). Membranes were blocked in 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in TBST buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. For detection of the Myc-tagged or HA-tagged proteins the membranes were incubated with the monoclonal 9E10 or 12CA5 antibody in 1% protifar in TBST buffer subsequently followed by incubation with peroxidase-conjugated rabbit antimouse secondary antibody (Jackson ImmunoResearch, West Grove, PA). Detection of endogenous PKB was performed using the monoclonal PKB/Akt antibody (Transduction Laboratories, Lexington, KY) or the polyclonal Akt-C20 (Santa Cruz Biochemical Corp., Santa Cruz, CA) subsequently followed by incubation with peroxidase-conjugated rabbit anti-mouse or donkey anti-goat (Jackson ImmunoResearch) secondary antibody, respectively. Endogenous PKC␣, PKC␦, and PKC were detected using the monoclonal PKC␣, PKC␦ (Transduction Laboratories), or polyclonal PKC antibody followed by incubation with peroxidaseconjugated rabbit anti-mouse or goat anti-rabbit secondary antibody (Jackson ImmunoResearch). For PKB detection with the phospho-specific Akt (Ser 473 ) antibody (New England Biolabs, Beverly, MA) polyvinylidene difluoride membranes were incubated with the polyclonal antibody followed by incubation with peroxidase-conjugated goat antirabbit secondary antibody. Proteins were visualized by Enhanced Chemiluminescence (Renaissance, NEN Life Science Products Inc., Boston, MA). For quantification of protein amounts a densitometer (Molecular Dynamics) and ImageQuant software were used.
In Vitro Kinase Assays for PKC, PKB, and GSK-3-PKC activity was measured with the ⑀-peptide (ERMRPRKRQGSVRRRV) as substrate as described previously (27,28). Immunoprecipitations were incubated with 45 l of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 ) containing 50 M ⑀-peptide, 0.2 mM EGTA, 50 M unlabeled ATP, and 3 Ci of [␥-32 P]ATP (Amersham International, United Kingdom). PKB activity was assayed with the Crosstide peptide (GRPRTSS-FAEG) as a substrate (10). Immunoprecipitations were incubated with 45 l of kinase assay mixture (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 1 mM dithiothreitol, 30 M Crosstide peptide, 1 M of the specific peptide inhibitor of cyclic AMP-dependent protein kinase (PKI) (Bachem, Bubendorf, Switzerland), 50 M unlabeled ATP, and 3 Ci of [␥-32 P]ATP). GSK-3 activity was measured using the GS peptide (YR-RAAVPPSPSLSRHSSPHQSEDEEE) (29). Cell lysates were incubated with 60 M GS peptide, 2 mM MgCl 2 , 100 M ATP, and 2 Ci of [␥-32 P]ATP. After incubation for 20 min at 30°C under continuous shaking, reactions were stopped by addition of 200 mM EDTA. Proteins were precipitated by the addition of 25% trichloroacetic acid and centrifuged for 1 min at 14,000 rpm. Supernatants containing the phosphorylated peptide were spotted onto p81 phosphocellulose filters (Whatman), washed three times with 1% (v/v) orthophosphoric acid, and analyzed by Cerenkov counting. Control experiments revealed that phosphorylation of the GS peptide is highly specific for GSK-3␤ and that neither PKB nor PKC is able to phosphorylate the peptide. 2 Under the conditions used the kinase assays are linear for at least 60 min.

PKC Associates with PKB in CHO Cells-In vitro binding
studies have recently shown that PKB associates with the ␣, ␦, and isoforms of PKC (5). In order to investigate the possible interaction of these PKC isoforms with PKB in vivo, we performed co-immunoprecipitation studies using CHO cells. Endogenous PKC␣, PKC␦, and PKC were immunoprecipitated from cell lysates and Western blot analysis shows that similar amounts of the three PKC isoforms were precipitated (Fig. 1B). The presence of PKB was analyzed using Western blot detection and, as shown in Fig. 1A, PKB is present in the PKC but not in the PKC␣ and PKC␦ immunoprecipitates. As a control, normal rabbit serum was incubated with lysates of CHO cells and only a faint band is visible possibly reflecting aspecific binding to the non-immune control (Fig. 1A).
The binding of PKB with PKC was subsequently investigated in more detail by transient expression of HA-tagged PKB and Myc-tagged PKC (Myc-PKC) in COS-1 cells. HA-PKB was immunoprecipitated using a monoclonal antibody against the HA-tag (12CA5) and co-immunoprecipitation of Myc-PKC was observed on Western blot using a monoclonal antibody against the Myc-tag (9E10) (Fig. 1C). To identify the domain of PKB that is necessary for the association with PKC in vivo, we generated HA-tagged PKB constructs lacking the AH domain (HA-PKB⌬AH) or comprising the AH domain (HA-PKBAH). Co-expression of these constructs with PKC revealed that the interaction of PKB with PKC depends entirely on the presence of the AH domain. This observation is in agreement with the in 2 R. P. Doornbos, unpublished observations. vitro data obtained by Konishi and co-workers (26). PKC could not be observed on a Western blot when PKB⌬AH was immunoprecipitated from cells expressing PKB⌬AH and PKC (Fig.  1C). As a control, the expression levels of the transiently expressed proteins were analyzed in total cell lysates and similar expression levels were found for all constructs (Fig. 1C). In conclusion, our observations clearly demonstrate that PKC associates with PKB in vivo. Furthermore, the in vivo association of PKB and PKC is mediated via the AH domain of PKB.
PKB Activity Is Required for Complex Dissociation-To in-vestigate the effect of PDGF on the PKB-PKC complex, COS-1 cells were transiently co-transfected with both HA-PKB and Myc-PKC. The cells were serum-starved overnight and either left untreated or stimulated with 25 ng/ml PDGF for 10 min. PKB was immunoprecipitated and co-immunoprecipitation of PKC was determined by Western blot analysis ( Fig. 2A). Upon PDGF treatment the interaction decreased with approximately 75% indicating that PDGF induces the dissociation of the complex.
As previously reported, PDGF induces the activation of PKB and, albeit to a lesser extent, also of PKC (27). In order to establish whether the activity of these kinases is involved in PKB⅐PKC complex formation, we analyzed the effect of kinasedead mutants of both PKB and PKC. In repeated experiments expression of kinase-dead PKC resulted in a reduction of complex formation which, however, can be explained by the reduction in PKC kd expression (Fig. 2B). In contrast, expression of kinase-dead PKB resulted in a dramatic increase in the PKB-PKC interaction ( Fig. 2A). This demonstrates that PKB activity induces the dissociation of the complex, whereas PKC activity seems not to be required for the regulation of the complex. As a control experiment, we incubated the same blot with anti-HA antibodies showing that similar amounts of HA-PKB were precipitated (Fig. 2B).
PKC Activity Is Not Affected by PKB in Vivo-In order to establish the physiological role for the PKB-PKC interaction we investigated the effect on the activity of both kinases. To test a possible role for PKB on PKC activity, COS-1 cells were transiently transfected with PKC alone or co-transfected with PKB. After stimulation of the cells with PDGF, PKC was immunoprecipitated and its activity was measured by an in vitro kinase assay using the ⑀-peptide as a substrate (28). PDGF stimulation resulted in an increase of PKC activity (Fig. 3) which is in agreement with previous studies (27). Coexpression of PKB did not affect activation of PKC upon PDGF treatment, demonstrating that PKB has no effect on the PDGFinduced activity of PKC (Fig. 3). To demonstrate that PKC and not PKB activity accounts for the observed change in ⑀-peptide phosphorylation we co-transfected PKC with kinasedead PKB. Similar results were obtained as with wild-type PKB showing that PKB activity does not influence the observed change in ⑀-peptide phosphorylation (Fig. 3).
PKC Is a Negative Regulator of PKB Activity-Using the same approach, we investigated whether PKC has an effect on PKB activity. For these experiments, COS-1 cells were transiently transfected with wild-type PKB or co-transfected with wild-type PKB and either wild-type PKC or kinase-dead PKC. After PDGF treatment, PKB was immunoprecipitated and its activity was measured by an in vitro kinase assay using Crosstide as substrate (10). Activity measurements showed that PKB activity was increased more than 3-fold upon stimulation of the cells with PDGF (Fig. 4A). However, PDGF-induced PKB activation was almost completely abolished when PKB was co-expressed with wild-type PKC (Fig. 4A). This indicates that PKC is able to inhibit PDGF induced activity of PKB. In contrast, PKB activity was increased more than 7-fold by PDGF when the cells were co-transfected with the kinasedead mutant of PKC (Fig. 4A). These data clearly show that PKB activity is negatively regulated by PKC.
An important step in the full activation of PKB is the phosphorylation of residues Thr 308 and Ser 473 by PDK1 and -2 (21). To establish whether the inhibition of PKB activation by PKC is due to a reduced increase in the phosphorylation of PKB we used a polyclonal antibody against PKB when phosphorylated on Ser 473 . As shown in Fig. 5, A and C, PDGF treatment induced an significant increase (p Ͻ 0.05) in Ser 473 phospho-FIG. 1. PKC associates with PKB in CHO cells. A, CHO cells were lysed and incubated with normal rabbit serum (non-immune) or antibodies against PKC␣, PKC␦, and PKC as described under "Materials and Methods." Immunoprecipitates and a cell lysate (lysate) from CHO cells were analyzed for the presence of PKB by Western blot using the monoclonal PKB/Akt (lysate, non-immune, and ␣-PKC) or the polyclonal Akt C-20 antibody (␣-PKC␣ and ␣-PKC␦). IgG indicates the heavy chain of the immunoglobulin. B, immunoprecipitates of PKC␣, PKC␦, and PKC were analyzed for the presence of these PKC isoforms using Western blot analysis. C, COS-1 cells were transiently transfected with HA-PKB and Myc-PKC, HA-PKBAH, and Myc-PKC or HA-PKB⌬AH and Myc-PKC as described under "Materials and Methods." PKB, PKBAH, or PKB⌬AH were immunoprecipitated from the lysates using the monoclonal HA antibody 12CA5 and subsequently separated by SDS-PAGE and immunoblotted onto polyvinylidene difluoride. Co-immunoprecipitation of PKC was analyzed using the monoclonal Myc antibody 9E10. Aliquots of each cell extract (30 l) were separated by SDS-PAGE and immunoblotted using both the monoclonal HA antibody 12CA5 and the monoclonal Myc antibody 9E10 to analyze both PKB and PKC expression levels.
rylation when PKB was the only transfected protein in COS-1 cells. In contrast, no significant increase in Ser 473 phosphorylation was observed upon PDGF treatment when PKC was co-expressed with PKB (Fig. 5, A and C). As a control, expression levels of total PKB protein were determined and as shown in Fig. 5B the amount of PKB in all lanes was equal indicating that the observed differences in phosphorylated PKB on Ser 473 was not due to differences in PKB expression levels. Taken together, from these experiments it can be concluded that PKC is a negative regulator of PDGF-induced PKB activity.
PKC Inhibits p110-CAAX-induced PKB Activity-As already mentioned, both PKB and PKC are activated by PDGF most probably through the PI 3-kinase signaling pathway. In order to find out whether the negative regulation of PKB by PKC is mediated by the PI 3-kinase/PKB signal transduction pathway we expressed a catalytically active membrane-targeted PI 3-kinase (p110-CAAX) together with PKB in COS-1 cells. p110-CAAX caused a significant, ligand-independent increase in PKB activity (Fig. 6A). In contrast, a catalytically inactive membrane-targeted PI 3-kinase (PLAP-CAAX) was unable to do so (Fig. 6A), which is in agreement with the work of Didichenko and co-workers (30). Co-expression of PKC with p110-CAAX and PKB reduced the p110-CAAX-induced PKB activity with almost 80% (Fig. 6A). Interestingly co-expression of PKC with PLAP-CAAX and PKB also resulted in a decrease in basal PKB activity (Fig. 6A). These observations demonstrate that basal activity of PKC is already sufficient to inhibit PKB. (10,11). In order to test whether this downstream effector of PKB is also affected by co-expression of PKC, we measured GSK-3 activities in cells expressing PKB alone, PKB and PKC, and PKB and kinase-dead PKC using a peptide phosphorylation assay (29). As expected, upon treatment of cells with PDGF the GSK-3 activity is decreased (Fig. 4B). However, when PKB is co-expressed with PKC the PDGF-induced reduction in GSK-3 activity is completely overcome (Fig. 4B). In contrast, cells co-expressing a kinase inactive PKC mutant exhibits a normal reduction in GSK-3 activity upon PDGF treatment (Fig. 4B). In addition, similar results were obtained when PKB was activated via the constitutively activated PI 3-kinase. While expression of p110-CAAX induces the activation of PKB, a significant decrease was found in the GSK-3 activity. Conversely, the expression of the catalytically inactive PLAP-CAAX did not Alternatively, COS-1 cells were transiently transfected with wild-type PKB and wild-type PKC, wild-type PKB and kinase-dead PKC, or kinase-dead PKB and wild-type PKC (B). PKB was immunoprecipitated from the lysates using the monoclonal HA antibody 12CA5 and subsequently separated by SDS-PAGE and immunoblotted onto polyvinylidene difluoride. Co-immunoprecipitation (IP) of PKC was analyzed using the monoclonal Myc antibody 9E10. The amount of immunoprecipitated PKB was analyzed using the monoclonal HA antibody 3F10. Expression levels of PKB and PKC were analyzed by Western blot detection using the monoclonal 3F10 and 9E10 antibodies, respectively (as described under "Materials and Methods").

FIG. 3. PKC activity is not affected by PKB. COS-1 cells overex-
pressing wild-type PKC (wt), wild-type PKC, and wild-type PKB or wild-type PKC and kinase-dead (kd) PKB were left untreated (gray bars) or stimulated with 25 ng/ml PDGF-BB (black bars) for 10 min as described under "Materials and Methods." PKC was immunoprecipitated from the lysates using the monoclonal Myc antibody 9E10 and its activity was assayed with the ⑀-peptide as substrate (see "Materials and Methods"). The results are presented as Ϯ S.E. for six determinations (three independent experiments) related to the activity of PKC in unstimulated cells (100%). Asterisks indicate p Ͻ 0.05 (Student's t test).
induce a reduction of GSK-3 activity (Fig. 6B). Expression of PKC completely abolished the p110-CAAX-induced decrease in GSK-3 activity, whereas co-expression of PKC with PLAP-CAAX did not affect GSK-3 at all (Fig. 6B). These results clearly show that both PDGF-and p110-CAAX-induced PKB activity result in a decrease in GSK-3 activity. Furthermore, these experiments demonstrate that the inhibitory effect of PKC on PKB activity is also reflected in the GSK-3 activity.
PKC Acts Directly on PKB to Inhibit Its Activity-The question that remains is where PKC exactly affects the PKB signaling pathway. To find out whether PKC might act on PKB itself, we expressed an active PKB mutant (PKB DD ) in which the two phosphorylation sites (Thr 308 and Ser 473 ) that are necessary for complete activation are replaced by aspartic acid resulting in a constitutively active form of PKB (21). This mutant exhibits high activity already in resting cells and as expected, PDGF treatment did not increase the activity any further (Fig. 7). Co-expression of PKC resulted in a reduction of the ligand-independent activity of the PKB DD mutant (Fig.  7). PDGF treatment did not contribute to this inhibitory effect since no difference could be observed between untreated and PDGF stimulated conditions (Fig. 7). As a control, a PKB mutant (PKB AA ) in which the two phosphorylation sites are mutated into alanine was expressed and its activity was measured. Very little activity was detected for this mutant and neither PDGF treatment nor PKC co-expression changed its activity any further (Fig. 7). Together, these observations show that PKC does not act upstream of PKB in the PI 3-kinase signaling pathway to inhibit its activity but strongly suggest that PKC acts at the level of PKB kinase causing the reduction of its activity. In addition, these data show that PI 3-kinase is not necessarily required for the inhibitory effect of PKB by PKC.

DISCUSSION
In this report we show that PKC binds in vivo to the serine/ threonine kinase PKB (c-Akt, RAC-PK). The PKB-PKC interaction was demonstrated by co-immunoprecipitation studies of both endogenous and transiently expressed PKC and PKB proteins. In contrast, we were not able to detect association of PKB with endogenous PKC␣ and PKC␦ in vivo in CHO cells. This latter observation is in disagreement with binding studies of Konishi and co-workers (5). These studies, however, were performed by in vitro binding studies using the PH domain of PKB fused to GST and lysates of COS-7 cells containing transiently expressed PKC isoforms. In addition, the PKC␦ associ- FIG. 4. PDGF-induced PKB signaling is negatively regulated by PKC. A, COS-1 cells overexpressing wild-type (wt) PKB, wild-type PKB and wild-type PKC, or wild-type PKB and kinase-dead (kd) PKC were serum starved for 16 h and left untreated (gray bars) or stimulated with 25 ng/ml PDGF-BB (black bars) for 10 min as described under "Materials and Methods." PKB was immunoprecipitated from the lysates using the monoclonal HA antibody 12CA5 and its activity was assayed with Crosstide as substrate (see "Materials and Methods"). The results are presented as average Ϯ S.E. for six determinations (three separate experiments) related to the activity of PKB in unstimulated cells overexpressing PKB (100%). Asterisks indicate p Ͻ 0.05 (Student's  t test). B, corresponding GSK-3 activity measurements. Total cell lysates of untreated and PDGF-stimulated cells were analyzed for GSK-3 activity using a peptide phosphorylation assay (see "Materials and Methods"). The results are presented as average Ϯ S.E. for four determinations (two independent experiments) and PDGF-stimulated values were related to their own control (100%). ation has only been demonstrated in heat-treated cells (16). These results suggest that the affinity of PKB for PKC is higher than for PKC␣ or PKC␦. Although we were not able to detect the association of PKB with PKC␣ or PKB␦ by co-immunoprecipitation studies we cannot exclude the association of these kinases in the in vivo situation. The association between PKB and PKC was observed in both serum-starved and PDGF-stimulated cells. Stimulation of the cells with PDGF resulted in a reduction in complex formation. An important question is how the association between PKB and PKC is regulated.
As shown in this paper, the PKB-PKC interaction is mediated by the AH domain of PKB. This was concluded from the observation that PKC co-precipitated with the AH domain while no co-precipitation was observed between PKC and the kinase-tail domain of PKB (PKB⌬AH). The AH domain of PKB largely consists of a PH domain, which has previously been identified as a lipid-binding domain (31). PH domains, however, including the PH domain of PKB, have also been described to bind to proteins such as PKC and the ␤␥ subunit of the heterotrimeric G-protein (5,32). The ␤␥ subunit has been shown to bind to the carboxyl-terminal ␣-helix region of the ␤ARK PH domain (33). In contrast, different isoforms of PKC including the Ca 2ϩ -dependent (␣, ␤I, and ␤II) and Ca 2ϩ -independent (⑀ and ) isoforms have been shown to interact with the second and third ␤-sheet of the PH domain of the tyrosine kinase Bruton tyrosine kinase (Btk) (34,35). PKC has been shown to bind to the first and second ␤-sheet of PKB (26). The ␤-sheets are part of the binding pocket of the PH domain for phosphoinositides. This raises the possibility that the PKB-PKC interaction is regulated by the PDGF-induced generation of D3-phosphoinositide lipids as they may compete with PKC for binding to the PKB-AH domain. This mechanism was implicated for the binding of PKC␤I to the PH domain of Btk (35). Alternatively, PKB itself may regulate the dissociation of the activity using a peptide phosphorylation assay (see "Materials and Methods"). The results are presented as average Ϯ S.E. for four determinations (two independent experiments) and related to the GSK-3 value of mock-transfected cells (100%). The expression levels of PKB were analyzed by Western blotting of total cell lysates (as described under "Materials and Methods").
FIG. 6. p110-CAAX induced PKB signaling is totally abolished by PKC. A, COS-1 cells overexpressing wild-type PKB, wild-type PKB together with either p110-CAAX or PLAP-CAAX in the presence or absence of PKC were serum starved for 16 h and left untreated. PKB was immunoprecipitated from the lysates using the monoclonal HA antibody 12CA5 and its activity was assayed with Crosstide as substrate (see "Materials and Methods"). Results are presented as average Ϯ S.E. for four determinations (two independent experiments) related to the activity of PKB in unstimulated cells overexpressing PKB (100%). Asterisks indicate p Ͻ 0.05 (Student's t test). The amount of immunoprecipitated PKB was analyzed using Western blot detection (as described under "Materials and Methods"). B, corresponding GSK-3 activity measurements. Total cell lysates were analyzed for GSK-3 FIG. 7. Constitutively active PKB is inhibited by PKC. A, COS-1 cells overexpressing wild-type (wt) PKB, constitutively active PKB DD , PKB DD , and PKC, constitutively inactive PKB AA or PKB AA and PKC were serum starved for 16 h and either left untreated or stimulated with 25 ng/ml PDGF-BB for 10 min. PKB was immunoprecipitated from the lysates using the monoclonal HA antibody 12CA5 and its activity was assayed with Crosstide as substrate (see "Materials and Methods"). Results are presented as average Ϯ S.E. for four determinations (two independent experiments) related to the activity of PKB in unstimulated cells overexpressing PKB (100%). Asterisks indicate p Ͻ 0.05 (Student's t test). complex. A huge increase was observed in the binding of PKC to a kinase-dead PKB mutant while no difference was found in the association with a PKC kinase-dead mutant. This indicates that PKB activity is very important for dissociation of the complex. The most straightforward explanation for this effect is that PKC is a substrate for PKB. In this situation the phosphorylation of PKC by PKB after stimulation of the cell with a growth factor would result in the dissociation of the complex. Alternatively, it is equally possible that another unknown protein in the complex, which is a substrate for PKB, is mediating this interaction. Current research is aimed at the determination of the role of inositol lipids and PKB substrates in complex formation between PKB and PKC.
An important observation of this study is the inhibitory effect of PKC on the PDGF-and p110-CAAX-induced activation of PKB. This effect is both measured on PKB activity and on the phosphorylation of serine 473 of PKB. Also the downstream signaling to GSK-3 is inhibited by PKC. These findings can be explained either by the inhibition of the upstream signaling of PKB or by a direct effect of PKC on PKB itself. Our results do not support the possibility that PKC inhibits PI 3-kinase, since PKC was shown to inhibit the constitutive active PKB mutant (PKB DD ) even without stimulation of the cell with a growth factor. The PKB DD mutant is no longer a substrate for the PDK-1 and -2 kinases and is active without stimulation of the cell by growth factors. A possible PI 3-kinase independent mechanism for inhibition of PKB by PKC could be that PKC activates PP2A, the serine/threonine-specific phosphatase. PP2A activity has been described to inhibit PKB activity (15). However, the fact that PKC inhibits the activity of the PKB DD mutant that cannot be dephosphorylated does not favor this model. In conclusion, our data strongly suggest that the inhibiting activity of PKC is expressed directly on PKB.
The question, however, remains how PKC negatively regulates PKB activity. One of the first steps in the activation process of PKB is the translocation of PKB to the membrane. This process could be sensitive to PKC activity. Our data show that PKC inhibits the constitutively active PKB mutant already in quiescent cells, making this possibility unlikely. On the other hand, we have observed that a kinase-dead PKC normally binds to PKB but fails to inhibit PKB activity. This suggests that PKB is inhibited by phosphorylation rather than binding. An obvious model is that PKB is a direct substrate of PKC and that phosphorylation of PKB results in the inhibition of enzyme activity. A similar mechanism has been found for the regulation of Btk activity by PKC. Btk has been shown to bind to different PKC isoforms and they inhibit Btk autophosphorylation activity by direct phosphorylation (34). Preliminary experiments in our laboratory indeed show that there is a PKC-dependent phosphorylation of PKB in vitro when PKB was immunoprecipitated from cells expressing both PKB and PKC. 2 More research is required to completely understand the mechanism by which PKB is inhibited by PKC.
Recently, PDK-1 has been described as the kinase that is responsible for the PI 3-kinase-dependent phosphorylation and activation of PKC (36,37). PDK-1 was also found to associate with PKC in unstimulated cells (36,37). This, together with our observation that PKB associates with PKC in quiescent cells, suggests that PDK-1 can form complexes with both PKB and PKC. This is an intriguing situation given the fact that PDK-1 stimulates the activation of PKB by direct phosphorylation and induces the inactivation of PKB via PKC. As shown in this paper, the dissociation of the PKB⅐PKC complex depends upon PKB activity, suggesting that the binding of the inositol lipids PI(3,4)P 2 and PI(3,4,5)P 3 to PKB results in the partial activation of PKB and subsequently in the dissociation of the complex. The same lipids stimulate PDK-1 resulting in the activation of PKC and the subsequent inactivation of PKB. On the other hand, the fraction of PKB that is not in complex with PKC can become completely activated also by PDK-1. This implies that both activity states of PKB, the inactive (PKB in complex with PKC) and the active state (PKB associated to the membrane), may be under the control of PDK-1 activity.
The link between PKC and the PKB pathway also has implications for the downstream effectors of PKB. As shown in this paper, the decrease in PKB activity by PKC leads to changes in GSK-3 activity. Furthermore, it has recently been shown that C2-ceramides inhibit PKB/Akt activity and induce apoptosis through an unknown mechanism (38,39). In addition, C2-ceramides have also been shown to decrease glucose uptake through inhibition of PKB activity (40). Since ceramides have been described to activate atypical PKC (41), it is tempting to speculate that these effects are mediated by PKC. In addition, PKC has been described to regulate MAPK activity through Raf-1 (42). So, it seems that PKC may regulate the activity of different proteins from different signaling pathways.
In summary, we have shown that PKC associates in vivo with PKB through its AH domain. Both PDGF-and p110-CAAX-induced activation of PKB was inhibited by co-expression of PKC, which was also reflected in GSK-3 activity. Our data demonstrate that PKC negatively regulates PKB signaling, an effect that is regulated by direct action on PKB.