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J Biol Chem, Vol. 274, Issue 13, 8589-8596, March 26, 1999


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

Robert P. DoornbosDagger §, Marga TheelenDagger §, Paul C. J. van der Hoeven, Wim J. van Blitterswijk, Arie J. VerkleijDagger , and Paul M. P. van Bergen en HenegouwenDagger parallel

From the Dagger  Institute of Biomembranes, Department of Molecular Cell Biology, Utrecht University, 3584 CH Utrecht, The Netherlands and  The Netherlands Cancer Institute, Department of Cellular Biochemistry, 1066 CX Amsterdam, The Netherlands

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PKCzeta but not PKCalpha and PKCdelta can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCzeta was also found in COS-1 cells transiently expressing PKB and PKCzeta , 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-PKCzeta 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 PKCzeta 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 PKCzeta . In contrast, co-expression of a kinase-dead PKCzeta 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 PKCzeta . A clear inhibitory effect of PKCzeta was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCzeta in vivo and that PKCzeta acts as a negative regulator of PKB.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (1-3). So far, three isoforms of PKB have been isolated: PKBalpha , PKBbeta , and PKBgamma (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 (p70S6K) (14).

PKB comprises a NH2-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 COOH-terminal 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 (Delta 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 (15-17). Thus, in vivo, PKB can be activated via at least two pathways: a PI 3-kinase dependent and a PI 3-kinase independent pathway.

The mechanism of PKB activation through the PI 3-kinase signaling pathway is not completely understood. In vitro, phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), one of the lipid products generated by PI 3-kinase, stimulates PKB activity by binding to the PH domain (19, 20). Furthermore, PKB activation was shown to be dependent on its phosphorylation of Thr308 and Ser473 (21). Phosphorylation of Thr308 is mediated by an upstream kinase, called phosphatidylinositol 3,4,5-triphosphate-dependent protein kinase-1 (PDK-1), while the kinase responsible for phosphorylation of Ser473, already designated as PDK-2, remains to be identified (22, 23). The proposed mechanism for PKB activation is that PI(3,4)P2 and phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) generated by PI 3-kinase recruit PKB to the plasma membrane where Thr308 is phosphorylated by PDK-1 and Ser473 by PDK-2 (24). The activation of PKB by both PI(3,4)P2 and PDK-1 and -2 makes the activation of PKB a multistep process.

Initial studies by Konishi and co-workers (25, 26) showed that the alpha , delta , and zeta  isoforms of PKC are able to interact with PKB in vitro. In this paper we show that PKB can only be co-immunoprecipiatated with PKCzeta and in addition we found that this interaction is under control of PKB activity. To understand the possible function of the PKB-PKCzeta association, we investigated whether the interacting kinases regulate the activity of the respective kinases. Although no effect was found of PKB on PKCzeta activity, both PDGF- and p110-CAAX-induced activation of PKB is abolished by co-expression of PKCzeta . The activity of GSK-3, a downstream target of PKB is also affected by PKCzeta co-expression. Finally, we found that the constitutive active PKB mutant (T308D/S473D) is inhibited by PKCzeta in a PDGF-independent fashion. The results obtained establish PKCzeta as a negative regulator of PKB activity.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression Constructs-- The pSG5 (Stratagene, La Jolla, CA) constructs containing HA-tagged wild-type bovine PKBalpha , PKBalpha "kinase dead" (K179A), PKBDD and PKBAA 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 (PKBDelta 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 PKCzeta and Myc-tagged kinase-dead PKCzeta 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% CO2. Transient transfections in COS-1 cells were performed at 40% confluency by a DEAE-dextran method. In short, DNA was diluted in 500 µg/ml DEAE-dextran (Sigma) in phosphate-buffered saline and added to the cells. Following a 30-min incubation at 37 °C, medium containing 80 µM 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 beta -glycerophosphate, 1 mM sodium vanadate, 50 mM sodium fluoride, and 10 µg/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 µg of protein) of the precleared extracts using 6 µg of the monoclonal anti-HA antibody (12CA5) coupled to protein G-Sepharose beads (Sigma), whereas Myc-PKCzeta was immunoprecipitated from precleared lysates by 1 µg of the monoclonal anti-Myc antibody (9E10) (Boehringer, Mannheim, Germany) coupled to protein A-Sepharose beads. Endogenous PKCzeta was immunoprecipitated from CHO cells using a polyclonal PKCzeta antibody (27) coupled to protein G-Sepharose beads, whereas PKCalpha and PKCdelta were immunoprecipitated by monoclonal PKCalpha and PKCdelta 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 MgCl2) prior to Western blot analysis or twice with high salt buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 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 anti-mouse 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 PKCalpha , PKCdelta , and PKCzeta were detected using the monoclonal PKCalpha , PKCdelta (Transduction Laboratories), or polyclonal PKCzeta antibody followed by incubation with peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibody (Jackson ImmunoResearch). For PKB detection with the phospho-specific Akt (Ser473) antibody (New England Biolabs, Beverly, MA) polyvinylidene difluoride membranes were incubated with the polyclonal antibody followed by incubation with peroxidase-conjugated goat anti-rabbit 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 PKCzeta , PKB, and GSK-3-- PKCzeta activity was measured with the epsilon -peptide (ERMRPRKRQGSVRRRV) as substrate as described previously (27, 28). Immunoprecipitations were incubated with 45 µl of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2) containing 50 µM epsilon -peptide, 0.2 mM EGTA, 50 µM unlabeled ATP, and 3 µCi of [gamma -32P]ATP (Amersham International, United Kingdom). PKB activity was assayed with the Crosstide peptide (GRPRTSSFAEG) as a substrate (10). Immunoprecipitations were incubated with 45 µl of kinase assay mixture (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 30 µM Crosstide peptide, 1 µM of the specific peptide inhibitor of cyclic AMP-dependent protein kinase (PKI) (Bachem, Bubendorf, Switzerland), 50 µM unlabeled ATP, and 3 µCi of [gamma -32P]ATP). GSK-3 activity was measured using the GS peptide (YRRAAVPPSPSLSRHSSPHQSEDEEE) (29). Cell lysates were incubated with 60 µM GS peptide, 2 mM MgCl2, 100 µM ATP, and 2 µCi of [gamma -32P]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-3beta and that neither PKB nor PKCzeta is able to phosphorylate the peptide.2 Under the conditions used the kinase assays are linear for at least 60 min.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PKCzeta Associates with PKB in CHO Cells-- In vitro binding studies have recently shown that PKB associates with the alpha , delta , and zeta  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 PKCalpha , PKCdelta , and PKCzeta 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 PKCzeta but not in the PKCalpha and PKCdelta 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).


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  		Fig. 1.   PKCzeta associates with PKB in CHO cells. A, CHO cells were lysed and incubated with normal rabbit serum (non-immune) or antibodies against PKCalpha , PKCdelta , and PKCzeta 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 alpha -PKCzeta ) or the polyclonal Akt C-20 antibody (alpha -PKCalpha and alpha -PKCdelta ). IgG indicates the heavy chain of the immunoglobulin. B, immunoprecipitates of PKCalpha , PKCdelta , and PKCzeta 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-PKCzeta , HA-PKBAH, and Myc-PKCzeta or HA-PKBDelta AH and Myc-PKCzeta as described under "Materials and Methods." PKB, PKBAH, or PKBDelta 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 PKCzeta was analyzed using the monoclonal Myc antibody 9E10. Aliquots of each cell extract (30 µl) 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 PKCzeta expression levels.

The binding of PKB with PKCzeta was subsequently investigated in more detail by transient expression of HA-tagged PKB and Myc-tagged PKCzeta (Myc-PKCzeta ) in COS-1 cells. HA-PKB was immunoprecipitated using a monoclonal antibody against the HA-tag (12CA5) and co-immunoprecipitation of Myc-PKCzeta 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 PKCzeta in vivo, we generated HA-tagged PKB constructs lacking the AH domain (HA-PKBDelta AH) or comprising the AH domain (HA-PKBAH). Co-expression of these constructs with PKCzeta revealed that the interaction of PKB with PKCzeta depends entirely on the presence of the AH domain. This observation is in agreement with the in vitro data obtained by Konishi and co-workers (26). PKCzeta could not be observed on a Western blot when PKBDelta AH was immunoprecipitated from cells expressing PKBDelta AH and PKCzeta (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 PKCzeta associates with PKB in vivo. Furthermore, the in vivo association of PKB and PKCzeta is mediated via the AH domain of PKB.

PKB Activity Is Required for Complex Dissociation-- To investigate the effect of PDGF on the PKB-PKCzeta complex, COS-1 cells were transiently co-transfected with both HA-PKB and Myc-PKCzeta . 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 PKCzeta 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.


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  		Fig. 2.   Regulation of the PKB-PKCzeta complex. COS-1 cells were transiently transfected with either wild-type PKB and wild-type PKCzeta , serum starved for 16 h and left untreated or stimulated with 25 ng/ml PDGF-BB for 10 min as described under "Materials and Methods" (A). Alternatively, COS-1 cells were transiently transfected with wild-type PKB and wild-type PKCzeta , wild-type PKB and kinase-dead PKCzeta , or kinase-dead PKB and wild-type PKCzeta (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 PKCzeta 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 PKCzeta were analyzed by Western blot detection using the monoclonal 3F10 and 9E10 antibodies, respectively (as described under "Materials and Methods").

As previously reported, PDGF induces the activation of PKB and, albeit to a lesser extent, also of PKCzeta (27). In order to establish whether the activity of these kinases is involved in PKB·PKCzeta complex formation, we analyzed the effect of kinase-dead mutants of both PKB and PKCzeta . In repeated experiments expression of kinase-dead PKCzeta resulted in a reduction of complex formation which, however, can be explained by the reduction in PKCzeta kd expression (Fig. 2B). In contrast, expression of kinase-dead PKB resulted in a dramatic increase in the PKB-PKCzeta interaction (Fig. 2A). This demonstrates that PKB activity induces the dissociation of the complex, whereas PKCzeta 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).

PKCzeta Activity Is Not Affected by PKB in Vivo-- In order to establish the physiological role for the PKB-PKCzeta interaction we investigated the effect on the activity of both kinases. To test a possible role for PKB on PKCzeta activity, COS-1 cells were transiently transfected with PKCzeta alone or co-transfected with PKB. After stimulation of the cells with PDGF, PKCzeta was immunoprecipitated and its activity was measured by an in vitro kinase assay using the epsilon -peptide as a substrate (28). PDGF stimulation resulted in an increase of PKCzeta activity (Fig. 3) which is in agreement with previous studies (27). Co-expression of PKB did not affect activation of PKCzeta upon PDGF treatment, demonstrating that PKB has no effect on the PDGF-induced activity of PKCzeta (Fig. 3). To demonstrate that PKCzeta and not PKB activity accounts for the observed change in epsilon -peptide phosphorylation we co-transfected PKCzeta with kinase-dead PKB. Similar results were obtained as with wild-type PKB showing that PKB activity does not influence the observed change in epsilon -peptide phosphorylation (Fig. 3).


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  		Fig. 3.   PKCzeta activity is not affected by PKB. COS-1 cells overexpressing wild-type PKCzeta (wt), wild-type PKCzeta , and wild-type PKB or wild-type PKCzeta 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." PKCzeta was immunoprecipitated from the lysates using the monoclonal Myc antibody 9E10 and its activity was assayed with the epsilon -peptide as substrate (see "Materials and Methods"). The results are presented as ± S.E. for six determinations (three independent experiments) related to the activity of PKCzeta in unstimulated cells (100%). Asterisks indicate p < 0.05 (Student's t test).

PKCzeta Is a Negative Regulator of PKB Activity-- Using the same approach, we investigated whether PKCzeta 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 PKCzeta or kinase-dead PKCzeta . 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 PKCzeta (Fig. 4A). This indicates that PKCzeta 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 kinase-dead mutant of PKCzeta (Fig. 4A). These data clearly show that PKB activity is negatively regulated by PKCzeta .


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  		Fig. 4.   PDGF-induced PKB signaling is negatively regulated by PKCzeta . A, COS-1 cells overexpressing wild-type (wt) PKB, wild-type PKB and wild-type PKCzeta , or wild-type PKB and kinase-dead (kd) PKCzeta 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 ± S.E. for six determinations (three separate experiments) related to the activity of PKB in unstimulated cells overexpressing PKB (100%). Asterisks indicate p < 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 ± S.E. for four determinations (two independent experiments) and PDGF-stimulated values were related to their own control (100%).

An important step in the full activation of PKB is the phosphorylation of residues Thr308 and Ser473 by PDK1 and -2 (21). To establish whether the inhibition of PKB activation by PKCzeta is due to a reduced increase in the phosphorylation of PKB we used a polyclonal antibody against PKB when phosphorylated on Ser473. As shown in Fig. 5, A and C, PDGF treatment induced an significant increase (p < 0.05) in Ser473 phosphorylation when PKB was the only transfected protein in COS-1 cells. In contrast, no significant increase in Ser473 phosphorylation was observed upon PDGF treatment when PKCzeta 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 Ser473 was not due to differences in PKB expression levels. Taken together, from these experiments it can be concluded that PKCzeta is a negative regulator of PDGF-induced PKB activity.


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  		Fig. 5.   PKCzeta attenuates PDGF-induced phosphorylation of PKB-Ser473. A, COS-1 cells overexpressing either wild-type (wt) PKB or wild-type PKB and wild-type PKCzeta were left untreated or stimulated with 25 ng/ml PDGF-BB for 10 min as described under "Materials and Methods." Aliquots of each cell extract were separated by SDS-PAGE and Ser473 phosphorylation was analyzed using the polyclonal phospho-specific Akt (Ser473) antibody. B, PKB expression levels were analyzed by Western blot detection using the monoclonal HA antibody 12CA5. C, Ser473 phosphorylation was quantified by densitometry and presented as average ± S.E. for three determinations related to the phosphorylation in unstimulated cells overexpressing PKB (100%). Asterisk indicate p < 0.05 (Student's t test).

PKCzeta Inhibits p110-CAAX-induced PKB Activity-- As already mentioned, both PKB and PKCzeta 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 PKCzeta 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 PKCzeta with p110-CAAX and PKB reduced the p110-CAAX-induced PKB activity with almost 80% (Fig. 6A). Interestingly co-expression of PKCzeta with PLAP-CAAX and PKB also resulted in a decrease in basal PKB activity (Fig. 6A). These observations demonstrate that basal activity of PKCzeta is already sufficient to inhibit PKB.


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  		Fig. 6.   p110-CAAX induced PKB signaling is totally abolished by PKCzeta . 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 PKCzeta 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 ± S.E. for four determinations (two independent experiments) related to the activity of PKB in unstimulated cells overexpressing PKB (100%). Asterisks indicate p < 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 activity using a peptide phosphorylation assay (see "Materials and Methods"). The results are presented as average ± 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").

GSK-3, a Downstream Target of PKB, Is Also Affected by PKCzeta -- It has previously been shown that GSK-3 is phosphorylated and inactivated by PKB in vitro and in vivo (10, 11). In order to test whether this downstream effector of PKB is also affected by co-expression of PKCzeta , we measured GSK-3 activities in cells expressing PKB alone, PKB and PKCzeta , and PKB and kinase-dead PKCzeta 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 PKCzeta the PDGF-induced reduction in GSK-3 activity is completely overcome (Fig. 4B). In contrast, cells co-expressing a kinase inactive PKCzeta 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 induce a reduction of GSK-3 activity (Fig. 6B). Expression of PKCzeta completely abolished the p110-CAAX-induced decrease in GSK-3 activity, whereas co-expression of PKCzeta 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 PKCzeta on PKB activity is also reflected in the GSK-3 activity.

PKCzeta Acts Directly on PKB to Inhibit Its Activity-- The question that remains is where PKCzeta exactly affects the PKB signaling pathway. To find out whether PKCzeta might act on PKB itself, we expressed an active PKB mutant (PKBDD) in which the two phosphorylation sites (Thr308 and Ser473) 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 PKCzeta resulted in a reduction of the ligand-independent activity of the PKBDD 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 (PKBAA) 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 PKCzeta co-expression changed its activity any further (Fig. 7). Together, these observations show that PKCzeta does not act upstream of PKB in the PI 3-kinase signaling pathway to inhibit its activity but strongly suggest that PKCzeta 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 PKCzeta .


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  		Fig. 7.   Constitutively active PKB is inhibited by PKCzeta . A, COS-1 cells overexpressing wild-type (wt) PKB, constitutively active PKBDD, PKBDD, and PKCzeta , constitutively inactive PKBAA or PKBAA and PKCzeta 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 ± S.E. for four determinations (two independent experiments) related to the activity of PKB in unstimulated cells overexpressing PKB (100%). Asterisks indicate p < 0.05 (Student's t test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report we show that PKCzeta binds in vivo to the serine/threonine kinase PKB (c-Akt, RAC-PK). The PKB-PKCzeta interaction was demonstrated by co-immunoprecipitation studies of both endogenous and transiently expressed PKCzeta and PKB proteins. In contrast, we were not able to detect association of PKB with endogenous PKCalpha and PKCdelta 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 PKCdelta association has only been demonstrated in heat-treated cells (16). These results suggest that the affinity of PKB for PKCzeta is higher than for PKCalpha or PKCdelta . Although we were not able to detect the association of PKB with PKCalpha or PKBdelta by co-immunoprecipitation studies we cannot exclude the association of these kinases in the in vivo situation. The association between PKB and PKCzeta 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 PKCzeta is regulated.

As shown in this paper, the PKB-PKCzeta interaction is mediated by the AH domain of PKB. This was concluded from the observation that PKCzeta co-precipitated with the AH domain while no co-precipitation was observed between PKCzeta and the kinase-tail domain of PKB (PKBDelta 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 beta gamma subunit of the heterotrimeric G-protein (5, 32). The beta gamma subunit has been shown to bind to the carboxyl-terminal alpha -helix region of the beta ARK PH domain (33). In contrast, different isoforms of PKC including the Ca2+-dependent (alpha , beta I, and beta II) and Ca2+-independent (epsilon  and zeta ) isoforms have been shown to interact with the second and third beta -sheet of the PH domain of the tyrosine kinase Bruton tyrosine kinase (Btk) (34, 35). PKCzeta has been shown to bind to the first and second beta -sheet of PKB (26). The beta -sheets are part of the binding pocket of the PH domain for phosphoinositides. This raises the possibility that the PKB-PKCzeta interaction is regulated by the PDGF-induced generation of D3-phosphoinositide lipids as they may compete with PKCzeta for binding to the PKB-AH domain. This mechanism was implicated for the binding of PKCbeta I to the PH domain of Btk (35). Alternatively, PKB itself may regulate the dissociation of the complex. A huge increase was observed in the binding of PKCzeta to a kinase-dead PKB mutant while no difference was found in the association with a PKCzeta 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 PKCzeta is a substrate for PKB. In this situation the phosphorylation of PKCzeta 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 PKCzeta .

An important observation of this study is the inhibitory effect of PKCzeta 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 PKCzeta . These findings can be explained either by the inhibition of the upstream signaling of PKB or by a direct effect of PKCzeta on PKB itself. Our results do not support the possibility that PKCzeta inhibits PI 3-kinase, since PKCzeta was shown to inhibit the constitutive active PKB mutant (PKBDD) even without stimulation of the cell with a growth factor. The PKBDD 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 PKCzeta could be that PKCzeta activates PP2A, the serine/threonine-specific phosphatase. PP2A activity has been described to inhibit PKB activity (15). However, the fact that PKCzeta inhibits the activity of the PKBDD mutant that cannot be dephosphorylated does not favor this model. In conclusion, our data strongly suggest that the inhibiting activity of PKCzeta is expressed directly on PKB.

The question, however, remains how PKCzeta 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 PKCzeta activity. Our data show that PKCzeta 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 PKCzeta 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 PKCzeta 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 PKCzeta -dependent phosphorylation of PKB in vitro when PKB was immunoprecipitated from cells expressing both PKB and PKCzeta .2 More research is required to completely understand the mechanism by which PKB is inhibited by PKCzeta .

Recently, PDK-1 has been described as the kinase that is responsible for the PI 3-kinase-dependent phosphorylation and activation of PKCzeta (36, 37). PDK-1 was also found to associate with PKCzeta in unstimulated cells (36, 37). This, together with our observation that PKB associates with PKCzeta in quiescent cells, suggests that PDK-1 can form complexes with both PKB and PKCzeta . 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 PKCzeta . As shown in this paper, the dissociation of the PKB·PKCzeta complex depends upon PKB activity, suggesting that the binding of the inositol lipids PI(3,4)P2 and PI(3,4,5)P3 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 PKCzeta and the subsequent inactivation of PKB. On the other hand, the fraction of PKB that is not in complex with PKCzeta can become completely activated also by PDK-1. This implies that both activity states of PKB, the inactive (PKB in complex with PKCzeta ) and the active state (PKB associated to the membrane), may be under the control of PDK-1 activity.

The link between PKCzeta and the PKB pathway also has implications for the downstream effectors of PKB. As shown in this paper, the decrease in PKB activity by PKCzeta 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 PKCzeta (41), it is tempting to speculate that these effects are mediated by PKCzeta . In addition, PKCzeta has been described to regulate MAPK activity through Raf-1 (42). So, it seems that PKCzeta may regulate the activity of different proteins from different signaling pathways.

In summary, we have shown that PKCzeta associates in vivo with PKB through its AH domain. Both PDGF- and p110-CAAX-induced activation of PKB was inhibited by co-expression of PKCzeta , which was also reflected in GSK-3 activity. Our data demonstrate that PKCzeta negatively regulates PKB signaling, an effect that is regulated by direct action on PKB.

    ACKNOWLEDGEMENTS

We gratefully thank Dr. Paul Coffer for kindly providing HA-tagged PKB cDNAs. Dr. Adri Thomas is thanked for help with the GSK-3 activity measurements. Dr. Marcus Thelen is acknowledged for kindly providing p110-CAAX and PLAP-CAAX cDNAs. Finally, we also thank Jose van der Wal for technical assistance and Drs. Jord Stam and Johannes Boonstra for critically reading the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Contributed equally to the results of this work.

parallel To whom correspondence should be addressed: Institute of Biomembranes, Dept. of Molecular Cell Biology, Utrecht University, 3584 CH Utrecht, The Netherlands. Tel.: 31-30-2533349; Fax: 31-30-2513655; E-mail: bergenp{at}bio.uu.nl.

2 R. P. Doornbos, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PKB, protein kinase B; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PI, phosphatidylinositol; GSK-3, glycogen synthase kinase-3; GS, glycogen synthase; PH, pleckstrin homology; AH, Akt homology; PDK, phosphatidylinositol-3,4,5-triphosphate-dependent protein kinase; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; Btk, Bruton tyrosine kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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