Ceramide Disables 3-Phosphoinositide Binding to the Pleckstrin Homology Domain of Protein Kinase B (PKB)/Akt by a PKC{zeta}-Dependent Mechanism

Ceramide is generated in response to numerous stress-inducingstimuli and has been implicated in the regulation of diversecellular responses, including cell death, differentiation, andinsulin sensitivity. Recent evidence indicates that ceramidemay regulate these responses by inhibiting the stimulus-mediatedactivation of protein kinase B (PKB), a key determinant of cellfate and insulin action. Here we show that inhibition of thiskinase involves atypical PKC ${zeta}$ , which physically interacts withPKB in unstimulated cells. Insulin reduces the PKB-PKC ${zeta}$ interactionand stimulates PKB. However, dissociation of the kinase complexand the attendant hormonal activation of PKB were preventedby ceramide. Under these circumstances, ceramide activated PKC ${zeta}$ ,leading to phosphorylation of the PKB-PH domain on Thr³⁴. Thisphosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate(PIP₃) binding to PKB, thereby preventing activation of thekinase by insulin. In contrast, a PKB-PH domain with a T34Amutation retained the ability to bind PIP₃ even in the presenceof a ceramide-activated PKC ${zeta}$ and, as such, expression of PKBT34A mutant in L6 cells was resistant to inhibition by ceramidetreatment. Inhibitors of PKC ${zeta}$ and a kinase-dead PKC ${zeta}$ both antagonizedthe inhibitory effect of ceramide on PKB. Since PKB confersa prosurvival signal and regulates numerous pathways in responseto insulin, suppressing its activation by a PKC ${zeta}$ -dependent processmay be one mechanism by which ceramide promotes cell death andinduces insulin resistance.

INTRODUCTION

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Introduction

Protein kinase B (PKB), also known as c-Akt, is a serine/threoninekinase that has been implicated in the control of diverse cellularfunctions, including glucose metabolism, gene transcription,cell proliferation, and apoptosis (16, 27, 34, 48). Three PKBisoforms ( ${alpha}$ , ß, and ${gamma}$ ) have been identified, and thesecan be activated rapidly in response to insulin and growth factorsin a phosphoinositide 3-kinase (PI3K)-dependent manner. PI3Kactivation results in the increased production of 3-phosphoinositides,e.g., phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃]and phosphatidylinositol 3,4-bisphosphate, which play a keyrole in the recruitment of PKB to the plasma membrane (5). TheN-terminal domain of all three PKB isoforms contains a pleckstrinhomology (PH) domain, which is considered critical in allowingthe kinase to interact with 3-phosphoinositides and possiblyother signaling proteins (13, 16, 19). Binding of 3-phosphoinositidesto the PH domain of PKB is also thought to induce conformationalchanges in the kinase that expose two key regulatory sites,Thr³⁰⁸ and Ser⁴⁷³ (3), allowing them to be phosphorylated bytwo upstream kinases. One of these, 3-phosphoinositide-dependentkinase-1 (PDK1), phosphorylates Thr³⁰⁸ (4, 44), whereas theidentity of the second kinase that phosphorylates Ser⁴⁷³ (putativelytermed PDK2) remains unknown, although a number of potentialcandidates have recently been proposed (for a review, see reference15).

The activation of PKB elicited by insulin and growth factorscan be reduced dramatically by stimuli such as tumor necrosisfactor alpha (TNF- ${alpha}$ ) and saturated fatty acids, such as palmitate(41), which have been implicated strongly in the pathogenesisof insulin resistance. Both TNF- ${alpha}$ and palmitate, as well as numerousother stress-inducing stimuli, such as heat shock, UV radiation,and oxidants, have been shown to promote an increase in theproduction of ceramide, a sphingomyelin-derived lipid molecule(28, 32). Cell-permeant analogues of ceramide have been shownto exert a profound inhibitory effect on insulin-stimulatedglucose transport in muscle and fat cells (26, 46), and thereis mounting evidence to support the idea that the lipid alsoacts as an intracellular effector molecule that promotes celldeath (32). We and others have shown that ceramide inhibitsinsulin-stimulated glucose transport by suppressing the hormonalactivation of PKB in L6 muscle cells and 3T3-L1 adipocytes (26,46). In these cell types, ceramide blocks the insulin-dependentrecruitment of PKB to the plasma membrane despite an increasein cellular 3-phosphoinositides, suggesting that ceramide maytarget additional elements that may either be required or regulatethe translocation and activation of PKB. Ceramide is known toregulate both directly and indirectly the activity of numeroussignaling molecules, such as mitogen-activated protein kinases(MAPKs), stress-activated protein kinases, phosphatases, andmembers of the PKC family, such as atypical PKC ${zeta}$ (10, 36, 37).The latter is of particular interest given that a number ofstudies have shown that PKC ${zeta}$ can interact with and inhibit PKB(9, 22, 33, 35). Moreover, the observation that a dominant-negativePKC ${zeta}$ attenuates ceramide's ability to inhibit PKB activationin smooth muscle cells (9) and that inhibition of PKC ${zeta}$ in neuroblastomacells suppresses apoptosis induced by ceramide analogues (8)provides a strong basis for suggesting that the lipid may dysregulatePKB-directed signaling via PKC ${zeta}$ . In the present study we testedthis proposition with a view to ascertaining the mechanism bywhich PKC ${zeta}$ may modulate PKB signaling in response to ceramide.

MATERIALS AND METHODS

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Materials and Methods

Materials. ${alpha}$ -Minimal essential medium, fetal bovine serum, and antimycotic-antibioticsolution were from Life Technologies. All other reagent-gradechemicals, insulin, DAPI (4',6'-diamidino-2-phenylindole), andprotamine-agarose beads were obtained from Sigma-Aldrich (Poole,United Kingdom). PKC ${zeta}$ and the PKC ${alpha}$ myristoylated-pseudosubstrateinhibitors, Ro 31.8220, SB-203580, rapamycin, dihydroceramide,and phorbol 12-myristate 13-acetate (PMA), were purchased fromCalbiochem-Novabiochem, Ltd. (Nottingham, United Kingdom), andceramide was obtained from Tocris (Bristol, United Kingdom).SB-212963 was a gift from GlaxoSmithKline (Harlow, United Kingdom).Antibodies against the PH domain of PKB ${alpha}$ , peptide substratesfor kinase assays, and recombinant PKB ${alpha}$ - ${Delta}$ PH protein were providedby Philip Cohen (MRC Protein Phosphorylation Unit, Universityof Dundee, Dundee, Scotland). Antibodies to phospho-PKC ${zeta}$ ⁴¹⁰,constructs encoding glutathione S-transferase (GST)-PKC ${zeta}$ , kinaseGST-dead GST-PKC ${zeta}$ , PKB, and the isolated PH domain of PKB wereprovided by Dario Alessi (MRC Protein Phosphorylation Unit,University of Dundee). A semipurified isolated PKB PH domainprotein was provided by Dan Van Aalten (University of Dundee).sn-2-Stearoyl-3-arachidonyl D-PtdIns(3,4,5)P₃ (PIP₃) and phosphatidylserine(PS) were gifts from Peter Downes (University of Dundee). Antibodiesto p38 MAPK, PKB ${alpha}$ , phospho-PKB³⁰⁸, and phospho-PKB⁴⁷³ were fromNew England Biolabs (Herts, United Kingdom); anti-phospho-GSK3was from Life Signaling Technologies; anti-c-myc was from Sigma-Aldrich;and anti-PKC ${zeta}$ was purchased from Santa Cruz Biotechnology (SantaCruz, Calif.). Horseradish peroxidase (HRP)-conjugated to anti-rabbitimmunoglobulin G (IgG), anti-mouse IgG, and anti-sheep/goatIgG were obtained from the Scottish Antibody Production Unit(Law Hospital, Carluke, Lanarkshire, Scotland). Protein A-Sepharosebeads were purchased from ICN Biomedicals (Basingstoke, Hants,United Kingdom). ATP and Hyperfilm MP autoradiography film werepurchased from Amersham Biosciences). Fugene 6 transfectionreagent, antihemagglutinin (anti-HA) antibody, and Completeprotein phosphatase inhibitor tablets were purchased from Boehringer-RocheDiagnostics (Basel, Switzerland).

Tissue culture and cellular fractionation. L6 muscle cells were grown as described previously in ${alpha}$ -minimalessential medium containing 2% (vol/vol) fetal bovine serumand 1% (vol/vol) antimycotic-antibiotic solution at 37°Cin an atmosphere of 5% CO₂ and 95% air (25). Plasma membraneswere isolated from L6 cells as described previously (25). Theprotein content of the isolated membrane fractions was determinedby the Bradford assay (11).

L6 cell transfection. Subconfluent L6 myoblasts were transfected with 1 µg ofpCMV5-PKC ${zeta}$ , pCMV5-kinase dead PKC ${zeta}$ , or the PCMV5 vector aloneby using Fugene 6 transfection reagent. After 48 h, muscle cellswere treated with 100 µM C₂-ceramide and 100 nM insulinprior to lysis. Cell lysates were subsequently subjected toanalysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblotting.

Phospho-PKBT34 antibody. A phosphospecific antibody recognizing PKB ${alpha}$ phosphorylated atThr³⁴ was raised in sheep against the peptide LKNDGTFIGYK (correspondingto residues 29 to 39 of full-length human PKB ${alpha}$ ; the underlinedresidue is phosphothreonine). The antibodies were affinity purifiedon activated Sepahrose covalently coupled to the phosphorylatedpeptide.

SDS-PAGE and immunoblotting. Cell lysates (50 µg) and isolated membrane fractions fromL6 cells (20 µg) were subjected to SDS-PAGE on 10% resolvinggels and transferred onto Immobilon-P or Hybond-C membranes(Millipore, Harts, United Kingdom) as described previously (25).Membranes were probed with primary antibodies to proteins ofinterest. For analysis of PKC ${zeta}$ phosphorylation on its activationloop residue (Thr⁴¹⁰), cell lysates (250 µg of protein)were initially subjected to incubation with protamine-agarosebeads to "pull down" and enrich PKC ${zeta}$ as described previously(6). PKC ${zeta}$ associated with protamine beads was resuspended inSDS sample buffer and resolved by SDS-PAGE prior to immunoblottingwith an phospho-specific antibody directed against Thr⁴¹⁰. Detectionof primary antibodies was performed with either HRP-labeledanti-rabbit IgG, HRP-labeled anti-mouse IgG, or HRP-labeledanti-sheep/goat IgG and then visualized by using enhanced chemiluminescence(Amersham Biosciences) on Kodak X-Omat film (Eastman-Kodak,Rochester, United Kingdom).

Analysis of PKB ${alpha}$ activity in L6 cells. L6 myotubes grown in 10-cm dishes were exposed to 5 µMRo 31.8220 for 2.5 h, 100 µM C₂-ceramide for 2 h, and100 nM insulin for 10 min. Cells were then harvested in lysisbuffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM EGTA, 1% [vol/vol]Triton X-100, 1 mM Na₃VO₄, 10 mM sodium ß-glycerophosphate,50 mM NaF, 5 mM Na₄P₂O₇, 1 µM microcystin-LR, 270 mM sucrose,1 mM benzamidine, 10 µg of leupeptin/ml, Complete proteinaseinhibitor cocktail [one tablet per 25 ml], and 0.1% [vol/vol]2-mercaptoethanol). PKB ${alpha}$ was immunoprecipitated from lysatesby using an antibody against the C-terminal domain, and kinaseactivity assayed by using the synthetic peptide substrate "crosstide"as described previously (17).

In vitro protein phosphorylation. Phosphorylation assays were performed by incubating 1 µgof recombinant PKB ${alpha}$ PH domain protein with 30 µl of assaybuffer (20 mM morpholinepropanesulfonic acid [pH 7.2], 25 mMß-glycerophosphate, 1 mM sodium orthovanadate, 1 mMdithiothreitol, 1 mM CaCl₂, 5 mM MgCl_2, 1 µM ATP, 5 µCiof [³²P]ATP, 4 pg of PS/ml) for 10 min at 30°C in the absenceor presence of 30 ng of recombinant PKC ${zeta}$ (Upstate Biotech) and100 µM ceramide. The reaction was stopped by the additionof Laemmli buffer; phosphorylated PH peptides and PKC ${zeta}$ were thenresolved by SDS-PAGE, and bands visualized by exposure to AmershamHyperfilm MP film. Alternatively, resolved samples were immunoblottedwith antibodies to the PH domain and PKC ${zeta}$ as described above.

PKC ${zeta}$ /PKB ${alpha}$ overlay assay. Protein-protein overlay assays were carried out by using recombinantGST-PKC ${zeta}$ , GST-PKB ${alpha}$ , and GST-PKB ${alpha}$ lacking the PH domain (PKB ${Delta}$ PH).Briefly, 0.1 µg of GST-PKC ${zeta}$ and 50 µg of L6 lysatewere subjected to SDS-10% PAGE and then immunoblotted. Immobilonmembranes were next incubated overnight at 4°C with 0.1µg of recombinant PKB and PKB ${Delta}$ PH proteins, as well as 500µg of L6 cell lysate. Membranes were then incubated withantibodies against PKC ${zeta}$ , PKB ${alpha}$ , and c-myc and probed with HRP-conjugatedanti-mouse and anti-rabbit antibodies, and immunoreactivitywas visualized by enhanced chemiluminescence as described above.

Protein-lipid overlay assay. To assess whether C₂-ceramide and PKC ${zeta}$ affect the phospholipid-bindingproperties of PKB, a protein-lipid overlay assay was carriedout with GST fusion proteins of PKC ${zeta}$ and PKB ${alpha}$ as described previously(23, 26). Briefly, 1 µl of PIP₃ (500 pM stock) dissolvedin chloroform-methanol-water (1:2:0.8) was spotted onto HybondC-Extra membrane (Amersham Pharmacia Biotech, Amersham, UnitedKingdom) and allowed to dry for 1 h at room temperature. Membraneswere blocked with 5% (wt/vol) bovine serum albumin in Tris-bufferedsaline-Tween (TBST; 10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1%[vol/vol] Tween 20) for 1 h at room temperature. Membranes werethen incubated overnight at 4°C in TBST containing 5% (wt/vol)bovine serum albumin, recombinant proteins, and C₂-ceramideat the concentrations indicated in the figure legends. Afterthis incubation period, membranes were washed with TBST beforeincubation with an anti-GST antibody (1:1,000). Membranes werethen probed with HRP-conjugated anti-mouse antibody, and theimmunoreactivity was visualized by enhanced chemiluminescence.

PKB ${alpha}$ PH domain mutagenesis. Site-directed mutagenesis was carried out by using the QuikChangekit (Stratagene). 3' and 5' primers (3'-CCTGAAAAACGACGGAGCATTTATAGGTTACand 5'-GTAACCTATAAATGCTCCGTCGTTTTTCAGG) were used to mutateThr³⁴ to an Ala in a pGEX4.1 construct encoding wild-type PKB ${alpha}$ PH domain. The resulting plasmid was transformed into XL1-Bluecells and then harvested by using a Qiagen plasmid Miniprepkit according to the manufacturer's protocol. The sequence ofthe mutated PH domain was verified by using an automated DNAsequencer (model 373; Applied Biosystems).

Analysis of cell viability. To assess cell viability in response to ceramide, subconfluentL6 myoblasts were transfected with 1 µg of the indicatedconstructs by using the Fugene 6 system as described above.After 48 h, muscle cells were incubated in the absence or presenceof either 100 µM C₂-ceramide or C₂-ceramide plus 5 µMRo 31.8220. Viable cells were classified as those that wereadherent, displayed trypan blue exclusion, and stained positivelywith DAPI. Stained cells were visualized by using an Axiovert200 fluorescence microscope and quantitated by counting individualnuclei from several randomly chosen visual fields.

Statistical analyses. One-way analysis of variance, followed by a Newman-Keuls posttest,was used to assess statistical significance. The data analysiswas performed by using GraphPad Prism software and consideredstatistically significant at P values of <0.05.

RESULTS

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Results

Ceramide inhibits the insulin-mediated phosphorylation and activation of PKB: effect of kinase inhibitors. In accord with our previous work (26), Fig. 1A and B show thatceramide treatment abolishes completely the insulin-inducedphosphorylation and activation of PKB. As a first step in attemptingto establish whether ceramide mediates this inhibition via PKCs,we investigated the effects of Ro 31.8220, a bisindolemaleimide,that potently inhibits conventional and novel PKCs in an ATP-competitivemanner with 50% inhibitory concentration values in the submicromolarrange (2, 43) and atypical PKCs in the micromolar range (43).At submicromolar concentrations, Ro 31.8220 had no significanteffect on the loss in PKB activation elicited by ceramide. However,this inhibition was progressively reversed when muscle cellswere incubated with increasing concentrations of Ro 31.8220in the micromolar range that inhibited atypical PKCs (Fig. 1A and B).Similar results were obtained with GF 109203X, anotherstructurally unrelated bisindolemaleimide (data not shown).

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FIG. 1. Representative blots showing the effects of ceramide and kinase inhibitors on the insulin-mediated phosphorylation of PKB and GSK3. L6 myotubes were incubated in the absence or presence of C₂-ceramide (Cer, 100 µM) for 2 h. In some experiments, cells were preincubated with Ro 31.8220 (Ro, at the concentrations indicated) for 30 min prior to incubation with ceramide. At the end of this incubation period cells were incubated with insulin (Ins, 100 nM) for a further 10 min before being lysed. (A and B) Cell lysates were then immunoblotted with a phospho-specific antibody directed against PKB-Ser⁴⁷³, PKB-Thr³⁰⁸, or PKB (A) and used for assaying PKB activity as described in the text (B). (C) L6 myotubes were incubated in the absence or presence (singularly or in combination) of kinase inhibitors at the indicated concentrations for 30 min prior to incubation with C₂-ceramide (100 µM) for 2 h and with insulin (100 nM) for 10 min. Cell lysates were immunoblotted with a phospho-specific antibody directed against PKB-Ser⁴⁷³, with blots being reprobed with an antibody against p38 MAPK (which was used as a marker for protein loading). (D) L6 myotubes were pretreated with Ro 31.8220 (Ro, 5 µM) for 30 min prior to incubation with C₂-ceramide (100 µM) for 2 h and insulin (Ins, 100 nM) for 10 min. Cell lysates were immunoblotted with phospho-specific antibodies directed against either GSK3 ${alpha}$ -Ser²¹, GSK3ß-Ser⁷, PKB-Ser⁴⁷³, or PKB.

The selectivity of both Ro 31.8220 and GF109203X has been questionedin recent years, since evidence exists that these inhibitorsalso suppress the activity of other kinases, including, forexample, S6K1, GSK3ß, MSK1, and MAPKAP-K1 (2, 20).To assess the possible involvement of these kinases in the ceramide-inducedinhibition of PKB, we preincubated L6 myotubes with Ro 31.8220,PD 98059 (which inhibits the classical MAPK [Erk] pathway),SB-203580 (which inhibits the p38 MAPK pathway), rapamycin (whichinhibits the mTOR pathway), SB-216763 (which selectively inhibitsGSK3 subtypes), and PD 98059 and SB-203580 together (which suppressactivation of MSK1) (20). The ability of these compounds toinhibit their respective target pathways in our cell systemwas verified in separate experiments (data not shown). Figure1C shows that, with the exception of Ro 31.8220 (when used at5 µM), none of the inhibitors tested could prevent theloss in PKB activation elicited by ceramide.

We hypothesized that if Ro 31.8220 was able to suppress theinhibitory effects of ceramide on PKB activation, then the inhibitorshould also reinstate PKB signaling to downstream targets, suchas GSK3 (17). Figure 1D shows that insulin induces phosphorylationof PKB and both GSK3 isoforms but that this was reduced substantiallyin ceramide-treated cells. However, this reduction was attenuatedsignificantly when muscle cells were preincubated with 5 µMRo 31.8220 prior to treatment with ceramide (Fig. 1D).

Classical and novel PKC isoforms are unlikely to mediate the loss in PKB activation by ceramide. In order to exclude the involvement of the classical and novelPKC isoforms as potential players in the regulation of PKB byceramide, we incubated L6 cells with 1 µM PMA for 15 min,2 h, and 20 h prior to incubation with ceramide. Short-termPMA treatment (up to 2 h) stimulates classical and novel PKCisoforms, whereas long-term PMA exposure (24 h) downregulatestheir expression (18, 39). Figure 2A shows that neither shortnor long-term incubation of muscle cells with PMA had any detectableeffect on ceramide's ability to inhibit the hormonal activationof PKB. This finding suggests that it is highly unlikely thatclassical or novel PKC isoforms are utilized by ceramide aseffector molecules that regulate PKB activation.

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FIG. 2. Effects of insulin, ceramide, and PMA on PKB and PKC ${zeta}$ phosphorylation in L6 myotubes. (A) L6 myotubes were pretreated with 1 µM PMA for the times indicated prior to incubation with C₂-ceramide (Cer, 100 µM) for 2 h and with insulin (Ins, 100 nM) for 10 min. Cells lysates were immunoblotted with a phospho-specific antibody directed against PKB-Ser⁴⁷³ or PKB-Thr³⁰⁸. (B) L6 myotubes were incubated in the absence or presence of Ro 31.8220 (Ro, 5 µM) for 30 min prior to treatment with C₂-ceramide (Cer, 100 µM) for 10 min. Myotubes were harvested, and plasma membranes were isolated by subcellular fractionation as described in the text. Plasma membranes (20 µg of protein) were subjected to SDS-PAGE and immunoblotted with antibodies against the ${alpha}$ 1 subunit of the Na/K-ATPase (a plasma membrane marker) and PKC ${zeta}$ . (C) In vitro activation of PKC ${zeta}$ was assessed by incubating 0.1 µg of recombinant PKC ${zeta}$ protein with C₂-ceramide (Cer, 100 µM) and Ro 31.8220 (Ro, 5 µM) in the presence of [ ${gamma}$ -³²P]ATP, PS (4 pg/ml), and 5 mM MgCl₂ at 30°C for 20 min as described in the text. PKC ${zeta}$ was subsequently resolved by SDS-PAGE and subjected to analysis by autoradiography. Loading of PKC ${zeta}$ on SDS gels was assessed by probing the transfer membrane with an antibody directed against PKC ${zeta}$ . (D) L6 myotubes were incubated with C₂-ceramide (Cer, 100 µM) for the times indicated and then lysed. Cells lysates were immunoblotted with a phospho-specific antibody directed against PKC ${zeta}$ -Thr⁴¹⁰ or PKC ${zeta}$ .

Ceramide activates PKC ${zeta}$ . The data presented in Fig. 1 and 2A provide prima face evidencethat ceramide may suppress the hormonal activation of PKB viaatypical PKCs. To test this possibility further, we determinedwhether ceramide activates PKC ${zeta}$ in L6 myotubes. Braiman et al.(12) have previously shown that activation of PKC ${zeta}$ in culturedrat skeletal muscle cells involves its increased associationwith the plasma membrane. We therefore isolated plasma membranesfrom L6 myotubes after treatment with ceramide and/or Ro 31.8220and then assessed PKC ${zeta}$ abundance by immunoblotting. Figure 2Bshows that ceramide increased the plasma membrane abundanceof PKC ${zeta}$ but that this was reduced in cells that had been pretreatedwith 5 µM Ro 31.8220. In contrast, the abundance of the ${alpha}$ 1 subunit of the Na/K-ATPase, a plasma membrane marker, wasunaltered. The observation that Ro 31.8220, an ATP competitiveinhibitor, suppresses ceramide-induced recruitment to the plasmamembrane implies that the basal activity of PKC ${zeta}$ may be requiredto support its translocation and subsequent activation at thecell surface. This suggestion is consistent with a previousstudy reporting that autophosphorylation of PKC ${zeta}$ on Thr⁵⁶⁰ iscrucial for supporting the activation of the kinase in responseto stimuli such as insulin (7). Since ceramide has been shownto directly activate PKC ${zeta}$ in vitro (10) and the kinase is autophosphorylatedupon activation (42), we also assessed whether ceramide enhanced³²P incorporation into PKC ${zeta}$ in vitro. Figure 2C shows that incubationof PKC ${zeta}$ with ceramide led to increased ³²P labeling, which wassuppressed significantly in the presence of Ro 31.8220. In additionto autophosphorylation, activation of PKC ${zeta}$ requires phosphorylationof its T-loop residue (Thr⁴¹⁰). A phospho-specific antibodydirected against this residue revealed detectable phosphorylationof this site even in unstimulated muscle cells, which was enhancedupon ceramide treatment of cells in a time-dependent manner(Fig. 2D).

A myristoylated PKC ${zeta}$ pseudosubstrate inhibitor and cellular expression of a kinase-dead PKC ${zeta}$ (kd-PKC ${zeta}$ ) overcome the loss in PKB activation elicited by ceramide. To further establish the involvement of PKC ${zeta}$ as a ceramide effectormolecule, we utilized a myristoylated PKC ${zeta}$ pseudosubstrate peptide,which has been shown to specifically inhibit PKC ${zeta}$ activity (43).Pretreatment of muscle cells with the peptide prior to incubationwith ceramide suppressed the ceramide-mediated loss in PKB phosphorylationon both Ser⁴⁷³ and Thr³⁰⁸ normally elicited by insulin (Fig.3A). In contrast, no such protection against the inhibitoryeffects of ceramide was afforded in cells preincubated witha myristoylated PKC ${alpha}$ pseudosubstrate peptide inhibitor (Fig.3B). Collectively, the data obtained by using the bisindolemaleimidecompounds and the peptide inhibitors implies that the catalyticactivity of PKC ${zeta}$ is required for mediating the inhibitory effectsof ceramide on PKB activation. To strengthen this proposition,we investigated whether the expression of a kd-PKC ${zeta}$ would antagonizethe inhibition exerted by ceramide. Figure 3C shows that, comparedto cells transfected with the expression vector alone or overexpressingwild-type PKC ${zeta}$ , cells expressing the kd-PKC ${zeta}$ displayed enhancedphosphorylation of PKB Ser⁴⁷³ in response to insulin. Furthermore,although ceramide treatment abolished insulin's ability to promotePKB phosphorylation in cells expressing the empty vector orthe wild-type PKC ${zeta}$ , the hormone was still capable of inducingPKB phosphorylation in cells expressing kd-PKC ${zeta}$ . The transfectionefficiency in these studies was $~$ 60%. Consequently, the hormonalactivation of PKB would still be susceptible to the inhibitionby ceramide in a significant proportion of the cells not expressingthe kd-PKC ${zeta}$ , which would account for the slightly lower intensityof the Ser⁴⁷³ phosphorylation signal on the immunoblot.

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FIG. 3. A myristoylated PKC ${zeta}$ pseudosubstrate peptide inhibitor and expression of a kinase-inactive PKC ${zeta}$ mutant attenuate the inhibitory effects of ceramide on the insulin-induced phosphorylation of PKB in muscle cells. (A) L6 myotubes were pretreated with PKC ${zeta}$ myr-pseudosubstrate peptide (40 µM) or (B) with PKC ${alpha}$ myr-pseudosubstrate peptide (50 µM) for 30 min prior to incubation with C₂-ceramide (100 µM) for 2 h and with insulin (100 nM) in the penultimate 10-min period prior to cell lysis. Cells lysates were immunoblotted with a phospho-specific antibody directed against PKB-Ser⁴⁷³ or PKB-Thr³⁰⁸. (C) L6 myoblasts were transfected with pCMV5 lacking or containing cDNA encoding kd-PKC ${zeta}$ or wild-type PKC ${zeta}$ as described in the text. Cells were then incubated with C₂-ceramide (100 µM) for 2 h prior to treatment with insulin (100 nM) for 10 min. Cell lysates were subsequently immunoblotted with a phospho-specific antibody to PKB-Ser⁴⁷³ or with an antibody to PKB.

PKC ${zeta}$ interacts with PKB in vitro and in vivo. To understand the mechanism by which PKC ${zeta}$ may negatively regulatePKB activation in response to ceramide, we assessed whetherthe two kinases could interact with each other. Immunoprecipitationof PKB from unstimulated L6 myotubes revealed that PKC ${zeta}$ couldbe coprecipitated (Fig. 4A). Interestingly, when PKB ${alpha}$ was immunoprecipitatedfrom cells after incubation with insulin, the amount of PKC ${zeta}$ coprecipitated was significantly less, indicating that insulinpromotes dissociation of the PKB-PKC ${zeta}$ complex. However, the interactionbetween the two kinases was enhanced after treatment of cellswith ceramide and, more importantly, the lipid abolished insulin'sability to induce dissociation of the kinase complex (Fig. 4A).

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FIG. 4. PKB and PKC ${zeta}$ physically interact with each other both in vivo and in vitro, and this interaction requires the PH domain of PKB. (A) L6 myotubes were incubated in the absence or presence of C₂-ceramide (100 µM) for 2 h and/or with insulin (100 nM) in the penultimate 10-min period prior to cell lysis. PKB ${alpha}$ was immunoprecipitated from cell lysates and resolved by SDS-PAGE prior to immunoblotting with antibodies to PKB or PKC ${zeta}$ . (B) Far-Western analysis was performed by resolving GST-tagged PKC ${zeta}$ (0.1 µg of protein) and whole-cell lysates from L6 cells (WCL, 50 µg of protein) by SDS-PAGE, followed by immobilization on polyvinylidene difluoride (PVDF) membranes. Membranes weresubsequently incubated overnight at 4°C with either 0.1 µg of recombinant PKB or 250 µg of whole-cell lysates prior to immunoblotting with antibodies directed against PKC ${zeta}$ (lane 1) or PKB (lanes 2 and 3). As a negative control, membranes retaining PKC ${zeta}$ were also probed with an antibody directed to c-myc (lane 4) after overnight incubation with whole-cell lysates. Expression of c-myc in L6 lysates was confirmed by probing whole-cell lysates with a c-myc antibody (lane 5). (C) To assess the importance of the PKB-PH domain for kinase interaction, far-Western analysis was performed by resolving 50 µg of whole-cell lysates and 0.1 µg of GST-PKC ${zeta}$ protein by SDS-PAGE, followed by immobilization of PKC ${zeta}$ on PVDF membranes. Membranes were subsequently incubated overnight at 4°C with either 0.1 µg of recombinant PKB or 0.1 µg of recombinant PKB lacking its PH domain (PKB ${Delta}$ PH) prior to immunoblotting with antibodies to PKC ${zeta}$ (lanes 1 and 2) or PKB (lanes 3 to 6). To confirm the presence of PKC ${zeta}$ on the membranes, lanes 3 to 6 were subsequently stripped and probed with an antibody to PKC ${zeta}$ .

In addition to the interaction observed in vivo, we also observedthat PKB and PKC ${zeta}$ can interact in vitro, as assessed by usinga far-Western (protein-protein overlay) assay. Recombinant PKBor PKB present within whole-cell lysates was capable of associatingwith PKC ${zeta}$ (Fig. 4B, lanes 2 and 3). In contrast, c-myc did notshow any detectable binding to PKC ${zeta}$ , suggesting that the observedinteraction between PKB and PKC ${zeta}$ was unlikely to be nonspecific(Fig. 4B, lane 5). Our in vivo and in vitro data are consistentwith previous work showing that PKC ${zeta}$ interacts with PKB via itsAkt homology domain (9, 22, 33, 35). Since ceramide inhibitsthe cell surface recruitment of the isolated PH domain of PKBin fibroblasts (45), we investigated whether the interactionbetween the two kinases requires the PH domain of PKB. Far-Westernanalysis revealed that only PKB possessing its PH domain wascapable of interacting with PKC ${zeta}$ (Fig. 4C).

Activated PKC ${zeta}$ suppresses the binding of PIP₃ to the PH-domain of PKB. Since the PH domain of PKB plays a critical role in 3-phosphoinositidebinding, it is plausible that the association of PKC ${zeta}$ with thisdomain inhibits PIP₃ binding, resulting in loss of PKB activation.A protein-lipid overlay assay, which allows a qualitative assessmentof the binding between PIP₃ and PKB (23), was performed to testthis possibility. Figure 5A shows that PKB binds to PIP₃ spottedon nitrocellulose filters and that this interaction was notaffected by the presence of ceramide (lane 3), an observationin line with previous work (26). PKC ${zeta}$ did not bind PIP₃, andthis interaction was not promoted even in the presence of ceramide(Fig. 5A, lanes 4 and 5). Moreover, the presence of PKC ${zeta}$ in thein vitro assay did not suppress the binding of PKB to PIP₃.However, in vitro activation of PKC ${zeta}$ , achieved by inclusion ofceramide in the incubation solution, led to a dramatic lossin PKB binding to PIP₃ (Fig. 5A, lane 7). To further substantiatethat activation of PKC ${zeta}$ interferes with the PKB-PIP₃ binding,additional experimental controls were performed in which ATP,PS and Mg²⁺ (required to support PKC ${zeta}$ activation) were eitherexcluded, or Ro 31.8220 (PKC inhibitor) was added to the incubationsolution. Under these conditions, the inclusion of ceramidedid not inhibit the interaction between PKB and PIP₃ (Fig. 5B,lanes 4 and 5).

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FIG. 5. Protein-lipid overlay assay showing that in vitro activation of PKC ${zeta}$ by ceramide leads to a loss in PIP₃ binding to PKB. (A) Nitrocellulose membranes were spotted with 1 µl of 500 pM PIP₃. Membranes were then incubated overnight at 4°C in TBST buffer containing 1 µM ATP, 4 pg of PS/ml, and 5 mM MgCl₂. This buffer also contained or lacked GST-PKB (0.5 µg/ml), GST-PKC ${zeta}$ (0.5 µg/ml), and/or C₂-ceramide (Cer, 100 µM) as indicated. Membranes were subsequently washed, and bound PKB was detected by probing with an anti-GST antibody. (B) To assess the importance of PKC ${zeta}$ activation in influencing the binding of PIP₃ to PKB, the experiment in panel A was repeated but with TBST buffer that either lacked ATP, PS, and MgCl₂ or which had been supplemented with Ro 31.8220 (Ro, 5 µM), as indicated.

PKC ${zeta}$ phosphorylates the PH domain of PKB. Since the interaction between PKC ${zeta}$ and PKB requires the PH domainof PKB and binding of PIP₃ to this domain can be suppressedby ceramide-activated PKC ${zeta}$ , we hypothesized that PKC ${zeta}$ may phosphorylatethe PH domain. To test this hypothesis, we assessed whetherPKC ${zeta}$ phosphorylates the isolated PKB PH domain in vitro. Figure6A shows that, in the absence of ceramide but in the presenceof ATP, PS, and MgCl₂, PKC ${zeta}$ induces a detectable incorporationof labeled phosphate into the PH domain (lane 3). The incorporationof label was increased by $~$ 3-fold upon activation of PKC ${zeta}$ by inclusionof ceramide in the assay mixture (lane 4).

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FIG. 6. In vitro activation of PKC ${zeta}$ by ceramide results in the phosphorylation of the isolated PH domain of PKB on Thr³⁴ with important consequences for PIP₃ binding. (A) The isolated PH domain of PKB (1 µg) was incubated in the absence or presence of 30 ng of PKC ${zeta}$ and/or C₂-ceramide (Cer, 100 µM) in buffer containing [ ${gamma}$ -³²P]ATP and cofactors required to support kinase activation for 20 min at 30°C as described in the text. Phosphorylated proteins were then resolved by SDS-PAGE and transferred to PVDF membranes prior to autoradiography and immunoblotting with anti-PH antibodies. (B) To identify putative PKC ${zeta}$ phosphorylation sites within the isolated PH domain of all three PKBisoforms, a web-based motif-scanning analysis tool was used (49). The aligned peptide sequences of all three PKB isoforms are shown and highlight the putative PKC ${zeta}$ phosphorylation site. (C) In vitro phosphorylation of wild-type (PH-wt) and T34A mutant PH (PH-T34A) domains was performed by incubating 30 ng of recombinant PKC ${zeta}$ with the appropriate PH peptide (1 µg of protein) in the presence of 1 µM [ ${gamma}$ -³²P]ATP and cofactors required for supporting kinase activation but in the absence or presence of C₂-ceramide (Cer, 100 µM) for 20 min at 30°C as indicated. PH peptides were then resolved by SDS-PAGE and subjected to analysis by autoradiography. Protein loading was subsequently assessed with an antibody directed against the PKB-PH domain. (D) Far-Western analysis was performed by resolving recombinant PKC ${zeta}$ (0.1 µg of protein) by SDS-PAGE and transferring it onto PVDF membranes. The membranes were then incubated overnight at 4°C with either 0.1 µg of PH-wt (lane 2) or 0.1 µg of PH-T34A peptide (lane 3) prior to immunoblotting them with antibodies to PKC ${zeta}$ (lane 1) or the PH domain of PKB (lanes 2 to 4). (E) Protein-lipid overlay was performed to assess PIP₃ binding to the PH-wt and T34A-PH peptides. Nitrocellulose membranes were spotted with 1 µl of PIP₃ and subsequently incubated overnight at 4°C in TBST buffer containing 1 µM ATP, 4 pg of PS/ml, and 5 mM MgCl_2. The incubation buffer either contained or lacked 0.5 µg of the appropriate PH peptide/ml, 0.5 µg of PKC ${zeta}$ /ml, and C₂-ceramide (Cer, 100 µM) as indicated. Membranes were washed, and bound PH protein was detected by probing samples with an anti-PH domain antibody.

To identify the site(s) phosphorylated within the PH domain,we utilized a peptide library-based searching algorithm thatidentifies sequence motifs likely to be phosphorylated by specificprotein kinases (49). High-stringency analysis revealed a singlesurface-accessible PKC ${zeta}$ phosphorylation site in all three PKBisoforms at residue 34 (Fig. 6B, Thr in PKB ${alpha}$ and Ser in PKBßand PKB ${gamma}$ ). To establish whether this site was phosphorylatedin vitro by PKC ${zeta}$ , we mutated Thr³⁴ in the PH domain of PKB ${alpha}$ toan alanine. Figure 6C shows that ceramide-activated PKC ${zeta}$ phosphorylatesthe wild-type PH peptide but not the T34A mutant peptide. Sincethe mutant peptide still interacts with PKC ${zeta}$ , the lack of anydetectable phosphorylation cannot be attributed to a loss inits physical interaction with the kinase (Fig. 6D). This findingimplies that, although Thr³⁴ is phosphorylated by PKC ${zeta}$ , it isunlikely to play a critical role in supporting the interactionbetween the two kinases.

Since activation of PKC ${zeta}$ inhibits PIP₃ binding to PKB (presumablydue to phosphorylation of site 34 within the PH domain), weinvestigated whether the T34A-PH protein retains the capacityto bind PIP₃ in the presence of active PKC ${zeta}$ . Figure 6E showsthat the wild-type PH protein binds PIP₃ (lanes 2 and 3)_, butits ability to do so is lost in the presence of ceramide-activatedPKC ${zeta}$ (lane 4). The T34A-PH protein also binds PIP₃ (Fig. 6E,lanes 5 and 6) but, unlike the wild-type protein fragment, itretains the ability to bind PIP₃ even in the presence of anactive PKC ${zeta}$ (Fig. 6E, compare lanes 4 and 7). It is plausiblethat the T34A-PH peptide may have a higher binding affinityfor PIP₃ than the wild-type PH fragment, which could potentiallyexplain the observed binding of PIP₃ to the mutated PH fragmentin the presence of activated PKC ${zeta}$ . However, using a sensitiveFRET-based assay that monitors the displacement of biotinylated-PIP₃from GST-tagged PH peptide by nonbiotinylated lipid (24), wecould not detect any significant differences in the PIP₃ displacementprofiles between the wild type and the T34A PH peptide (datanot shown).

PKB is phosphorylated on Thr³⁴ in response to ceramide in vivo. To establish whether ceramide induces phosphorylation of PKBon Thr³⁴ in intact cells, we probed cell lysates with a phospho-specificantibody against this site. Figure 7A shows that the antibodydetected an immunoreactive protein band corresponding to PKBfrom ceramide-treated cells but not from untreated cells. Thespecificity of this signal was verified by demonstrating thatantisera that had been preadsorbed with the antigenic phospho-peptidefailed to detect the phosphorylated kinase. We subsequentlyinvestigated whether the ceramide-induced phosphorylation ofthis site could be influenced by insulin and Ro 31.8220. Figure7B shows that insulin per se had no effect on Thr³⁴ and, moreover,does not appear to antagonize phosphorylation of this site inresponse to ceramide. However, incubation of cells with 5 µMRo 31.8220 prior to treatment with ceramide led to a noticeablereduction in the phosphorylation of Thr³⁴.

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FIG. 7. Ceramide induces phosphorylation of PKB on Thr³⁴ in intact cells and a PKB T34A mutant is ceramide resistant. (A) L6 myotubes were incubated in the absence or presence of C₂-ceramide (Cer, 100 µM) for 2 h prior to cell lysis. Cell lysates were resolved by SDS-PAGE prior to immunoblotting with an antibody to PKB, a phospho-specific antibody to PKB-Thr³⁴, or anti-PKB-Thr³⁴ that had been preadsorbed the antigenic phospho-peptide (100 µg/ml). (B) Myotubes were treated as in panel A but, in addition, were also exposed to insulin (Ins, 100 nM for 10 min) or pretreated with Ro 31.8220 (Ro, 5 µM) prior to incubation with Cer. Cells were lysed and immunoblotted with antibodies to PKB or PKBThr³⁴. (C) HA-tagged PKB (wild type) and HA-tagged PKB T34A were transiently transfected into L6 cells as described. Cells were exposed to C₂-ceramide (Cer, 100 µM, 2 h) and or insulin (Ins, 100 nM, 10 min) prior to cell lysis and immunoprecipitation with an anti-HA antibody. Precipitated kinases were resolved by SDS-PAGE and immunoblotted with antibodies to PKB-Ser⁴⁷³ or anti-HA. Phospho-PKB-Ser⁴⁷³- and HA-immunoreactive bands were quantified and are expressed as a ratio (lower panel).

We postulated that if phosphorylation of PKB on Thr³⁴ contributesto its inhibition by ceramide, then a PKB T34A mutant shouldbe resistant to ceramide. To test this, we transiently expressedHA-tagged wild-type PKB and HA-tagged PKB T34A into L6 cells.Cells expressing these kinases were then incubated with ceramideand/or insulin prior to immunoprecipitation and immunoblottingwith an antibody to PKB-Ser⁴⁷³ or HA. Figure 7C shows that wild-typePKB is phosphorylated on Ser⁴⁷³ in response to insulin but thatthis was reduced by incubation of cells with ceramide. The PKBT34A mutant was also phosphorylated on Ser⁴⁷³ by insulin treatmentbut, in contrast to the wild-type kinase, phosphorylation ofthis site was retained in cells incubated with ceramide. Owingto slight variations in the amounts of the HA-tagged kinasesthat were immunoprecipitated among the different experimentaltreatments, the immunoblot data were quantified and normalizedby expressing them as ratios of the signal intensities of Ser⁴⁷³to HA (Fig. 7C, lower bar panel).

Physiological implications. Previous work has shown that PKC ${zeta}$ plays a key role in inducinggrowth arrest in vascular smooth muscle cells in response toceramide and that a key feature of this mechanism is a lossin PKB activation (9). L6 myotubes are terminally differentiatedsyncytia and, as such, display minimal cell cycle or growthactivity. Thus, to assess the importance of PKC ${zeta}$ in ceramide-inducedcell death, we transfected wild-type PKC ${zeta}$ , kd-PKC ${zeta}$ , and the PKBT34A mutant into L6 myoblasts. PKB activation is also diminishedby ceramide in mononucleated muscle cells (data not shown) butmyoblasts, unlike myotubes, exhibit significant cell cycle andgrowth activity and display a far greater sensitivity to ceramide;consequently, they are more suited for cell viability studies.Based on nuclear counting of adherent viable cells in severalrandomly chosen visual fields, ceramide treatment led to an $~$ 60% loss in the number of myoblasts expressing the empty expressionvector compared to an untreated cell population (Fig. 8A and B).The sensitivity to ceramide was enhanced in cells overexpressingthe wild-type PKC ${zeta}$ on the basis that cell viability was reducedfurther ( $~$ 80%, Fig. 8A and B). However, the increase in cellloss was not apparent when control (empty vector) or wild-typeexpressing cells were incubated with the inactive C2-dihydroceramideanalogue (data not shown). Moreover, the effects of ceramidecould be significantly reduced by pretreating cells with 5 µMRo 31.8220 (Fig. 8A and B). Interestingly, expression of thekd-PKC ${zeta}$ in L6 myoblasts conferred significant resistance to celldeath induced by ceramide (Fig. 8A and B). These findings areconsistent with the notion that PKC ${zeta}$ plays an important rolein reducing cell survival rate in response to ceramide. SincePKB activation would be suppressed under circumstances whenPKC ${zeta}$ is activated by ceramide (Fig. 1), we subsequently assessedwhether expression of a PKB T34A mutant offered any protectionagainst the death-inducing effects of ceramide. Figure 8C and Dshow that the ability of ceramide to promote cell loss waslower in cells expressing the PKB T43A mutant compared to thatof a control cell population transfected with the empty expressionvector. Interestingly, we also noted in separate experimentsthat myoblasts stably expressing a constitutively active membrane-targetedPKB that is resistant to inhibition by ceramide (26, 46) alsoexhibit a higher survival potential in the presence of ceramide(data not shown).

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FIG. 8. Effects of ceramide on muscle cell viability. L6 myoblasts transiently transfected with pCMV5 lacking or containing cDNA encoding wild-type PKC ${zeta}$ , kd-PKC ${zeta}$ , or PKB T34A were incubated with either vehicle alone (dimethyl sulfoxide), C₂-ceramide (Cer, 100 µM, 2 h), or C₂-ceramide (100 µM, 2 h) plus Ro 31.8220 (5 µM, added 15 min prior to ceramide). Ceramide promotes detachment and death of myoblasts, and thus viable cells were those that remained adherent, displayed trypan blue exclusion, and stained positively for DAPI. (A and C) Representative images of DAPI-stained L6 cells; (B and D) quantitative analysis of viable cells from five randomly chosen visual fields. Cell loss was expressed as a percentage change in cell number relative to that from the appropriate untreated experimental cell population. Asterisks signify significant changes (P < 0.05) between the indicated bars or compared to an untreated cell population, as determined by one-way analysis of variance.

DISCUSSION

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Discussion

Previous work from our group has shown that C₂-ceramide inhibitsthe hormonal activation of PKB in L6 muscle cells by suppressingits cell surface recruitment despite the fact that insulin enhancescellular PIP₃ levels (26). This finding is in agreement witha similar study showing that ceramide blocks the stimulus-inducedrecruitment of the PH domain of PKB in NIH 3T3 fibroblasts (45).Collectively, these observations suggest that ceramide may eitherdirectly regulate PKB recruitment via interaction with its PHdomain or alternatively targets an ancillary molecule(s) involvedin regulating its translocation and activation. There is noevidence supporting the former possibility in the literatureand, indeed, we have shown previously that ceramide does notassociate with the PH domain of PKB nor does it compete or interferewith PIP₃ binding to the kinase (26). Here we show that anothermember of the AGC family of kinases, atypical PKC ${zeta}$ , interactswith the PH domain of PKB and that it is activated by ceramide.We present novel data showing that activation of PKC ${zeta}$ resultsin phosphorylation of Thr³⁴ in the PH domain of PKB ${alpha}$ and thatthis leads to a loss in PIP₃ binding to the kinase PH domainin vitro. Suppressing PKC ${zeta}$ activation by using chemical or peptide-basedinhibitors or by expressing a dominant-interfering kd-PKC ${zeta}$ , alleviatesthe inhibitory effect of ceramide on PIP₃ binding to the PHdomain of PKB and its activation by insulin.

The finding that PKC ${zeta}$ interacts with PKB is not unprecedented.Konishi et al. first reported this interaction in COS-7 cellsnearly a decade ago (33). Subsequently, work by other investigatorshas shown that these two kinases can form stable complexes inCHO (22), breast cancer (35), and vascular smooth muscle (9)cells and that, within these complexes, PKC ${zeta}$ appears to negativelyregulate PKB activity. The interaction between the two kinaseswould be expected to be regulated in order to allow the positiveflow of signals through the PI3K/PKB pathway in response tostimuli such insulin and growth factors. This proposition isindeed supported by the finding that insulin and platelet-derivedgrowth factor induce dissociation of PKB and PKC ${zeta}$ in L6 (Fig.4A) and COS-1 cells (22), respectively. Precisely how dissociationof the complex is achieved is understood poorly, but it hasbeen suggested that PKB activity is a key requirement for thisevent, whereas activation of PKC ${zeta}$ appears not to be crucial inthis regard (22). Nevertheless, it is difficult to negate thepossibility that PKC ${zeta}$ activity may serve to stabilize or preventdissociation of the complex based on the observation that ceramide,which activates PKC ${zeta}$ , not only enhances the association betweenthe two kinases but also blocks insulin's ability to dissociatethe complex (Fig. 4A). In addition, the demonstration that PKCinhibitors and the expression of an inactive PKC ${zeta}$ alleviate thenegative regulation of PKB strengthens the idea that PKC ${zeta}$ activityexerts a powerful influence on PKB signaling (Fig. 1 and 3)(22, 35).

A key issue that has remained poorly understood concerns themechanism by which ceramide inhibits PKB activation. In somecell types, such as PC12 and C2C12 cells, ceramide activatesa protein phosphatase 2A-like activity, which dephosphorylatesPKB on Thr³⁰⁸ and Ser⁴⁷³ in an okadaic acid-sensitive manner(14, 40). However, in L6 muscle cells and 3T3-L1 adipocytes,okadaic acid does not antagonize ceramide action on PKB (26,46). The data presented here indicate that ceramide suppressesPKB activation by modulating the activity of PKC ${zeta}$ and by stabilizingits interaction with PKB. Recently, both mutational analysisand a high-resolution crystal structure of the isolated PKB-PHdomain complexed to PIP₃ have revealed that the key amino acidresidues that interact with the inositol head group of PIP₃are localized within the first two beta sheets of the PH domain(47). Since PKC ${zeta}$ also binds to this region (33), it is conceivablethat its association with PKB results in the competitive exclusionof PIP₃ with a subsequent loss of PKB activation. However, ourlipid-overlay data (Fig. 5A) indicate that in the absence ofceramide, PKC ${zeta}$ , and PIP₃ do not compete for PKB binding. Moreover,previous work has shown that PKB molecules can form homotypiccomplexes via interactions formed between their PH domains atregions that overlap with those important for 3-phosphoinositidebinding (19). Despite the apparent overlap in the lipid andprotein binding regions, it has been proposed that these complexescan be activated, implying that their formation per se is unlikelyto hinder PIP₃ binding to the PH domain (16, 19). However, whenPKB and PKC ${zeta}$ were incubated in the presence of ceramide therewas a dramatic loss in PIP₃ binding to PKB (Fig. 5A). Our findingsindicate that this loss stems from ceramide's ability to activatePKC ${zeta}$ , which then phosphorylates the PKB PH domain at Thr³⁴. Thesignificance of this phosphorylation in regulating PIP₃ bindingto PKB is underscored by the finding that a T34A PH domain mutantretains the ability to bind PIP₃ and to be activated by insulinin intact cells even in the presence of ceramide (Fig. 6E and7C). Since binding of PIP₃ to PKB is considered a prerequisitefor recruiting the kinase to the plasma membrane prior to phosphorylationby PDK1 and PDK2, the inability to bind this lipid in ceramidetreated cells would account for the loss in cell surface PKBrecruitment previously reported (26). Thr³⁴ is located withina region of the PH domain that is five and seven amino acidsdownstream, respectively, from Arg²⁵ and Arg²³, which play acritical role in PIP₃ binding (47) (Fig. 9). Initial analysisof the crystal structure of the PKB-PH domain suggests thatThr³⁴ is sufficiently removed from these critical arginine residuesto have any direct effect on PIP₃ binding. However, it is plausiblethat phosphorylation of Thr³⁴ may instigate changes in the conformationof the PKB-PH domain that either prevent PIP₃ binding throughsteric exclusion or, alternatively, affects the affinity ofthe kinase for the 3-phosphoinositide as a result of space-chargerepulsions from the negative phosphate groups.

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FIG. 9. Structure of PKB ${alpha}$ PH complexed to Ins(1,3,4,5)P₄. A ribbon structure of the PH domain of PKB ${alpha}$ , depicting the localization of Thr³⁴ relative to that of Arg²⁵ and Arg²³, which play a critical role in the binding of the inositol head group, is shown. Also labeled are the seven ß strands (labeled ß1 to ß7). The ribbon drawing is based on the crystal structure proposed by Thomas et al. (47).

Our observation (Fig. 4A) and that of others (9, 22) that PKC ${zeta}$ can form stable complexes with PKB prompts us to wonder whatrelevance this interaction may have in unstimulated cells. Onerecent study has proposed that this interaction may be importantfor recruiting a "PDK2-like" activity to this complex, whichphosphorylates the regulatory Ser residue in the C terminusof PKB (29). If this model of PKB activation is accepted, thenPKC ${zeta}$ may normally serve to deliver PKB to the cell surface andplace it in close proximity to its upstream kinases prior todissociating from the complex. However, this "delivery" processmay become impaired when PKC ${zeta}$ is activated by ceramide as a resultof its ability to block the interaction between PIP₃ and thePKB-PH domain, a step, which, as already indicated, is essentialfor recruiting PKB to the cell surface. The finding that ceramideinhibits the cell surface recruitment of PKB but fails to suppressthe activity of a membrane-targeted form of the kinase in muscleand fat cells is consistent with such a proposition (26, 46).

Reports showing that PKC ${zeta}$ can be activated by insulin and growthfactors in a PI3K-dependent manner (1, 43) support a positiverole for the kinase in cell signaling. However, evidence presentedhere and by others (9, 22) suggests that PKC ${zeta}$ can also regulatecell signaling in a negative fashion. Such diversity in PKC ${zeta}$ function has been documented previously, and there is now growingrecognition that the kinase acts as a molecular switch which,depending on the activating stimulus, either promotes or inhibitscell signaling (37). Since the ability of PKC ${zeta}$ to modulate PKBsignaling is stimulus dependent, establishing the mechanismby which different stimuli modulate PKC ${zeta}$ , including their siteof action, is likely to be important for understanding the controlof PKB-regulated cell functions. Ceramide production at theplasma membrane can be enhanced significantly in response tonumerous stress-inducing stimuli such as cytokines (e.g., TNF- ${alpha}$ ),heat shock, and oxidants (32). Indeed, the localization of theTNF- ${alpha}$ receptor and sphingomyelinases within caveola-like microdomainsin the plasma membrane (30) provides for an extremely effectiveway of producing a highly localized concentration of ceramide(21). Approximately 70% of the cellular ceramide is thoughtto be produced in such membrane domains (21), which may serveto localize proteins that bind the lipid with high affinity,such as PKC ${zeta}$ (37). This proposition is supported by evidencethat PKC ${zeta}$ interacts with caveolin and that PKC isoforms can accumulatein caveolae (38). Thus, it is tempting to speculate that anincrease in ceramide production in caveola-like microdomainsmay not only help localize and activate PKC ${zeta}$ but also sequesterthe PKB-PKC ${zeta}$ complex. If the complex does localize to caveola-likedomains and is spatially segregated from the upstream PKB kinases,then this may provide a mechanism for maintaining PKB in a repressedstate. Testing these possibilities will be important investigativegoals for future work.

Ceramide's ability to suppress PKB activation has importantimplications for numerous cellular responses. It is well documented,for example, that ceramide has negative effects on cell growthand survival (36), which in many cell types has been linkedto an inhibition in PKB activation (26, 40, 41, 46, 50). InL6 skeletal muscle cells and in vascular smooth muscle cells(9), this inhibition is mediated via activation of PKC ${zeta}$ by ceramidewith important consequences for cell viability. Indeed, theability of PKC inhibitors and of ceramide-resistant forms ofPKB to impede ceramide-induced cell death (Fig. 8) implies thatthe regulation of PKB by PKC ${zeta}$ is likely to be of physiologicalsignificance especially under circumstances when intracellularceramide synthesis may be modulated (e.g., in response to TNF- ${alpha}$ ,free fatty acids, hyperosmotic stress, and UV radiation, aswell as certain anti-cancer drug therapies) (31, 36, 51). Thus,a greater understanding of the physical interplay between PKBand PKC ${zeta}$ is likely to be of significant value for developingnovel strategies aimed at controlling cell growth, survival,and death.

In summary, the work presented here demonstrates that in ourexperimental system PKB interacts with PKC ${zeta}$ via its PH domainand that this association is reduced upon treating muscle cellswith insulin. However, when muscle cells are incubated withC₂-ceramide not only is insulin's capacity to dissociate thiskinase complex impaired, but the hormone fails to induce activationof PKB. Under these circumstances, ceramide stimulates PKC ${zeta}$ ,which phosphorylates the PH domain of PKB on Thr³⁴. Phosphorylationof this site suppresses the binding of PIP₃ to the PH domainof PKB, thereby severely limiting its activation in responseto insulin. Since PKB is thought to play a key role in regulatingcell survival and insulin action, the ability of ceramide tosuppress PKB signaling via regulation of PKC ${zeta}$ provides one potentialmechanism by which the lipid may promote cell death and induceinsulin resistance.

ACKNOWLEDGMENTS

We are grateful to Dario Alessi, Philip Cohen, and Peter Downesfor making available some of the reagents used in our studies.We thank Daan van Aalten for providing the structural imageof the isolated PH domain of PKB in complex with the head groupof PIP₃, and we thank Alex Gray (DSTT) and Maria Deak (MRC phosphorylationunit) for technical input in some of the experimental work.

This work was supported by the MRC, the Diabetes Research andWellness Foundation, and Diabetes UK.

FOOTNOTES

* Corresponding author. Mailing address: Division of Molecular Physiology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, United Kingdom. Phone: (44) 1382-344969. Fax: (44) 1382-345507. E-mail: h.s.hundal{at}dundee.ac.uk.

REFERENCES

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