MOLECULAR AND CELLULAR BIOLOGY,0270-7306/98/$04.0010

Feb. 1998, p. 839–845 Vol. 18, No. 2

Copyright © 1998, American Society for Microbiology

Activation of Protein Kinase C Triggers Its Ubiquitinationand Degradation†

ZHIMIN LU,1 DAVID LIU,2 ARMAND HORNIA,1 WAYNE DEVONISH,1 MICHELE PAGANO,2

AND DAVID A. FOSTER1*

Department of Biological Sciences, Hunter College and the Graduate School of the City University of New York,New York, New York 10021,1 and Department of Pathology, New York University Medical Center, New York,

New York 100162

Received 13 August 1997/Returned for modification 25 September 1997/Accepted 20 October 1997

Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion ofphorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has beenimplicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. Wereport here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms a, d, and« in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasomeinhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which resultsfrom the depletion of PKC d. Consistent with the involvement of the ubiquitin-proteasome pathway in thedegradation of PKC isoforms, ubiquitinated PKC a, d, and « were detected within 30 min of TPA treatment.Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC,suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activa-tion of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, akinase-dead ATP-binding mutant of PKC a could not be depleted by TPA treatment. These data are consistentwith a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasomepathway.

Tumor promotion by phorbol esters involves the selectiveamplification of cells previously mutated in an appropriategrowth-stimulatory gene (3, 17). Phorbol esters exert theireffects on the protein kinase C (PKC) family of genes, whichconsists of genes that encode at least nine distinct isoforms thatare responsive to tumor-promoting phorbol esters (9). Phorbolesters first activate phorbol ester-responsive PKC isoforms, butupon prolonged treatment, these isoforms are proteolyticallydegraded (16). Using a cell culture model system in which cellsoverexpressing c-Src were transformed by phorbol ester treat-ment, we recently demonstrated that the tumor-promoting ef-fect of the phorbol ester 12-O-tetradecanoylphorbol-13-ace-tate (TPA) on these cells was due to the depletion of PKC d(7). These data suggested that PKC d may function as a tumorsuppressor. Consistent with this hypothesis, PKC d was inacti-vated by tyrosine phosphorylation in cells transformed by v-Src(19) and v-Ras (2). Thus, regulation of PKC d at the level ofactivity and expression may be a very important cell growthcontrol mechanism.

PKC a has been reported to become ubiquitinated in re-sponse to bryostatin 1, an activator of PKC that prevents tumorpromotion in mouse skin by TPA (6). The ubiquitin-protea-some pathway is a nonlysosomal degradation system that con-trols the timed destruction of cell cycle-regulatory proteins,including the tumor suppressor p53; the cyclin-dependent ki-nase inhibitor p27; the cyclins; the oncogene products c-Myc,

c-Jun, and c-Fos; and the transcription factors NF-kB and E2F(reviewed in reference 13). This pathway involves the covalenttagging of proteins with ubiquitin, followed by proteasome-mediated degradation of tagged proteins. Conjugation of ubiq-uitin to substrate proteins requires three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2),and a ubiquitin ligase (E3). Both the E2 and E3 proteinsbelong to large families of proteins, and it is believed thatdifferent combinations of E2 proteins with different E3 ligasesdefine a high substrate specificity. In this study, we have inves-tigated the role of the ubiquitin-proteasome pathway in thedownregulation of PKC isoforms in response to the tumor-promoting phorbol ester TPA.

MATERIALS AND METHODS

Cells and cell culture conditions. Rat 3Y1 cells or rat 3Y1 cells expressingeither v-Src or c-Src were maintained in Dulbecco’s modified Eagle mediumsupplemented with 10% bovine calf serum (HyClone). Cell cultures were madequiescent by growing them to confluence and then replacing the medium withfresh medium containing 0.5% newborn calf serum for 1 day. Cells expressing thekinase-dead PKC a were generated as described previously (7). The kinase-deadPKC a clone was generated by a mutation to the ATP-binding site as describedpreviously (15).

Materials. The PKC inhibitors staurosporine, bisindolylmaleimide II, rot-tlerin, and Go6976 were obtained from Calbiochem. Monoclonal antibodies forPKC a, ε, and z were obtained from Transduction Laboratories; a polyclonalantibody for PKC d was obtained from Santa Cruz. A monoclonal antibody forubiquitin was obtained from Zymed.

Cell lysate preparation and subcellular fractionation. Cells grew to approxi-mately 90% confluence in 100-mm-diameter culture dishes and were then shiftedto Dulbecco’s modified Eagle medium containing 0.5% serum for 24 h. Cellswere washed three times with ice-cold isotonic buffer (phosphate-buffered saline,containing 136 mM NaCl, 2.6 mM KCl, 1.4 mM KH2PO4, and 4.2 mM Na2HPO4,pH 7.2). For subcellular fractionation, cells from 100-mm-diameter dishes werewashed and then scraped into 2 ml of homogenization buffer (20 mM Tris-HCl[pH 7.5], 5 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 2 mM dithiothreitol, 200 mMphenylmethylsulfonyl fluoride, 10 mg of aprotinin per ml, 10 mg of leupeptin perml). Cells were then disrupted with 20 strokes in a Dounce homogenizer (type Bpestle), and the lysate was centrifuged at 100,000 3 g for 1 h. The supernatant

* Corresponding author. Mailing address: Department of BiologicalSciences, Hunter College and the Graduate School of the City Uni-versity of New York, 695 Park Ave., New York, NY 10021. Phone:(212) 772-4075. Fax: (212) 772-5227. E-mail: foster@genectr.hunter.cuny.edu.

† This paper is dedicated to Erwin Fleissner on the occasion of hisretirement as the dean of sciences and mathematics at Hunter College.

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was collected as the cytosolic fraction. The membrane pellet was suspended inthe same volume of homogenization buffer with 1% Triton X-100. After incu-bation for 30 min at 4°C, the suspension was centrifuged at 100,000 3 g for 1 h.The supernatant was collected as the membrane fraction. For whole-cell lysates,cells were treated with 3 ml of homogenization buffer containing 1% TritonX-100 followed by centrifugation at 100,000 3 g for 1 h. The supernatant wascollected and used as the whole-cell lysate.

Immunoprecipitation and Western blot analysis. Extraction of proteins fromcultured cells was performed as previously described (7) with a modified bufferconsisting of 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 1 mMdithiothreitol, 0.5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, leupeptin(12 mg/ml), aprotinin (20 mg/ml), 100 mM sodium vanadate, 100 mM sodiumpyrophosphate, 1 mM sodium fluoride, 10 mM ethylmethylmaleimide, and 50mM hemin. Cell extracts were clarified by centrifugation at 12,000 rpm, and thesupernatants (1,500 mg of protein/ml) were subjected to immunoprecipitationwith anti-PKC d, a, and ε antibodies. After overnight incubation at 4°C, proteinA-agarose beads were added and left for an additional 3 h. Immunocomplexeswere then subjected to Western blot analysis as described previously (7). Westernblot analysis with antiubiquitin antibody was performed with modifications de-scribed by Avantaggiati et al. (1).

RESULTS

Proteasome inhibitors block depletion of PKC isoforms. Toinvestigate whether the ubiquitin-proteasome pathway is in-volved in the downregulation of PKC in response to phorbolesters, we first examined the effect of proteasome inhibitorson TPA-induced PKC depletion. MG101 and MG132, whichinhibit proteasome function (11, 12), prevented the TPA-induced depletion of the a, d, and ε PKC isoforms, the onlyTPA-responsive isoforms present in these cells (Fig. 1). E64,which shares with MG101 and MG132 the ability to inhibitcalpain protease, but not the proteasome, had no effect onTPA-induced PKC depletion. We also examined the effectof these compounds on PKC z, a PKC isoform that is ex-pressed in these cells but is not responsive to phorbol esters(9). As shown in Fig. 1, neither MG101 nor MG132 had any

effect on PKC z. These data implicate the ubiquitin-protea-some pathway in the phorbol ester-induced depletion ofPKC.

PKC isoforms become ubiquitinated upon TPA treatment.The data in Fig. 1 demonstrate that compounds which in-hibit proteasome function inhibit TPA-induced downregu-lation of PKC. Therefore, it is predicted that the affectedPKC isoforms should become ubiquitinated in response toTPA. In Fig. 1, it was also observed that the anti-PKC dantibody recognized several higher-molecular-weight spe-cies within 30 min after TPA treatment. The appearance ofthese higher-molecular-weight species of PKC d is consis-tent with the rapid ubiquitination of PKC d in response toTPA. To investigate directly whether PKC isoforms werebeing ubiquitinated in response to TPA, we performedWestern blot analysis of PKC isoform immunoprecipitationswith antiubiquitin antibody. As shown in Fig. 2, ubiquitina-tion of PKC a, d, and ε, but not PKC z, was detected within30 min of TPA treatment. By 6 h, the ubiquitinated PKCisoforms were no longer detectable. However, when MG101was used to inhibit proteasome, the ubiquitinated isoformswere still present 6 h after TPA treatment (Fig. 2). Inter-estingly, 24 h of treatment with MG101 alone resulted in asignificant accumulation of ubiquitinated forms to a limitedextent for PKC a and substantially for PKC ε (Fig. 2),suggesting that ubiquitination may occur in response tophysiological stimuli as well as TPA. These data demon-strate that PKC isoforms a, d, and ε rapidly become ubiqui-tinated in response to TPA treatment and that their disap-pearance is blocked by inhibition of proteasome.

Degradation and ubiquitination of PKC are dependentupon PKC kinase activity. To begin to investigate the mech-anism for activation of ubiquitination and proteasome deg-

FIG. 1. Proteasome inhibitors prevent TPA-induced depletion of PKC. 3Y1 cells overexpressing c-Src were treated with TPA (400 nM) for the indicated times, andPKC depletion was monitored by Western blot analysis as described previously (7). The effect of MG101, MG132, or E64 (all at 50 mM) was determined by addingthese compounds 30 min prior to addition of TPA as shown. The levels of PKC d, a, ε, and z were determined by using antibodies specific for these isoforms.

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radation, we asked whether the kinase activity of PKC wasimportant for degradation. We first investigated the effect ofPKC inhibitors on the TPA-induced PKC downregulationand ubiquitination. In Fig. 3A, it is shown that the PKCinhibitors staurosporine and bisindolylmaleimide II pre-vented downregulation of PKC isoforms a, d, and ε. Inter-estingly, Go6976, which specifically inhibits PKC a (8), pre-vented TPA-induced downregulation of the a isoform only,and rottlerin, a more specific inhibitor of PKC d (4), pre-vented TPA-induced downregulation of the d isoform only.We also investigated the effect of the PKC inhibitors on theubiquitination of PKC isoforms a and d, and as expected,the PKC inhibitors also prevented TPA-induced ubiquitina-tion of these PKC isoforms with the same specificity ob-served for inhibition of downregulation (Fig. 3B). The PKCinhibitors did not inhibit translocation to the membrane ofthe PKC isoforms (Fig. 3C). Thus, the effects observed inFig. 3A and B were not due to a lack of membrane associ-ation. These data indicate that TPA-induced downregula-

tion and ubiquitination of the PKC isoforms require anactive kinase activity. Consistent with a requirement foractivation of PKC for downregulation, the inactive phorbolester 4a-phorbol 12,13-didecanoate, which does not activatePKC (14), did not lead to the downregulation of PKC (Fig.3D), nor did it result in the ubiquitination of PKC d (Fig.3E).

If PKC kinase activity is required for downregulation, then akinase-dead PKC mutant should be resistant to downregula-tion in response to TPA. An ATP-binding site mutant of PKCa (15) that was kinase dead was introduced into the c-Src-overexpressing cell line, and the ability to downregulate PKC awith TPA was examined. As shown in Fig. 4A, this PKC amutant was completely resistant to downregulation by TPA.Since the kinase-dead PKC a mutant could still be stimulatedto associate with the membrane in response to TPA (Fig. 4B),the lack of degradation was not due to lack of membranelocalization. Since PKC d and ε were both activated and down-regulated in these cells, activation of the ubiquitin-proteasomepathway by these PKC isoforms was apparently specific for theactivated isoforms only. These data further support the con-clusion that activation of the kinase activity of PKC is neces-sary for ubiquitination and downregulation.

PKC is ubiquitinated and downregulated in response to DGin a proteasome- and kinase-dependent mechanism. Phorbolesters bind to PKC at the site that binds the physiologicalactivator diacylglycerol (DG) (9). As shown in Fig. 2, the pro-teasome inhibitor MG101 stimulated an increase in the ubi-quitinated PKC isoforms a and ε, suggesting that ubiquitina-tion is a physiological response and not an artifact of phorbolester treatment. We therefore wished to investigate whetherubiquitination and downregulation of PKC occur in responseto DG. As shown in Fig. 5, the a and d isoforms and to a lesserextent the ε isoform were all downregulated in response to theDG dioctoylglycerol (DiC8). This downregulation was sensi-tive to both proteasome and PKC inhibitors (Fig. 5A). ThePKC a-specific Go6976 prevented downregulation of the aisoform specifically. We also wished to determine whether DGstimulated ubiquitination of PKC isoforms. We added DiC8 tothe 3Y1 cells and examined ubiquitination as in Fig. 2. In Fig.5B, it is shown that DiC8 stimulated ubiquitination of PKC d.The ubiquitination of PKC d was inhibited by the PKC inhib-itors staurosporine, bisindolylmaleimide II, and rottlerin butnot by the proteasome inhibitor MG101 or the PKC a inhibitorGo6976 (Fig. 5B). These data suggest that PKC isoforms be-come ubiquitinated and downregulated by the physiologicalstimulus of DG as well as by the tumor-promoting stimulus ofTPA and that downregulation is dependent upon an activekinase.

TPA-induced transformation of 3Y1 cells overexpressing c-Src is blocked by proteasome inhibitors. In cells overexpress-ing c-Src, TPA treatment causes the appearance of transfor-mation that is due to the depletion of PKC d (7). We thereforeinvestigated whether inhibitors of the ubiquitin-proteasomepathway could prevent the transformed phenotype induced byTPA in the c-Src-overexpressing cells by preventing the deple-tion of PKC d. As shown in Fig. 6A, the proteasome-specificinhibitor MG101 prevented the morphological transformationof the c-Src-expressing cells induced by TPA, whereas thenonspecific protease inhibitor E64 did not prevent the mor-phological transformation induced by TPA. The proteasomeinhibitors had no effect on the transformed phenotype inducedby v-Src (Fig. 6A). The ability of MG101 to prevent the TPA-induced morphological transformation was not likely due toany effects that proteasome inhibition have upon cell cycleprogression (10), since aphidicolin, which blocks cells at the

FIG. 2. PKC becomes ubiquitinated upon TPA treatment. The c-Src-overex-pressing 3Y1 cells were treated with TPA (400 nM) for the indicated times. PKCd, a, ε, and z were then immunoprecipitated (IP), and the level of ubiquitinatedPKC was determined by Western blot analysis with an antiubiquitin antibody.The effect of MG101 (50 mM) on ubiquitination of untreated cells and cellstreated with TPA is shown. Numbers on the left are molecular weights inthousands.

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G1/S boundary of the cell cycle (5), had no effect on theTPA-induced morphological transformation (data not shown).In addition, MG101 had no effect on the translocation of thePKC isoforms induced by TPA (Fig. 6B). Thus, the effectobserved in Fig. 6A is not due to the inability to translocatePKC isoforms to the membrane. These data suggest that PKCd is downregulated by the ubiquitin-proteasome pathway andthat this pathway is critical for the TPA-induced tumor pro-motion, as reported previously (7).

DISCUSSION

In this report, we have shown that downregulation of PKC inresponse to tumor-promoting phorbol esters is via theubiqutin-proteasome pathway. In response to TPA, PKC iso-forms a, d, and ε all became ubiquitinated within 30 min and

FIG. 3. Degradation of PKC is dependent upon PKC kinase activity. (A) 3Y1cells overexpressing c-Src were treated with TPA (400 nM) for 6 h to deplete thecells of PKC. This was then performed in the presence of the PKC inhibitorsstaurosporine (Stauro), bisindolylmaleimide II (Bis), Go6976, and rottlerin(Rott) at the indicated concentrations, and PKC levels were determined byWestern blot analysis as for Fig. 1. (B) The effect of the PKC inhibitors onTPA-induced ubiquitization of PKC a and d was determined as for Fig. 2. (C)The ability of TPA to induce translocation of the PKC isoforms from the cytosolto the membrane in the presence of PKC inhibitors was investigated by Westernblot analysis of the PKC isoforms present in the cytosolic and membrane frac-tions before and after TPA treatment. (D and E) The effect of the inactivephorbol ester 4a-phorbol 12, 13-didecanoate (4a-PDD) (400 nM) on the induc-tion of PKC isoform downregulation (D) and ubiquitination of PKC d (E) wasexamined as for panels A and B, respectively.

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were degraded within 6 h in 3Y1 rat fibroblasts. Proteasomeinhibitors prevented TPA-induced PKC downregulation butnot ubiquitination of the PKC isoforms. Ubiquitination anddownregulation of PKC isoforms were dependent on an activePKC kinase. We previously demonstrated that the downregu-lation of PKC d was responsible for the tumor-promoting ef-fects of TPA on 3Y1 cells overexpressing c-Src (7). Consistentwith PKC d downregulation being important for the tumor-promoting effects observed previously, the proteasome in-hibitor MG101, which prevented PKC d downregulation inresponse to TPA, also prevented the TPA-induced transfor-mation of the c-Src-overexpressing cells. Thus, the data pre-sented here implicate the ubiquitin-proteasome pathway inphorbol ester-induced tumor promotion.

Interestingly, treatment of 3Y1 cells with MG101 inducedthe appearance of PKC polyubiquitinated forms, especially forPKC ε, which tends to be the most constitutively activatedisoform in these cells (18). This suggested that ubiquitinationof PKC is a physiological response and is not unique to theresponse to phorbol esters. Consistent with this hypothesis,ubiquitination and downregulation were observed in responseto an exogenously provided DG. DG was less potent than TPAat inducing ubiquitination and downregulation of PKC; how-ever, this was most likely because DG can be metabolically

converted to other lipids such as phosphatidic acid and mono-acylglycerol.

The data presented here do not demonstrate the completemechanism of activation of the ubiquitin-proteasome pathway;however, it is apparently regulated at the level of ubiquitina-tion. Of special interest is the requirement for the kinase ac-tivity of the PKC isoforms. Compounds that inhibit activationof PKC prevented PKC downregulation and ubiquitination inresponse to TPA. Additionally, a kinase-dead PKC a was com-pletely resistant to TPA-induced downregulation. Since phor-bol esters still lead to the activation and downregulation ofPKC isoforms d and ε in cells expressing the kinase-dead PKCa, ubiquitination is apparently isoform specific and the activa-tion of one PKC isoform does not stimulate ubiquitination anddownregulation of other inactive PKC isoforms. Moreover,since the cells expressing the kinase-dead PKC a likely stillexpress wild-type PKC a, which would be activated by TPA, itis not likely that PKC a activates a PKC a-specific ubiquitina-tion system, because this would result in the degradation of thekinase-dead PKC a. Since the defect in the kinase-dead PKCa mutant that was not degraded in response to TPA was in theATP-binding site, activation of the ubiquitin-conjugating sys-tem is likely stimulated by a conformational change in PKCthat involves ATP binding or hydrolysis. This suggests a suicidemodel for regulation of PKC where upon activation, PKCbecomes ubiquitinated and thereby targeted for degradation ina negative feedback control mechanism.

FIG. 4. A kinase-dead mutant of PKC a is not downregulated by TPA. Thec-Src-overexpressing 3Y1 cells were stably transfected with a mutant PKC a genewhich has a mutation in the ATP-binding site (15). (A) The mutant PKC-a-overexpressing cells were then treated with TPA (400 nM,) and the levels of PKCa, d, and ε in these and the parental cells were determined at 6 and 72 h later asfor Fig. 1. (B) The ability of TPA to induce translocation of the PKC isoformsfrom the cytosol to the membrane in the parental cells and in the cells expressingthe kinase-dead PKC a was determined as for Fig. 3C.

FIG. 5. PKC is downregulated and ubiquitinated in response to DG in aproteasome- and kinase-dependent mechanism. (A) c-Src-overexpressing 3Y1cells were treated with DiC8 (10 mg/ml) for the indicated times in the presenceof either E64 (50 mM), MG101 (50 mM), bisindolylmaleimide II (Bis) (1 mM), orGo6976 (2 mM), and the levels of PKC a, d, and ε were then determined byWestern blot analysis as for Fig. 1. (B) The effect of DiC8 on the ubiquitinationof PKC d was determined in the presence of the proteasome inhibitor MG101(50 mM) and the PKC inhibitors staurosporine (Stauro) (50 nM), bisindolylma-leimide II (1.0 mM), Go6976 (2.0 mM), and rottlerin (80 mM) as for Fig. 2.

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ACKNOWLEDGMENTS

We thank Robert Krauss and Neil Rosen for comments on themanuscript. We thank Shigeo Ohno for providing the PKC a kinase-dead mutant. We thank Erwin Fleissner for the continued support andencouragement our lab received during his tenure as dean.

This investigation was supported by grants from the National Insti-tutes of Health (CA46677), the Council for Tobacco Research (3075),and the American Cancer Society (BE-243) (to D.A.F.) and by aResearch Centers in Minority Institutions (RCMI) award from theDivision of Research Resources, National Institutes of Health (RR-03037), to Hunter College. M.P. is supported in part by NIH grantCA66229-02.

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