okadaic acid interferes with phorbol-ester-mediated down-regulation of protein kinase c-α, c-δ and...

Eur. J. Biochem. 249, 92-97 (2997) 0 FEBS 1997

Okadaic acid interferes with phorbol-ester-mediated down-regulation of protein kinase C-a, C-6 and C-E Andrea GATTI' and Phillip J. ROBINSON2 ' Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

Cell Signalling Unit, Children's Medical Research Institute, New South Wales, Australia

(Received 2 Junell4 July 1997) - EJB 97 0785/1

A prolonged cell exposure of all examined cell types to tumour-promoting phorbol esters leads to a substantial inactivation and degradation of protein kinase C (PKC), a phenomenon known as down- regulation. With a combination of one- and two-dimensional immunoblot analyses we have previously shown the existence in PC12 cells of distinct PKC-a forms that differentially respond to cell treatment with phorbol ester [Gatti, A. & Robinson, P. J. (1996) J. Biol. Chem. 271, 31718-317221. Using the same experimental model, in the present study we investigated a possible relationship between PKC-a phosphorylation and its down-regulation. The exposure of PC12 cells to okadaic acid, a potent inhibitor of biologically relevant protein phosphatases, was found to partially protect PKC-n against phorbol-ester- mediated down-regulation. Further, a similar protective effect of okadaic acid was observed for PKC-8 and PKC-E, which are also expressed in PC12 cells. These results indicate that the tumour-promoting activity of okadaic acid itself may be due to a sustained phosphorylation of PKC.

Keywords: protein kinase C ; phorbol ester; two-dimensional immunoblot ; down-regulation ; PC12 cells.

Protein kinase C (PKC) is a serineithreonine kinase which is activated in response to a variety of extracellular and intracellular signals via stimulation of one or a combination of many of its isoenzymes (PKCs). The existing, closely related, PKCs differ in their tissue or intracellular distribution and individual biochemical characteristics [l]. All the identified PKCs share a common requirement for phosphatidyl-I-serine (PtdSer) and differ in their sensitivity to other activators such as Caz+ and 1,2-diacylglycerol. Three distinct groups of PKCs, designated as conventional isotypes (cPKCs, a, pI, PI and y ) , novel isotypes (nPKCs, 6, E ,

v, p and 8) and atypical isotypes (aPKCs, [ and Ah), have been defined on the basis of similarities in sequence and functional properties 12). If not otherwise indicated, in this paper we will refer to PKC as to members of the cPKCs and nPKCs groups.

Both phorbol esters and physiological activators cause translocation of PKC from the cytosolic to the membrane com- partment with subsequent kinase activation and phosphorylation of endogenous substrates. Phorbol ester is believed to differ from the physiological PKC activators primarily because of the prolonged duration of its action : while the Ca2+/diacylglycerol- induced translocation of PKC to the cell membrane is short- lived, phorbol-ester-promoted membrane association is sustained. The differential duration of this effect may well underlie some of the differences in the biological activity of these PKC activators. In spite of the finding that PKC is the major intracellular target for tumour-promoting phorbol esters [3], the mechanism by which a prolonged cell exposure to phorbol ester ini-

Correspondence to A. Gatti, Department of Biochemistry, University

E-mud: [email protected] Abbreviations. cPKCs, conventional protein kinase C isotypes;

nPKCs, novel protein kinase C isotypes; aPKCs, atypical protein kinase C isotypes ; OA, okadaic acid ; PhMeSO,F, phenylmethylsulfonyl fluoride; TPA, 12-0-tetradecanoylphorbol 13-acetate.

Enzyme. Protein kinase C (EC 2.7.1.37).

of California, Riverside, California 92521, USA

tially activates PKC and ultimately promotes its down-regulation is poorly understood.

Although the kinetics of PKC activationhactivation follows an isotype- and substrate-dependent pattern, a common mode of function is shared by all the phorbol-ester-sensitive PKC isotypes. Phorbol ester binding promotes the activation of PKC, which is then followed by an attenuation of the catalytic function and ultimately by a proteolytic degradation of PKC itself. The in vitro increased susceptibility of the active conformation of PKC to various proteases has suggested that the activation of site-specific proteases could be the primary cause of down-regulation [4]. However, down-regulation of PKC-a has recently been reported to be the consequence of a non-specific degrada- tive process, rather than being the effect of cleavage by site- specific proteases [5, 61.

The molecular mechanism by which a sustained PKC association with cell membranes is ultimately responsible for PKC down-regulation could be based on the conformational change resulting from the interaction of PKC with its lipid cofactor. Under certain in vitro conditions, PtdSer by itself was found to negatively affect the catalytic activity of PKC [7] and render it more susceptible to proteolytic attack [8, 91. Such a PtdSer-mediated inactivation of PKC is enhanced by the co-application of phorbol ester and reduced by ATP and divalent cations such as Caz+ and Mg". This is consistent with the general observation that the enzymatically active form of PKC is very unstable and requires stabilising factors to avoid rapid degradation. The negative modulation of PKC activity by PtdSer has been shown to result from a direct interaction of the phospholipid with the catalytic domain of the kinase [lo]. Accordingly, in untreated intact cells (i.e. in the presence of MgATP/Ca2' and in the absence of phorbol ester), PtdSer is believed to preferentially interact with the regulatory domain of PKC and kinase activation occurs prior to any down-regulation. In spite of the doubts raised by these observations on the relevance of PKC down-regulation, several

Gatti and Robinson ( E m J. Biochem. 249) 93

examples of PKC down-regulation upon a physiological cell stimulation have been reported [ l l , 121.

With a two-dimensional immunoblot procedure, we have recently demonstrated a multiple phosphorylation of the PKC-a isotype expressed in PC12 cells [13]. Changes in the phosphorylation state of PKC-u were found after short, as well as prolonged, cell exposure to phorbol ester [14]. In the present study, we used a potent phosphatase inhibitor, okadaic acid (OA), to interfere with the phorbol-ester-regulated conversion between differentially phosphorylated forms of PKC-a. In addition to a partial protection of PKC-a from down-regulation, OA was also found to impart on PKC-6 and PKC-e a substantial resistance to phorbol ester.

EXPERIMENTAL PROCEDURES

Cell culture. PC12 cells were cultured in Dulbecco's modi- fied Eagle medium in the presence of 100 U/ml penicillin and 0.1 mg/ml streptomycin and supplemented with 5 % foetal calf serum and 10% heat-inactivated horse serum. Cells were main- tained in a 37 "C incubator in a water-saturated atmosphere containing 7% CO,.

Preparation of cell homogenates. Culture plates were placed on ice and washed three times with ice-cold 20 mM Tris/ HCl, 1 mM phenylmethylsulfonyl fluoride (PhMeSO,F), 1 mM EGTA, pH7.4. All subsequent steps were performed at 4°C. Cells were scraped into lysis buffer containing 20 mM Tris/HCI, 1 mM PhMeSO,F, 1 mM EGTA, 1 % Triton X-100,50 mM NaF, 50 nM calyculin A (Boehringer Mannheim), 40 pM leupeptin (pH 7.4) and then lysed with 10 passes through a 26-gauge needle. Cell lysates were centrifuged for 10 min at 500 X g. The resulting pellet was discarded, while the supernatant (post- nuclear cell homogenate) was loaded onto an equilibrated 2-ml DEAE-cellulose (DE-52, Whatman) column to partially purify PKC, as previously <described [ 131.

Preparation of subcellular fractions. Culture plates were placed on ice and washed three times with ice-cold 20 mM Trid HCl, 1 mM PhMeSO,F, 1 mM EGTA, pH7.4. All subsequent steps were performed at 4°C. Cells were removed from culture dishes with a teflon-coated cell scraper and sonicated with an ultrasonic probe in the same washing buffer supplemented with 40 pM leupeptin and 50 nM calyculin A. After cell lysis, subcellular fractions were obtained by ultracentrifugation at 100000 X g for 1 h. The resulting supernatant is designated as soluble fraction. The pellets was resuspended in 20 mM TrislHCl pH 7.4, 1 pM EGTA, 0.75 mM CaCl,, 1 pM PhMeSO,F, 40 pM leupeptin, 50 nM calyculin A and 1 % (masshol.) n-octyl p-D-gluco- pyranoside. The resuspended pellet was dissociated by repeated forceful aspiration through a yellow tip of an Eppendorf pipett- man, shaken for 30min and then centrifuged at 13OOOXg for 15 min. The supernatant of the latter centrifugation is designated particulate fraction.

SDSPAGE and two-dimensional electrophoresis. DEAE- cellulose extracts were electrophoresed on 7.5 % SDYPAGE [ 151. When indicated, the samples were subjected to two-dimensional electrophoresis essentially as previously described [ 141. Briefly, samples were diluted 1 : 1 with buffer for isoelectric focusing, composed of 9.5 M urea, 5 % (masshol.) Chaps, 5 % (by vol.) 2-mercaptoethanol, 1.2% (by vol.) pH 5-8 plus 0.8% (by vol.) pH 3- 10 Pharmacia ampholines, and then additional solid urea was added (1 mg/ml). Isoelectric focusing (7000 V h) on 7.5-cm-long gel tubes in the first dimension was followed by 10% SDSlPAGE as second dimension on a Bio-Rad mini- gel.

A

Soluble fraction

Particulate fraction

B

Anti- PKC a 8 E 5

-116 z -77 -5

x J 0 -49 c;J

-116

-77 -5

X 2

-49 0

Anti-PKC-6 Anti-PKC-& Anti-PKC-i

- + I 1 - + I I - + ' -116 z -77 x

9

7

2

w

- 49 Fig. 1. Immunoblot analysis of PC12 cell subcellular fractions. (A) EGTA-extracted soluble and particulate fractions of PC12 cells were prepared as described in Experimental Procedures. Samples (50 pg protein) from each subcellular fraction were subjected to SDSPAGE and immunoblot analysis with the following monoclonal antibodies to PKC : anti-PKC-u from Amersham (MC5), anti-PKC-6, anti-PKC-e and anti- PKC-( from Gibco/BRL. Immunoreactive forms are indicated with arrows. The specificity of the irnmunorecognition is substantiated by the selective removal of the indicated signals upon inclusion of the irnrnuniz- ing peptide i n incubations with primary antibody (B). Migration of molecular mass markers is indicated on the right. Results shown are representative of three independent experiments. (B) Cell homogenates from PC12 cells were subjected to immunoblot and probed with the indicated antibodies. Note that (+) indicates that the specific immunizing peptide has been included in incubations with primary antibody at 1 : 1 (mass/ mass) ratio to antibody, while (-) indicates that the peptide has not been added.

Immunoblot. Proteins from SDS/PAGE and two-dimensional electrophoresis were transferred onto nitrocellulose membrane [16]. The nitrocellulose was blocked with phosphate-buf- fered saline (NaCVP,) containing 1 % polyvinylpyrrolidone

94 Gatti and Robinson (Eur: J . Biochenz. 249)

Control TPA TPNOA Con tro I 25 nM TPA

-80 kDa

anti-PKC-CX (Amersham)

anti-PKC-CX (Transduction Laboratories)

1 8 2 kDa -80 kDa

-80 kDa

Fig.2. Immunoblot analysis of partially purified PKC-a in PC12 cells treated with TPA and TPA/OA. Homogenates from untreated PC12 cells (control) or cells treated for 1 h with 1 pM TPA in the absence (TPA) or presence of 100nM OA (TPA/OA) were prepared as described in Experimental Procedures. Aliquots of DEAE-cellulose extracts were subjected to SDS/PAGE and subsequent immunoblot analysis with the antibody to PKC-u from Amersham (top) and from Transduc- tion Laboratories (bottom). In order to maximise the resolution of differentially migrating bands, the SDS/PAGE run time was increased and the electrophoresis was terminated only shortly before the 49-kDa marker migrated off the end of the gel. When expressing immunoreactivity as percentage of that obtained from untreated cells, densitometric analysis of the blot probed with Amersham and Transduction antibody revealed 13.8% and 10.5%, respectively, for cells treated with TPA, or 31 % and 32% for cells treated with TPA plus OA. The experiment was performed three times, with less than 10% variation between the experiments.

40000 and then incubated with the primary antibody in NaCl/ P,. Immunoreactivity was detected with peroxidase-conjugated secondary antibody (Dakopatts) by using the ECL method and the Amersham instructions. The monoclonal antibody to PKC-a from Amersham (MC5) and from Transduction Laboratories were used at the working concentration of 1 pg/ml and 0.15 pg/ ml, respectively. Note that by using PC12 cells the immunorecognition by MC5 is specific for the PKC-a isotype [13]. Rabbit anti-peptide anti-PKC-S, anti-PKC-c and anti-PKC-( from Gibco/BRL were all used at the working concentration of 5 pg/ ml. In some cases, the immunizing isozyme-specific peptides (commercially supplied by Gibco/BRL) were included in incubations with primary antibody to block the isozyme immunorecognition.

RESULTS To assess the intracellular distribution of the PKC isotypes

expressed in PCI 2 cells, EGTA-extracted subcellular fractions were subjected to immunoblot and probed with antibodies for three cPKCs ( a , p, and 1' isoforms), two nPKCs (6 and E isoforms) and one aPKC isotype (( isoform). Under our conditions of cell lysis and subcellular fractionation, the presence of PKC- (1, but not of PKC-/l or PKC-y (data not shown), was detected in the soluble fraction, while no cPKCs were detectable in the particulate fraction. The iminunoreactive PKC-6, -c and -[ forms were present in both soluble and particulate fractions with appar-

l u M TPA 1 y M TPA + 100nM OA

-80 kDa

I b c ( I 7 PH 4 7 PH 4

Fig. 3. Two-dimensional immunoblot analysis of partially purified PKC-a. Intact PC12 cells were exposed for 1 h to the indicated concentrations of TPA and OA and then lysed as indicated in Experimental Procedures. After DEAE-cellulose chromatography, samples were diluted in buffer for the first dimension and resolved by isoelectric focusing (7000 V h). The second dimension in 10% SDSE'AGE was terminated shortly before the dye (bromophenol blue) migrated off the end of the gel. Proteins were electrophoretically transferred onto nitrocellulose membrane and immunorecognition was performed with antibody to PKC-u from Transduction Laboratories. Only the relevant sections of the X-ray films are shown. Arrows indicate differentially migrating PKC-a forms that were designated with letters a, b, c, etc. according to their decreasing pl. Results shown are representative of three independent experiments.

ent molecular masses of 76, 90 and 76kDa, respectively (Fig. IA). Note that the specificity of the isozyme immunorecognition was confirmed by the selective displacement of the relevant signal upon inclusion of the immunizing peptide in incubations with primary antibody (Fig. 1 B).

To investigate whether phosphorylation of PKC plays a role in the down-regulation process, intact PC12 cells were incubated for 1 h in culture medium (control) and medium supplemented with 1 pM of 12-0-tetradecanoylphorbol 13-acetate (TPA) in the absence or in the presence of 100 nM OA (Fig. 2). After cell lysis, cell homogenates were subjected to DEAE-cellulose chromatography to partially purify PKC-a. The extracts were then analysed by SDS/PAGE and the electrophoretic profile of PKC- a was assessed via immunoblot with two distinct antibodies to PKC-a: MC5 from Amersham and the monospecific antibody from Transduction Laboratories. As previously described [ 141, MC5 recognises two differentially migrating species of PKC-a (80 and 82 kDa) in untreated PC12 cells, with the pool of slower-migrating forms being uniquely resistant to phorbol-ester-induced down-regulation. The antibody to PKC-a from Transduction Laboratories preferentially reacts with the 80-kDa PKC-rx form. A prolonged treatment with a high concentration of phorbol ester caused a dramatic reduction in the recovery of the major PKC-a form (80 kDa), while co-application of 100 nM OA with phorbol ester clearly reduced the extent of PKC-a down-regulation (Fig. 2).

DEAE-cellulose extracts from untreated PC12 cells (Fig. 3A), cells treated for 1 h with 25 nM TPA (Fig. 3B), and cells treated for 1 h with 1 pM TPA in the absence (Fig. 3C) or presence (Fig. 3 D) of 100 nM OA were then subjected to two- dimensional immunoblots. As assessed by the use of the antibody to PKC-a from Transduction Laboratories, exposure of PC12 cells to 25 nM TPA caused an acidic shift in the ratio of the PKC-a forms with subsequent accumulation of highly phosphorylated species such as those corresponding to spots c, d and

Gatti and Robinson ( E m J. Biochem. 249) 95

A TPA " 2 h r ) 0 10nM 25f lM 75 nM 200nM

I I I I I t

Anti-PKC-Cl

Anti-PKC- &

B TPA ( 1 2 h r ) o 10nM 2 5 n M 75nM

I I I I I

Anti-PKC-6

Ja2 kDa -a0 kDa

-90 kDa

JS0 kDa -76 kDa

Fig. 4. Immunoblot analysis of PKC-u, PKC-E (A) and PKC-6 (B) in PC12 cells. Homogenates from untreated PC12 cells or cells treated for 12 h with the indicated concentration of TPA were prepared as described in Experimental Procedures. Samples were directly subjected to SDS/ PAGE and subsequent immunoblot analysis with antibodies to PKC-a or PKC-E (A) or to PKC-8 (B). Note that the 80-kDa band in B is likely to be an immunoreactive protein other than PKCd, given its differential resistance to phorbol-ester-mediated down-regulation. The position of molecular mass standards is indicated on the right. Similar results were obtained in two additional experiments.

e. By contrast, cell exposure to 1 pM TPA induced a preferential loss of the PKC-a forms corresponding to spot b, some minor changes in the recovery of spots c and d and no apparent changes in spots a and e. Co-application of 100 nM OA in the TPA-containing cell culture medium caused a selective protection of the PKC-a form corresponding to spot c. Similar changes in the 80-kDa form of PKC-a were also revealed when using the MC5 antibody from Amersham (data not shown), except that this antibody also recognises an additional 82-kDa form of PKC- a, as previously described [14]. Note that under our experimental conditions (i.e. cell lysis in the presence of a mixture of phosphatases inhibitors), cell exposure to OA alone did not alter the overall recovery of PKC immunoreactivity (data not shown).

When examining the phorbol-ester-mediated down-regulation of other PKC isotypes we found that both PKC-c (Fig. 4A) and PKC6 (Fig. 4B: 76 kDa) were down-regulated at approxi- mately 10-fold lower concentrations of TPA than PKC-a. Al- though l h exposure of PC12 cells to l pM TPA was sufficient to cause a substantial loss of both PKCd (Fig. 5, top) and PKC- c (Fig. 5 , bottom), we found that the co-application of 100 nM OA in the cell culture medium greatly reduces down-regulation of these nPKCs. Note that in the immunoblot experiments focusing on PKC-t the specific immunoreactivity was resolved as a doublet, with OA preferentially protecting the slower-migrating band.

Anti-PKC- 6

Anti-PKC-&

- 116 kDa

-77 kDa

- 116 kDa

-77 kDa

Fig.5. Immunoblot analysis of PKC-6 and PKC-E in PC12 cells treated with TPA and TPA/OA. Homogenates from untreated PC12 cells (control) or cells treated for 1 h with 1 pM TPA in the absence (TPA) or presence of 100nM OA (TPA+OA) were prepared as described in Experimental Procedures. Samples were directly subjected to SDWPAGE and subsequent immunoblot analysis with antibody to PKC- 6 (top) or PKC-E (bottom). Immunoreactive forms are indicated by the arrows. Note that the immunoreactive band above the PKC-8 signal is a product of non-specific cross-reaction (see legend of Fig. 4), while the antibody to PKC-e specifically recognizes two differentially migrating forms, as indicated by the double arrow. When expressing immunoreactivity as percentage of that obtained from untreated cells, densitometric analysis of the blot probed with antibodies to PKC-6 and PKC-c revealed 37.5% and 35.370, respectively, for cells treated with TPA, and S9.896 or 75.1 % for cells treated with TPA plus OA. The experiment was performed three times, with less than 10% variation between the experiments.

DISCUSSION A role for PKC has been demonstrated in cell transforma-

tion, tumorigenesis and metastasis, with PKC activation repre- senting one of the best studied biochemical correlates of carcino- genicity [17]. A potential index of the in vivo PKC activation is the measure of its phosphorylation state [18]. Phorbol ester activation and increased phosphorylation are believed to be re- quired for subsequent PKC degradation [ 191. Accordingly, some [20, 211, but not all [22], inactive PKC mutants (kinase de- fective) have been found to be characterised by an abnormal resistance to phorbol-ester-mediated down-regulation. Given the possibility of resolving differentially phosphorylated forms of PKC-a [13, 141, in this study we set out to investigate whether an inhibition of PKC dephosphorylation by OA could interfere with its phorbol-ester-induced down-regulation.

To focus on the in vivo action of OA during PKC down- regulation and to avoid the well known toxic effects of prolonged cell exposure to OA, we treated cells with a high concentration of TPA (1 pM) for a limited time (1 h). Under these conditions we could accomplish a substantial down-regulation of PKC (Figs 2 and 5 ) without significantly altering the cell viabil- ity (as assessed by use of trypan blue; data not shown). Using an experimental approach based on two-dimensional immunoblot analysis we previously found that down-regulation in PC12 cells is restricted to the 80-kDa species of PKC-a, as this is the only form which undergoes membrane translocation upon phorbol ester exposure in these cells [14]. The results presented here indicate that the onset of down-regulation is paralleled by a shift in

96 Gatti and Robinson ( E m J . Biochem. 249)

the ratio of the variously phosphorylated 80-kDa forms of PKC- a. In particular, while the major immunoreactive form (spot b) is dramatically down-regulated, other minor immunoreactive species such those corresponding to spots a and e appear to be scarcely affected by phorbol ester. PKC activation, by differentially altering the sensitivity of PKC-a forms towards the endogenous phosphatases, might shape the process of down-regulation. When testing the hypothesis that the rate of down-regulation of PKC-a is regulated by endogenous phosphatases, OA was found to induce a preferential protection of the PKC-a form corresponding to spot c against phorbol-ester-mediated degradation.

According to these two-dimensional inimunoblot data, upon cell exposure to phorbol ester, PKC-a undergoes an early increase in phosphorylation that is likely to occur in parallel to the catalytic activation and, in the case of prolonged exposure, is followed by dephosphorylation and protein degradation. This is in agreement with the reported effect of another PKC activa- tor, bryostatin, which was found to concomitantly cause the accumulation of autophosphorylated, active PKC-a and its dephos- phorylated, inactive counterpart [6].

In intact cells PKC-a has been reported to be dephosphory- lated by protein phosphatases 2A [23), one of the two biologically relevant protein-serinekhreonine phosphatases which are known to be inhibited by OA. In our conditions OA could protect the overall content of PKC-a from down-regulation by shift- ing the balance of the PKC-a forms towards the accumulation of more highly phosphorylated forms (i.e. spot c), which appear to be less sensitive to phorbol-ester-mediated down-regulation.

Our finding that a differential resistance against down-regulation is dependent on the degree of phosphorylation is consistent with the recent report that the non-phosphorylated form of PKC-(1 represents a better substrate for ubiquination than the autophosphorylated form 161. The hypothesis that the more phosphorylated forms of PKC are more resistant to down-regulation is also supported by our immunoblot analysis of PKC-E (Fig. 5 , bottom), wherein the slower-migrating band of the doublet appears to be preferentially protected by OA.

Multi-site phosphorylation has been widely reported to play an important role in the regulation of PKC. The inactive, imma- ture form (76 kDa) of PKC is phosphorylated by a series of steps involving an unidentified activating kinase (at Thr497 in PKC- a ) and several autophosphorylation events (at Thr638 followed by Ser657 in PKC-a) to produce a mature kinase (80 kDa) that can be activated by phospholipids and calcium [24]. The major- ity of the PKC in a resting cell exists in this mature, cytosolic, form. We have recently shown that mature PKC may undergo further autophosphorylation and transphosphorylation steps and that such events seem to occur in intact cells [13, 141. The role of the latter changes of PKC phosphorylation state is not known, except for the indication that a site-specific autophosphorylation protects PKC-a from phorbol-ester-mediated membrane translocation and down-regulation [14]. Mature PKC-a can be dephos- phorylated and inactivated by phosphatase 2A and this is known to accompany phorbol-ester-mediated down-regulation 1231. I t has been suggested that the rate of PKC-a down-regulation is not dependent on the rate of its dephosphorylation, since a PKC- a phosphorylation site mutant (Thr638-Ala) that is hyper-sensitive to TPA-induced dephosphorylation is down-regulated at a similar rate to wild-type PKC-a [23]. However, a preferential protection from down-regulation by orthovanadate on PKC-a and by OA on PKC-c has been recently reported [25].

The data presented here on three distinct PKC isotypes, whose phorbol-ester-mediated down-regulation is counteracted by OA, suggest that a correspondence between phosphatase-I/ 2A-mediated PKC dephosphorylation and down-regulation is likely to retlect a general regulatory mechanism for all cPKCs

and nPKCs, rather than being isotype-specific. It remains to be determined whether, in phorbol-ester-exposed cells, dephosphorylation of PKC directly triggers its down-regulation, or whether changes in the state of phosphorylation of other proteins are involved.

In spite of its inability to induce PKC activation, OA is known to act as an agonist in the mouse skin model of tumour promotion [26] and this effect has been attributed to the sustained phosphorylation of PKC substrates [27]. Our report that OA interferes with the negative modulation of distinct PKC isotypes extends such an hypothesis by suggesting that the tumour- promoting activity of OA is, at least in part, due to the stabilisa- tion of the phosphorylated status of PKC itself. This implies that OA can indirectly influence the phosphorylation state of PKC substrates by counteracting the dephosphorylation of PKC and ultimately preserving its catalytic activity.

This work was supported by the Cancer Council (NSW, Australia). A. Gatti was partially supported by Fonduzione dell’lstiruto Eurupeo di Oncologia and Fondazione ltaliana per la Ricerca sul Cancru. We thank Dr Xin Wang for help with purification of cPKCs, Luigi D’Offizi for expert assistance in preparation of figures and Prof. Jolinda Traugh for critical reading of the manuscript.

REFERENCES 1. Nishizuka, Y. (1996) Studies and perspectives of protein kinase C,

Science 233, 305-312. 2. Tanaka, C. & Nishizuka, Y. (1994) The protein kinase C family for

neuronal signaling, Annu. Rev. Neurosci. 17, 551 -567. 3. Castagna, M., Takai, Y., Kaibuchi, K., Aano, K., Kikkawa, U. &

Nishizuka, Y. (1982) Direct activation of calcium-activated, phospholipid-dependent protein kinase C by tuniour-promoting phorbol esters. J . B i d . Chenz. 257, 7847-7851.

4. Kishimoto, A,, Mikawa. K., Hashimoto, K., Yasuda, I . , Tanaka, S., Tommaga, M., Kuroda, T. & Nishizuka. Y. (1989) Limited prote- olysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain), J . Biol. Chen?. 264, 4088-4092.

5. Junco, M., Webster, C., Crawford, C., Bosca, L. & Parker, P. J. (1994) Protein kinase C V3 domain mutants with differential sensitivity to m-calpain are not re. nt to phorbol-ester-induced down-regulation, Eur: J . Biochem. 223, 259 -263.

6. Lee, H. W., Smith, L., Pettit, G. R., Vinitsky, A. & Smith, J. B. (1996) Ubiquitination of protein kinase C-rx and degradation by the proteasome, J. Bid . Chem. 271, 20973-20976.

7. Inagaki, M., Hagiwara, M., Saitoh, M. & Hidaka, H. (1986) Protein kinase C negatively modulated by phorbol ester, FEBS Left. 202,

8. Kishimoto, A,. Kajiakawa, N., Shiota, M. & Nishizuka. Y. (1083) Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease, J .

9. Newton, A. & Koshland, D. (1989) High cooperativity, specificity and multiplicity in the protein kinase C-lipid interaction, J. Biol. Chem. 264. 14909-14915.

10. Huang, K. & Huang, F. (1990) Differential sensitivity of protein kinase C isozymes to phospholipid-induced inactivation, J. B i d . Cheni. 265, 738-744.

11. Stabel, S. & Parker, P. J. (1991) Protein kinase C, Pharniucol. Thee

12. Kiley, S., Parker, P. J.. Fabbro, D. & Jaken, S. (1991) Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4Cl cells, J. B i d . Chem. 266, 23 761 -23 768.

13. Gatti, A., Wang, X. & Robinson, P. J. (1996) Protein kinas, C-u is multiply phosphorylated in response to phorbol ester stimulation of PC12 cells, Biuchirn. Biophys. Acfo 1313, 11 1-1 18.

14. Gatti, A. & Robinson, P. J. (1996) Unique phosphorylation of protein kinase C-n in PC12 cells induces resistance to translocation and down-regulation, J . Biol. Clzem. 271, 31718-31 722.

15. Laemmli. U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nnture 227. 680-685.

277-280.

B i d . Chtm. 258, 1156-1164.

51, 71 -9.5.

Gatti and Robinson (ELM J . Biochem. 249) 97

16. Towbin, H., Staehelin, T. & Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Nut/ Acad. Sci. USA 76, 4350-4354.

17. Mason, J. M., Langenbach, R., Shelby, M. D., Zeiger, E. & Tennant, R. W. (1990) Ability of short-term tests to predict carcinogenesis in rodents, Annu. Rev. Pharmacol. Toxicol. 30, 149- 168.

18. Ohno, S . , Mizuno, K., Adachi, Y., Hata, A,, Akita, Y., Akimoto, K., Osada, S . , Hirai, S. & Suzuki, K. (1994) Activation of novel protein kinases C6 and Ct: upon mitogenic stimulation of quiescent rat 3Y1 fibroblasts, J . Biol. Chem. 269, 17495-17501.

19. Molina, C. A. & Ashendel, C. L. (1991) Tumor promoter 12-0- tetradecanoylphorbol 13-acetate and sn-1,2-dioctanoylglycerol increase the phosphorylation of protein kinase C in cells, Cancer Re.r. 51, 4624-4630.

20. Ohno, S., Konno, Y., Akita, Y., Yano, A. & Suzuki, K. (1990) A point mutation at the putative ATP-binding site of protein kinase Cn abolishes the kinase activity and renders it down-regulation- insensitive, J. Bid. Chem. 265, 6296-6300.

21. Zhang, J. I., Wang, L., Petrin, J. , Bishop, W. R. & Bond, R. W. (1996) Characterization of site-specific mutants altered at protein kinase C f l isozyme autohosphorylatiou sites, Proc. Natl Acad. Sci. USA 90, 6130-6134.

22. Pears, C. & Parker, P. J. (1991) Down-regulation of a kinase de- fective PKC-n, FEBS Lett. I , 120- 122.

23. Hansra, G., Bornancin, F., Whelan, R., Hemmings, B. A. & Parker, P. J. (1996) 12-0-Tetradecanoylphorbol-I 3-acetate-induced dephosphorylation of protein kinase Cn correlates with the presence of a membrane-associated protein phosphatase 2A hetero- trimer, J . Biol. Chem. 271, 32785-32788.

24. Newton, A. C. (1995) Protein kinase C: structure, function and regulation, J . Bid. Chem. 270, 28495-28498.

25. Lee, H. W., Smith, L., Pettit, G . R. &Smith, J. B. (1997) Bryostatinl and phorbol ester down-modulate protein kinase C-n and -8 via the ubiquitin/proteasome pathway in human fibroblasts, Moi. Pharmncol. 51, 439-447.

26. Suganuma, M., Fujiki, H., Suguri, H., Yoshizawa, S . , Hirota, M., Nakayasu, M., Ojjika, M., Wamatsu, K., Yamada, K. & Sugimura, T. (1988) Okadaic acid: an additional non-phorbol-12-tetradeca- noate-1 3-acetate-type tumour promoter, Proc. Natl Acad. Sci. USA 85, 1768-1771.

27. Cohen, P., Holmes, C. E B. & Tsukitani, Y. (1990) Okadaic acid: a new probe for the study of cellular regulation, Trend.7 Biochem. Sci. 15, 98-102.

okadaic acid interferes with phorbol-ester-mediated down-regulation of protein kinase c-α, c-δ and...

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