protein kinase C is synthesized on membranes as a 74-. kDa protein that can .... phosphatase inhibitors; PKA, catalytic subunit of cyclic AMP-de- pendent protein ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 264, No. 23, Issue of August 15, pp. 13902-13909,1989 Printed in U.S. A.
Biosynthesis and PosttranslationalModifications of Protein Kinase C in Human Breast Cancer Cells* (Received for publication, December 16, 1988)
Christoph BornerS, Ireos Filipuzzi, Markus Wartmann, Urs Eppenberger, and Doriano Fabbrog From the Laboratory of Biochemistry-Endocrinology,Department of Research, and Department of Gynecology and Obstetrics, University Medical School, CH-4031 Basel, Switzerland
Several forms of protein kinase C with molecular with the posttranslational processing that converts the masses of 74-, 77-, and 80-kDa were detected subin 74-kDa protein kinase C precursor into the 77- and cellular fractions of human breast cancer MDA-MB- 80-kDa formsof the enzyme. 231 cells which express the a-type protein kinase C. is Several linesof evidence indicated that the 74-kDa the precursor of the 77- and 80-kDa protein kinase C forms. (i)Pulse-labelingexperimentsrevealed that The Ca2+-and phospholipid-dependent proteinkinase (proprotein kinase Cis synthesized on membranesas a 74- tein kinase C), the major receptor for tumor promoting phorkDa protein that can be chased into the 77- and the bo1 esters, functionsas a transducer of the second messengers 80-kDa protein kinase C forms.(ii)The primary trans- calcium and diacylglycerol by coupling the agonist-induced lation productof protein kinase C displayed an apparent molecular sizeof 74-kDa as determined by in vitro breakdown of inositol phospholipids with a varietyof cellular translation of poly(A)+RNA from MDA-MB-231 cells. functions (1-3). Tumor-promoting phorbol esters such as (iii) Incubation with serinelthreonine-specificprotein phorbol 12-myristate 13-acetate (PMA)’ fully substitute for phosphatases (potato acid phosphatase and phospha- diacylglycerol (4). In contrast to diacylglycerol, tumor protase 1or 2A) resulted in the complete dephosphoryla- moters are only slowly metabolized in the cells and cause a tion of the 77-kDa to the 74-kDa protein kinase C protracted stimulation of protein kinase C that ultimately results in the down-regulation of protein kinase C by proteform. Protein kinase C appears to be synthesized in membranes as an unphosphorylated and presumably olysis (5-9). Protein kinase C belongs to a multigene family inactive 74-kDa formthat is converted into the active whose members display different molecular sizes (10-13). In 77- and 80-kDa protein kinase C by post-translational addition, all members of the protein kinase C family have been reported to undergo posttranslational modifications that modification involving at least two phosphorylation steps. The first phosphorylation is probably achieved change its molecular weight as well as itsenzymatic properties ki- (14-21). Protein kinase C is a phosphoprotein whose molecby a specific, yet unidentified, “protein kinase C nase’’ since the 74-kDa protein kinase C species did not ular weight can slightly vary following either autophosphoundergo autophosphorylation and was neither a sub- rylation (14-17) or treatment withphosphatases (18, 19) strate for the purified protein kinase C, S6 kinase, concomitant with changes in the catalytic state of protein phosphorylasekinase,casein kinase 11, nor for the kinase C activities. Furthermore, pulse-chase experiments of catalytic subunit of CAMP-dependent protein kinase. rat glioma 328 cells demonstrated that thenewly synthesized Except for phosphorylase kinase and the catalytic sub- protein kinase C form undergoes posttranslational modificaunit of the CAMP-dependent protein kinase, phospho- tions thatresult in a protein kinase C with a slightly increased rylation of the 77-kDa protein kinase C form with molecular size (16, 20). These data indicate that posttranslaC (autophosphorylation), S6 purified protein kinase tional phosphorylations may be involved in modulating prokinase or casein kinase I1 shifted the molecular mass of the 77-kDa protein kinaseC to 80-kDa. Prolonged tein kinase C activity as well as its molecular size in intact exposure of MDA-MB-231 cells to phorbol 12-myris- cells. Exposure of various human breast cancer cells to PMA tate 13-acetate not only leads to a complete downinhibits cell proliferation to various extents (22) and causes a regulation of protein kinase C activity but also to an a total loss of protein kinase C activity withoutaffecting protein accumulation of 74-kDa protein kinase C due to kinase C synthesis(23-25). During the prolonged PMA-treatretarded conversion of the 74-kDa into the 77- and 80-kDa protein kinaseC forms in these cells. Our data ment, human breast cancercells continuously synthesize and accumulate inactive membrane-bound protein kinase C forms indicate that tumor promoters additionally interfere of 74- and 80-kDa at rates thatappear to inversely correlate * This work was supported in part by the Swiss National Science with the degree of PMA-induced growth inhibition in the Foundation Grant 3.344.0.86, by the Regional Cancer League (Basel), and by CIBA-Geigy Ltd. (Basel). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Comprehensive Cancer Center, College of Physicians & Surgeons, Columbia University, 701 W. 168th St., New York, NY 10032. § T o whom correspondence and reprint requestsshould be addressed: Laboratory of Molecular Tumor Biology (Hormonlabor), Dept. of Research, Frauenklinik Basel, Schanzenstrasse 46, CH-4031 Basel, Switzerland. Tel.: 061-57-57-57 (ext. 2366 or 2323).
’
The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; DMEM, Dulbecco’s minimum essential medium; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; HEPES, 442-hydroxyethyl)-l-piperazineethanesulfonicacid; MES, 2[N-morpholino]ethanesulfonic acid; MOPS, 3-[N-morpholino]propanesulfonic acid; PhI, phosphatase inhibitors; PKA, catalytic subunit of cyclic AMP-dependent protein kinase; PKC, Ca2+-activated, phospholipid-dependent kinase; PPlc, catalytic subunit of protein phosphatase 1; PP2Ac, catalytic subunit of protein phosphatase 2A; PS, L-a-phosphatidylL-serine; SDS, sodium dodecyl sulfate; W7, N-(6-aminohexyl)-5chloro-l-naphthalene-sulfonamide.
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Biosynthesis of Protein Kinase C
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respective cell lines (24). These data suggest that PMA not mination of protein kinase C activity with histone H1 (Sigma, type only induces the down-regulationof protein kinase C but also V-S) as substrate were performed as described before (23-25, 33). Immunoprecipitation-Immunoprecipitation of protein kinase C leads to an accumulationof inactive 74- and 80-kDa protein from subcellular fractions was performed in the presence or absence kinase C forms. The present study demonstrates that proteinof 1 pgof unlabeled purified protein kinase C and resolved on 8% kinase C is synthesized as a presumably inactive, non-phos- SDS-polyacrylamide gels (24, 34). Quantification of fluorographs or autoradiographs was performed as described before (35, 36). phorylated 74-kDa protein that is converted into the active Dephosphorylation of Protein Kinase C-Dephosphorylation of pu77- and 80-kDa protein kinase C forms by posttranslational rified protein kinase C or of protein kinase C immunocomplexed to processes which involve at least two phosphorylation steps. EXPERIMENTALPROCEDURES
proteinA-Sepharose was carried out in a total volume of 50 pl. Immunocomplexes were washed twice in either buffer A (50mM TrisHCI, pH 7.0, 1 mM EDTA, 50 mM P-mercaptoethanol, 20 pg/ml leupeptin, 2 pg/ml aprotinin) or buffer B (10 mM MES, pH 5.5,l mM MgC12, 50 mM NaC1, 0.1 mM dithiothreitol, 100 pg/ml leupeptin, 10 pg/ml trypsin inhibitor, 0.1% aprotinin). Catalytic subunits of protein phosphatase 1 (PPlc, 100 units/ml) or 2A (PP2Ac, 1000 units/ml) were added to the immunocomplexed or purified protein kinase C in buffer A containing 1 mM MnClz and 10 pg/ml fatty acid-free bovine serum albumin to a final assay concentration of 2 units/ml and 20 units/ml, respectively. Potato acid phosphatase was added to immunocomplexed protein kinase C in buffer B to a final concentration of 25 units/ml. Theincubation with phosphatases was performed in the presence or absence of sonicated 100 pg of PS and1p~ PMA. Control incubations were performed for 60 min in the absence of phosphatases or in the presence of phosphatase inhibitors. After incubation for indicated times at 30 "C, the dephosphorylation reaction was stopped by the addition of phosphatase inhibitors NaF,Na3VOr,and KHZPO, at final concentrations of 50 mM, 100 pM, and 10 mM, respectively, followed by rapid washingof the immunocomplexes with 20 mM TrisHC1, pH 7.4. The immunocomplexed proteinkinase C was then analyzed by SDS-polyacrylamide gel electrophoresis as described (24). Optimal assay conditions for protein phosphatase ICand 2Ac activities were tested by the release of phosphate from 32P-labeled phosphorylase a (26). Phosphorylation of Protein Kinase C by Protein Kinases-Immunoprecipitated protein kinase C was mixed with various highly purified protein kinases in a total volume of 100 pl in the presence or absence of 100 pg of PS and1p~ PMA containing 20 pg/ml leupeptin and 2 pg/ml aprotinin.The following assaybuffers andprotein kinases were used. Protein kinase C (5000 units/mg) (15): 20 mM Tris-HC1, pH 7.4, 10 mM Mg(NO&, and 50 units/ml protein kinase c . S6 kinase (4000 units/ml) (29): 50 mM MOPS, pH 7.0, 2 mM dithiothreitol, 20 mM MgC12, 10 mM p-nitrophenylphosphate, and40 units/ml S6 kinase.Phosphorylasekinase (500 units/mg) (37): 60 mM Na-@-glycerophosphate, pH 7.0, 0.05% a-mercaptoethanol, 20 mM Tris-HC1, pH 6.8, 0.6 mM EDTA, 0.9 mM CaC12, 3.3 mM Mgacetate, and 10 units/ml phosphorylase kinase. Catalytic subunit of CAMP-dependent protein kinase (4000 units/ml) (38): 70 mM MESNaOH, pH 6.9, 75 mM NaCl, 0.5 mM EDTA, 2.5 mM Mg-acetate, 1.2 mg/ml fatty acid-free bovine serum albumin, and 10 units/ml catalytic subunit of CAMP-dependent protein kinase. Casein kinase I1 (5000 units/ml) (28): 50 mM Tris-HC1, pH 7.1, 100 mM NaC1, 2 mM MgC12, 2 mM spermidine, and 20 units/ml casein kinase 11. Incubations were carried out with 100 p M ATP in eitherpresence or absence of 10 pCi of [Y-~'P]ATPfor 20 min at 32 "C. Reactions were stopped by rapidly washing the immunocomplexes three times in 20 mM TrisHCI, pH 7.4 containing 20 mM EDTA followed by dissociation and electrophoresis on 8%SDS-polyacrylamide gels. I n Vitro Translation of Poly(A)+ RNA from MDA-MB-231 CellsPoly(A)+RNA was obtained from MDA-MB-231 human breast cancer cells by the acid guanidinium thiocyanatephenolchloroform extraction (39) followed by selection of total RNA on oligo(dT)-cellulose (40). 1 pg of poly(A)+ RNAwas translated in vitro for 1 h at 30 "C in reticulocyte lysates containing RNase inhibitor (10 units), calf liver tRNA (5 pg), 100 mM K-acetate, and 30 pCi of [35S]methionine (15 mCi/ml, >800 Ci/mmol, Du Pont-New England Nuclear). Translation was stopped with 1% SDS followed by immunoprecipitation of protein kinase C as described above. Other Analytical Methods-Protein was determined by the method of Bradford (41) using the Bio-Rad reagents and bovine serum albumin as standard. Silver staining was performed exactly as described (42).
Materials-Phosphorylase kinase, phosphorylase a, L-a-phosphatidyl-L-serine (PS), protein A-Sepharose CL-4B, PMA, N-(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide (W7), p-nitrophenylphosphate, ovalbumine, fatty acid-free bovine serum albumin, histone H1 (type V-S), trypsin inhibitor, spermidine, leupeptin, aprotinin, iodoacetamide, EDTA, EGTA, SDS, Triton X-100, Nonidet P-40, dithiothreitol, and potatoacid phosphatase were from Sigma.Sodium deoxycholate, HEPES, MOPS, and 0-mercaptoethanol were bought from Merck and oligo(dT)-cellulose from Pharmacia LKB Biotechnology Inc. Molecular weight markers for electrophoresis were provided by Bethesda Research Laboratoryand X-Omatfilms by Kodak. [32P]Orthophosphoricacid (carrier free),~ - [ ~ ~ S ] m e t h i o n(>800 i n e Ci/ mmol, translation grade), [9,10-3H]myristicacid (22.4 Ci/mmol), and [9,10-3H]palmitic acid (28.5 Ci/mmol) were purchased from Du PontNew England Nuclear. Rabbit reticulocyte lysate, RNase inhibitor, calf liver tRNA, ~ - [ ~ ~ S ] m e t h i o n(1200 i n e Ci/mmol), and [Y-~'P]ATP (30 Ci/mmol) were obtained from Amersham Corp. Ionomycin was from Behring Diagnostics. Staurosporine was a generous gift of T. Meyer, Ciba-Geigy Ltd., Basel. Full lengthbovine brain a-PKC cDNA (XbPKC306, 10) was a kind gift of P. J. Parker, Ludwig Institute, London. Pure catalytic subunits of protein phosphatase1 (PPlc) and 2A (PP2Ac) from rabbit skeletal muscle were kindly provided by P. Cohen,University of Dundee,Scotland(26). The following pure protein kinases were obtained from the Friedrich Miescher Institute, Basel: B. Hemmings, catalytic subunit of CAMP-dependent protein kinase (PKA) from rabbitskeletal muscle (27); S. Ferrari, casein kinase I1 from bovine brain (28); S. Kozma and G. Thomas, S6 kinase from Swiss 3T3 cells (29). Cell Labeling-The MDA-MB-231 human breast cancer cells were obtained from the Mason Research Institute (Rockville, MD) and cultured as previously described (30). Prior to each experiment cells were washed three times in Dulbecco's minimum essential medium (DMEM, Amimed, Basel) and once in the appropriate labeling medium in the absence of phosphatase inhibitors. Labeling of the cells was always performed in the absence of phosphatase inhibitors. Cells (2-5 X 10') were labeled for indicated times with 100 pCi/ml [35S] methionine (1200 Ci/mmol) in methionine-free Dulbecco's minimum essential medium (met-free DMEM) as described before (24). For pulse-chase experiments, cells were pulsed for 2 or 5 min with 200 pCi/ml [35S]methionine in met-free DMEM, rapidly washed three times in culture medium containing 5 mM unlabeled methionine and then chased for indicated times in DMEM containing 5% fetal calf serum (GIBCO) and 5 mM unlabeled methionine. 32P-Labeling of cells was carried out for 4 h with 100 pCi/ml of [32P]orthophosphoric acid in phosphate-free DMEM (Amimed, Basel) as described (31). Labeling with [3H]myristic or [3H]palmitic acid was performed by the method of Buss and Sefton (32) for 16 h. Subcellular Fractionation-Subcellular fractionation was performed as described before (25)with the following modifications. Cells were disrupted by sonication in 1.5 ml of homogenization buffer (HBI: 20 mM Tris-HC1, pH 7.4, 2 mM EGTA, 2 mM EDTA, 6 mM 0mercaptoethanol, 20 pg/ml leupeptin and 2 pg/ml aprotinin) and centrifuged for 30 min at 200,000 X g yielding the cytosol and the membrane pellet. The latter was resuspended in HB1 containing 1% Nonidet P-40 (w/v), sonicated, and subsequently recentrifuged for30 min at 200,000 X g. This supernatant isreferred to as theNonidet P40 membrane extract whereas the SDS-membrane extract was obtained by dissolving the residual membrane pellet in HB1 containing 1%SDS (w/v). Totalcellular extract was prepared by disrupting cells in HB1 containing 1% SDS followed by immediate boiling. When indicated phosphatase inhibitors (PhI: 50 mM NaF, 100 p~ Na3V04) were present in all buffers during subcellularfractionation. RESULTS Determination of Protein Kinase C Activity and Preparation of Phosphorylation States of Protein Kinase C in MDA-MBAnti-protein Kinase C Antibodies-Purification of protein kinase C from MDA-MB-231 human breast cancer cells, generation of poly- 231 Cells-As determined by hydroxyapatite chromatography, clonal antibodies against porcine brain protein kinase C, and deter- the MDA-MB-231 human breast cancer cells express the a-
Biosynthesis of Protein Kinase C
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A
35s
A
32P
a b c d e
+ -
+ M
CY
B
+ CY
35s
a
b c d e
+ M
l
C
68-
b c d e
32P
m
94e
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S DS
+ -+ CY
+ M
CY
-
+ -
breastcancer cells ( A ) or MDA-MB-231 cells exposed to 300 nM PMAfor 48 h ( B ) were labeledfor 6 h with 100pCi/ml [3sS] methionine ("S) orfor 4 h with 100 pCi/ml["P]orthophosphoric acid ("P) in the absence of PhI. Immunoprecipitationof protein kinase C from cytosol (CY) and membrane ( M ) fractions prepared in the absence (-) or presence (+) of phosphatase inhibitors were performed as described under "Experimental Procedures." Arrows, 77-kDa; dots, 80-kDa; open circles, 74-kDa protein kinase C.
isoform of protein kinase C? In these cells protein kinase C is present as a phosphoprotein with a molecular mass of 80 kDa (23, 24). Subcellular fractionationinthe absence of phosphatase inhibitors (PhI) led to a rapid and partial dephosphorylation of the 80-kDacytosolic protein kinase C concomitant with a M , shift to 77, whereas the M , of the membrane-bound enzymeremainedunaffected (Fig. lA).This rapid dephosphorylation in the absence of PhI occurred only during subcellular fractionation and did not depend on the presence of PhI during the labeling of cells? The 77-kDa protein kinase C form displayed lower affinities for cofactors and substrates as compared with the 80-kDa protein kinase C form.' Following complete down-regulation of protein kinase C activity by PMA, there was a continuous synthesis of two membrane-bound, presumably inactive 74- and 80-kDa protein kinase C forms (Fig. 1B). As compared with the membrane-bound 80-kDa protein kinase C of control cells, the membrane-associated 80-kDa species of PMA-treated cells contained 2-3-fold more [3'P]phosphate whereas no "P was covalently attached to the74-kDa protein kinase C (Fig. 1, A and B ) . These datasuggest that themembrane-associated 74-kDa protein kinase C does not contain phosphate or incorporates phosphate at a slow rate (Fig. 1B). Biosynthesis of Protein Kinase C in MDA-MB-231 CelhA protein kinase C form with a M , of 74 was immunoprecipitated from the SDS-membrane extract of MDA-MB-231 cells pulse-labeled with [35S]methionine for 2 min (Fig. 2A, lanes a). If the pulse was extended to 5 min, the 74-kDa protein kinase C was alsoimmunodetected intheNonidetP-40membrane extract aswell as in the cytosol (Fig. 2 A , lanes b ) . The 74-kDa protein kinase C was chased out from the SDSmembrane fraction whereas in thecytosol and NonidetP-40membrane extract the74 kDa was chased within 15 min into C. Borner, I. Filipuzzi, and D. Fabbro, unpublished results.
CY
C
M
FIG. 1. Characterization of different forms of protein kinase C in MDA-MB-231 cells. Control MDA-MB-231 human
NP-40
5 mw-
0
43-
"
SDS
NP- 40
CY
FIG. 2. Biosynthesis of protein kinase C. MDA-MB-231 cells were pulse-labeled with 200 pCi/ml [35S]methioninefor 2 (lanes a ) or 5 min (lanes b ) . Cells pulse-labeled for 5 min were chased for 15 min (lanes c) or 30 min (lanes d ) or 45 min (lanes e ) with 5 mM unlabeled methionine. Cells were not treated ( A ) or treated with 300 nM PMA for 30 min ( B ) or 48 h (C)prior to the pulse-chase experiment. The cytosolic (Cy), Nonidet P-40-membrane (NP-40), and SDS-membrane (SDS)fractions were prepared in the presence of phosphatase inhibitors and immunoprecipitated as described under "Experimental Procedures." Arrows, 74-kDa; dots, 80-kDa PKC forms; circles, 50kDa protein (putative catalytic unit of protein kinase C).
the 80-kDa protein kinase C (Fig. 2.4, lanes c-e). If similar pulse-chase experiments were performed on MDA-MB-231 cells treated for 30 min with 300 nM PMA, onlyminor amounts of the 74-kDa protein kinase C were found in the cytosol (Fig. 2B, lanes b ) . Under these conditions the conversion to the 80-kDa protein kinaseC species mainly occurred in the Nonidet P-40-membrane extract with but a slower time course (Fig. 2B, Nonidet P-40, lanes c-e). In addition, a 50kDa protein was specifically immunoprecipitated from SDSmembrane extracts (Fig. 2B, SDS).Preliminary results from tryptic peptide analysisindicatethatthe 50-kDa protein represents the catalytic unit of protein kinaseC.' In the SDSmembrane extract of MDA-MB-231 cells exposed for 48 h to 300 nM PMA immunoprecipitationof the 50-kDa protein was decreased by 40-60% (Fig. 2C, SDS). In addition, the timeof conversion of the 74-kDa to the 80-kDa protein kinase C in the Nonidet P-40-membranefraction was further reduced as compared with the respective subcellular fraction of MDAMB-231 cells treated for 30 min with PMA (Fig. 2, B+C, NP40). This delayed conversion apparently contributes to the accumulation of the 74-kDa protein kinase C form in the Nonidet P-40-membrane extracts of cells exposed for a prolonged time to PMA(Fig. 2C, NP-40). In VitroTranslation of Protein Kinase C-As shown in Fig. 3, in vitro translation of poly(A)+ RNA from MDA-MB-231 human breast cancer cells resulted in the specific immunoprecipitation of a single protein kinase C band (Fig. 3, lanes
Biosynthesis of Protein Kinase C
13905
a b c d be f
94
7 I 9 4 . 804 \\
:"
7
FIG.3. I n vitro translationof protein kinase C. Autoradiograph obtained after immunoprecipitationof in uitro translation of 1 pg of MDA-MB-231 poly(A)+ RNA(lanes b ) . Immunoprecipitations in the absence of poly(A)+ RNA (lanes d ) or in the presenceof 1 pg of poly(A') RNA but competed with 1 pg of purified unlabeled protein kinase C (lane e). Immunoprecipitation of cytosolic protein of phosphatase inhibitors from kinase C prepared in the absence MDA-MB-231cellspulse-labeledwith["S]methioninefor5 min (lane a ) followed by a chase for 30 min (lanec ) . Immunoprecipitation of the membrane extract from MDA-MB-231 cells exposed for 48 h to 300 nM PMA and labeled for 6 h with [35S]methionine (lane f). Arrows, 74- and 80-kDa protein kinase C, respectively; dots, 77-kDa protein kinase C. 0 15 45 sb b and e). The in uitro translation product of protein kinase C corresponded in its M , to the pulse-labeled 74-kDa protein FIG.4. Dephosphorylation of immunocomplexed 77-kDa kinase C of MDA-MB-231 cells (Fig. 3, lane a) as well as to protein kinase C by potato acid phosphatase (PAP), PPlc, a n d the membrane-bound 74-kDa protein kinase C form that is PP2Ac. Protein kinase C was immunoprecipitated from the cytosol synthesized under the continuous presence of PMA (Fig. 3, of MDA-MR-231 cells labeled for 6 h with ["SS]methionine (3sS)or lane f). A protein kinase C of 74 kDa was also obtained by in [J2P]orthophosphoricacid ("'Pp) in the absence of phosphatase inhibuitro transcription of the full length a-PKC cDNA cloned itors. The 77-kDa protein kinase C immunocomplexed to protein A,.( 20 units/ml), P P l c (A, 2 into pSP64 followed byimmunoprecipitation of the translated Sepharose was incubated with PAP or PP2Ac( 0 , 2 0 units/ml) in the presence of 100 pg of PS product' (10). Based on thesefindings the primary translation units/ml), and 1 p~ PMA for 15.30 (lanes b ) , 45,and 60 min (lanes c ) a t 30 "C product of protein kinase Cappears tohave a M , of 74. Taken or for 60 min but in the presenceof phosphatase inhibitors (lanes a ) . together these data strongly suggest that the 74-kDa protein Immunoprecipitable protein kinase C forms were quantified ( B and kinase C form which accumulates in membranes of human C ) by densitometric scanning of the autoradiographs ( A ) . Arrows indicate 74- and 77-kDa protein kinaseC forms. breast cancer cells treated with PMA may representthe inactive precursor of the 77- and 80-kDa protein kinase C a b c d e f g forms. Dephosphorylation of Protein Kinase C by Potato Acid Phosn K -4: PKC 74 K phatase, PPlc andPP2Ac"Since the 77- and 80-kDa protein FIG.5. Dephosphorylation of purified 77-kDa protein kikinase Cs are phosphoproteins we assumed that dephosphorylation of the 77-kDa protein kinase C form may generate nase C by the catalytic subunit ofprotein phosphatase 1. 100 ng (lanes b-d, f and g) or 50 ng (lane e ) of 77-kDa protein kinase C the 74-kDa species identified as the primary translation prod- purified from the cytosol of MDA-MB-231 cells was incubated for 1 uct of protein kinase C(Figs. 2 and 3). Therefore, the 77-kDa h at 30 "Cwith (lanes b-f) or without (lane g) 2 units/ml P P l c in the protein kinase Cwas immunoprecipitated from the cytosol of presence of either phosphatase inhibitors(lane b ) or 1 mM Mg(NOd2, MDA-MB-231 cells labeled with either [35S]methionine or 0.3 mM ATP, 100 pg of PS, and 1 p~ PMA (lane c) or 100 pg of PS ["P]orthophosphoric acid. Incubation of the "P-labeled 77- and 1 p~ PMA (lones d and e).100 ng of protein kinase C without additions (lane f).Lane a shows 2 units/ml P P l c alone. At the end kDa protein kinase C immunocomplexed to protein A-Seph- of the incubation the reactions were analyzed ona 8%SDS-polyacrylarose in the presence of 100 pg of PS and 1 p~ PMA with amide gel followed by silver staining. either PAP,P P l c or PP2Ac resulted inthe release of 32Pfrom the 77-kDa protein kinase C in a time-dependent manner as well as the release of 32Pfrom the 77-kDa enzyme ruling (Fig. 4). After 1 h about 90, 75, and 30% of the 32Pwere out proteolysis as a possible cause for the M , shift (Fig. 4A, removed from the 77-kDa protein kinase C by PPlc, PP2Ac, lanes a). or potato acid phosphatase, respectively (Fig. 4B). Thus, P P l c Similar results were obtained when highly purified 77-kDa was most effective (2 units/ml) in removing covalently at- protein kinase Cfrom MDA-MB-231 cells was dephosphoryltachedphosphates from the 77-kDa protein kinase C. A ated by PPlc. The phosphatase treatment generated a protein similar extent of dephosphorylation of the 77-kDa protein of 74-kDa that was paralleled by the loss of the 77-kDa protein kinase C was achieved with PP2Ac but at 10 times higher band (Fig. 5). The dephosphorylation of the 77-kDa protein phosphatase concentrations(20 units/ml). Potato acid phos- kinase C was strictly dependent on the presence of PS and phatase, however, showed very little activity against the 77- PMA (Fig. 5, lanes d-f). Again, the inclusion of phosphatase kDa protein kinase C species even a t high phosphatase con- inhibitors prevented the formation of the 74-kDa band (Fig. centrations (>25 units/ml). As shown by quantification of the 5, laneb). Unfortunately, but consistentwith previous reports autoradiographs (Fig. 4B), the decrease in the 32Pcontent of (43), preincubation of the 77-kDa protein kinase C with the the 77-kDa protein kinase C occurred concomitant with an PS and PMA resulted in a time-dependent inhibition of its increase of the [35S]methionine-labeled74-kDa protein kinase protein kinase activity (80%after 1 h) even in the absence of C form (Fig. 4A).Extensive dephosphorylation of the 77-kDa PPlc.' Although the addition of 1mM M e and 0.3 mM ATP protein kinase C, therefore, generates a74-kDa protein kinase to the incubation mixture protected protein kinase C from C which is almost devoid of phosphate (Fig. 4A). Addition of beinginactivated by PS and PMA, it also prevented the phosphatase inhibitorsduringthephosphatasetreatment formation of the 74-kDa protein kinase C species (Fig. 5, lane abolished both the formation of the 74-kDa protein kinase C c). Thus,although PS/PMA positively influenced the dephos-
=b
Biosynthesis of Protein Kinase C
13906
phorylation of the 77-kDa protein kinase C to the 74-kDa form without affecting the activity of P P l c i t inhibited the catalytic activity of protein kinase C. For this reason it was impossible to determine protein kinase C activity of the 74kDa protein kinase C following dephosphorylation. Phosphorylation of Protein Kinase C by Protein KinasesSincedephosphorylation of the 77-kDa proteinkinase C yielded a74-kDaspeciesvarioushighlypurified protein kinases (phosphorylase kinase, CAMP-dependent protein kiC) were nase,casein kinase 11, S6 kinase,proteinkinase tested for their ability to phosphorylate the 74-kDa protein kinase C. Therefore, MDA-MB-231 cells were pulse-labeled with ["S]methionine for 5 min and thecytosolic fraction was subjected to immunoprecipitation. Under theselabeling conditions a mixture of radiolabeled 74-kDa and unlabeled 77kDa proteinkinase C is immunocomplexed to protein ASepharose (Fig. 6, lanes a). None of the protein kinases were capable of converting the 3sS-labeled 74-kDa protein kinase C into the 77-kDa enzyme form irrespective of the presence of PS and PMA (Fig. 6B).Similar resultswere obtained when the radiolabeled 74-kDa protein kinaseC species immunoprecipitated from membranes of long-term PMA-treatedcells or when the 74-kDa protein kinase C obtained by dephosphorylation of the 77-kDa protein kinase C by P P l c were used as substrates for the above protein kinases.* By contrast, incubation of the immunocomplexes with purified S6 kinase (S6K),protein kinase C (PKC),or casein kinase I1 (CSK ZZ) in the presence of [y"P]ATP resulted in the appearance of an 80-kDa protein kinase C phosphoprotein (Fig. 6A). Generation of the "P-labeled 80-kDa protein kinase C form is rather due to the phosphorylation of the unlabeled 77 kDa than to the phosphorylationof the 74-kDa protein kinase C form that is present in the immunocomplex. The most efficient incorporation of 32Pinto the 77-kDa protein kinase C form was achieved by PKC followed by casein kinase I1 and S6 kinase. Interestingly, the catalytic subunit of CAMP-dependent protein kinase (PKA) phosphorylated the 77-kDa protein kinase C species without promoting a shift in its M , A
PPK
PKA
s6K
ba cbd cba cb c
CSK II a b c
whereas phosphorylase kinase (PPK) did not phosphorylate the 77-kDa protein kinaseC (Fig. 6A). All these phosphorylations were specific for the respective purified protein kinase since protein kinaseC was irreversibly inactivated by deoxycholate during immunoprecipitation and, thus, unable to autophosphorylateinits immunocomplexed state (Fig. 6A). Taken together these data suggest that (i) theconversion of the 74-kDa into the 77-kDa protein kinase C appears to be mediated by proteinkinase(s)otherthanthose employed herein and that (ii) the 77-kDa protein kinase C form is not only a substrate for protein kinase C (autophosphorylation) but also for casein kinase11, S6 kinase, and PKA albeit toa less extent. Autophosphorylation of the 74-kDa Protein Kinase C-Although the presenceof deoxycholate was absolutely required for a specific immunoprecipitation of protein kinase C (24); the detergent irreversibly inactivated the immunocomplexed enzyme. In order to test the ability of the 74-kDa protein was, kinase C to undergo autophosphorylation, the precursor therefore, subjected to phosphorylation in a cell-free extract before immunoprecipitation. Acytosolic fraction of MDAMB-231 cells containing the ["S]methioninepulse-labeled 74-kDa and the unlabeled 77-kDa protein kinase C was incubated in the presenceof PS, PMA, [y"'P]ATP, and phosphataseinhibitors. Followingendogenous phosphorylation the immunoprecipitates revealed a phosphorylated 80-kDa protein kinaseC whichis apparently the result of a PS/PMAdependent autophosphorylation of the unlabeled 77-kDa enzyme rather thanof the ["S]methionine pulse-labeled 74-kDa precursor (Fig. 7). The "S-labeled 74-kDa protein kinase C did not incorporate [:'2P]phosphate as shown by a short exposure of the x-ray film.' This finding indicates that the74kDa protein kinase C is incapable to undergo a PS/PMAdependent autophosphorylation and is not phosphorylated by other protein kinases present in thecell-free extracts. Moreover, the active 77-kDaprotein kinaseC form whichis present in the cytosolic fraction of MDA-MB-231 cells seems unable to phosphorylate the74-kDa protein kinase C precursor. Effects of Protein Kinase Inhibitors on thePhosphorylation
PKC a b c
B
A a
b ac b
c
F
B
PPK a
b c
PKA S6K d b c b c
CSKll PKC b c b c
94 i,. m 0
FIG.6. Phosphorylation of immunocomplexed protein kinase C by S6 kinase ( S 6 K ) . phosphorylase kinase ( P P K ) , protein kinase C, casein kinase I1 (CSK II), and catalytic subunit of PKA. MDA-MB-231 human breast cancer cells were pulse-labeled with 200 pCi/ml [?3]methionine for 5 min. Cytosol was preparedin the absence of phosphatase inhibitors followed by immunoprecipitation of protein kinase C. Protein kinase C immunocomplexed to protein A-Sepharose was incubated for 20 min at 30 "C with S6K, phosphorylase kinase, protein kinase C (PKC), casein kinase 11, or the catalytic subunit of PKA with (lanes c ) or without (lanes b ) 100 p g of PS and 1 pM PMA in the presence of 100 p M ATP ( R ) or 100 p~ ATP supplemented with 10 pCi of [y-'*P]ATP ( A ) . Phosphorylation with 100 p~ ATP in the absence of any exogeneous protein kinase ( A , left panel). Lanes a, immunoprecipitation of "Spulse-labeled 74-kDa protein kinase C not subjected to phosphorylation. Lanes d, immunoprecipitation of "S-labeled protein kinase C. The autoradiograph showing the phosphorylation with protein kinase C (PKC) was exposed for only %5 of the time as compared with the others. Arrows, 74- and 80-kDa; dots, 77-kDa protein kinase C.
-l
- 94
"
t,
. . a m
68-
"
43 -
"
t:
0
.
4
-68
- 43 90d
FIG.7. Autophosphorylation of the 74-kDa protein kinase C form. Cytosols of MDA-MB-231 cellspulse-labeled for 5 min with 200 pCi/ml [%]methionine were prepared in the absence of phosphatase inhibitors. Protein kinase C was immunoprecipitated from cytosols (400 pg) that were subjected to phosphorylation with [y3*P] ATP in the presence (lanes b ) or absence (lanes c ) of 100 pg of PS and 1 p~ PMA for 20 min at 32 "Cas described under "Experimental Procedures."The autoradiographwas developed either 24 h ( A ) or 90 days after ( B )the experiment. Lanes a show the immunoprecipitates of cytosolic proteins not subjected to the phosphorylation assay. Arrows, ["S]methionine pulse-labeled 74-kDa; dots, "P-labeled 80kDa protein kinase C.
Biosynthesis Kinase of Protein a
b c d e
” 4 9 ‘ 2
-94
g z 1
1
-68
68-
FIG. 8. Incubation of MDA-MB-231 cells with W7,staurosporine, and ionomycin. MDA-MB-231 cells were exposed to 1% Me2S0 (dimethyl sulfoxide) (lane a ) or to 1 pM of W7 (lone b ) , staurosporine (lane c), or ionomycin (lane d ) for 6 h at 37 “C followed by labeling of cells with 100 pCi/ml [“S]methionine. Total extracts were prepared in 1% SDS, immunoprecipitated, and the immunoprecipitates analyzed for radiolabeled 74- or 77-kDa protein kinase C species. The pulse-labeled 74-kDa protein kinase C is shown in lane e.
State of Protein Kinase Cin MDA-MB-231 Cells-The results obtained by phosphorylation of protein kinase C in cell-free extracts suggested that the phosphorylation of the 74-kDa protein kinase C may occur within defined cellular compartments. In an attempt to inhibit intracellular protein kinases the MDA-MB-231 cells were exposed for 6 to h various protein kinase inhibitors. During the last2 h of treatment cells were labeled with [35S]methioninefollowed by preparation of total extracts with SDS andimmunoprecipitation of protein kinase C. However, neither W7, a putative specific inhibitor for Ca2+calmodulin-dependent protein kinases nor staurosporine, a potent butunspecific inhibitor for a varietyof protein kinases (44) led to the accumulation of the 74-kDa protein kinase C form (Fig. 8, lanes b and c). Also, no 74-kDa protein kinase C form was accumulated by increasing the intracellular Ca2+ concentrations with ionomycin (Fig. 8, lane d). These data suggest that thephosphorylation of the 74-kDa to the77-kDa protein kinase Cform either occurs rapidlyor in intracellular compartments that are not accessible to the protein kinase inhibitors. DISCUSSION
According to the results obtained in this study different the forms of protein kinase C found in MDA-MB-231 human breast cancer cells appear tobe generatedby posttranslational modifications of one specific isozyme type rather than from the expression of different protein kinase Cgenes. All human breast cancer cells tested express the a-type protein kinase Cz and synthesize a membrane-bound inactive 74-kDa protein kinase C form following down-regulation of the active 77/80kDaproteinkinaseCactivity by PMA (24). Thisstudy demonstrates that this 74-kDa protein kinase C represents the precursor of the 77- and 80-kDa protein kinase C forms. As determined by in vitro translation of poly(A)+ RNA from MDA-MB-231 cells or by in vitro transcription/translation of the full length a-type protein kinase C cDNA (lo), the 74kDa protein is the primary translation product of protein kinase C. Consistent withthese findings isthat protein kinase C is initially synthesized as a 74-kDa protein in tight association with membrane components (SDS-membrane extract) resistant to solubilization by non-ionic detergents such as NonidetP-40 or Triton X-100. Subsequently, the 74-kDa species is transferred into othercellular compartments (Nonidet P-40-membrane extract, cytosol) and rapidly converted into 77- and 80-kDa protein kinase C forms. A similar shift in the molecular size during protein kinase C synthesis was observed in rat glioma 328 cells (16, 20). The appearance of an immunoprecipitable 50-kDa protein during the PMAexposure, which presumably represents the catalytic unit of protein kinase C, indicates rapid proteolysis of the 74-kDa protein kinase C at its siteof synthesis by the neutral Ca2+-
C
13907
activated protease calpain (6, 7,45-47). Nevertheless, substantial amounts of the newly synthesized 74-kDa protein kinase C precursor, whose rate of synthesis appears unaffected by PMA, seem to escape proteolysis. This is presumably due to either therapid transfer of the 74-kDa protein kinase Cto other cellular compartments or to the desensitization of the proteasesystem.PMAalso markedly reduced the time of conversion of the 74-kDa protein kinase C precursor to the 77- and 80-kDa species with the net effect that the 74-kDa protein kinase C accumulates in the membrane fractions of human breast cancer cells treated with PMA. Since PMA seems not toaffect protein kinase CmRNA synthesis nor its translation (20, 48) these findings suggest that PMA interferes with posttranslational steps that convert the 74-kDa protein kinase C precursorinto the77/80-kDa enzyme forms. Although the NH2-terminaldomain of a-type protein kinase C contains potential acylation sites (49, 50), neither myristoylation nor palmitoylation of the 74-, 77-, or 80-kDa protein kinase C formscould be detected in control as well as phorbol ester-treated MDA-MB-231 cells.2 On the other hand,several lines of evidence suggested that the 74-kDa protein kinase C undergoes posttranslational phosphorylations: (i) As compared with the 74-kDa, both the 77- and 80-kDaprotein kinase C forms are phosphoproteins (14-19, 23, 24). (ii) Autophosphorylation of the 77-kDa form has been shown to increase its M , to 80-kDa (17, 24). (iii) The80-kDa protein is rapidly and partially dephosphorylated to the77-kDa protein kinase C species (24). (iv) Treatmentof a 78/80-kDa protein (18) or immunoprecipitated (19) kinase C doublet purified from rat brain with potato acid phosphatasegenerateda faster-migrating doublet of 74/76-kDa. As shown in this report, a 74-kDa protein kinase C form can be obtained by complete dephosphorylation of the 77-kDa protein kinase C by the catalytic subunitsof protein phosphatase 1 and 2A as well as, to a less extent, by potato acid phosphatase. Interestingly, the dephosphorylation of the 77-kDa protein kinase C is strictly dependent on the presence of the cofactors PS and PMA. Since PS and PMA are known to induce a conformational change on the 77-kDa protein kinaseC they may facilitate the access of phosphatases tophosphorylation sites within the protein kinase C molecule (51). Determination of the catalytic state of the 74-kDa protein kinase C species following dephosphorylation was precluded due to the fact that preincubation with PS/PMA irreversibly inactivatedthe enzyme activity (43). The finding that Mg-ATP prevented the inactivation of protein kinase C by PS/PMA and also completely abolished the release of phosphate from the 77kDa protein kinase C as well as its conversion to the 74-kDa form strongly indicates that thephosphorylation sites recognized by the phosphatases may reside in the ATP-binding site of the 77-kDa protein kinase C. Indeed, within the ATPbinding site, that is present in all protein kinase C members (conserved region C3) and which containsthe consensus sequence required for the ATP binding, there is a potential phosphorylation site (R-K-G-T)for serine/threonine-specific protein kinases (10-13). However, neither of the purified protein kinases testedsuchas caseinkinase 11, catalytic subunit of PKA, S6 kinase, phosphorylase kinase, and the active 77-kDa protein kinase C were capable of phosphorylating the immunocomplexed 74-kDa precursor in vitro. No phosphorylation occurred upon addition of cytosolic or Nonidet P-40-membrane extracts to the immunocomplexed 74kDa protein kinase Cform under a variety of conditions such as presence of PhI or PS/PMA, suggesting that a putative specific protein kinase C kinaseis either notlocalized in these subcellular compartments ormay not efficiently phosphoryl-
Biosynthesis Kinase of Protein
13908
C
kinase C form (Fig. 9). The 77-kDa enzyme is then further phosphorylated to the 80-kDa proteinkinase C by a variety of serinelthreonine-specific protein kinases suchas S6 kinase, casein kinase 11, and/or protein kinase C (Fig. 9). Based on these observations it is reasonable to define the 74-kDa protein kinase C species which accumulates in membranes of 80 kD 74 kD 77 kD cells pretreated with PMA as the non-phosphorylated precurPUC PKC PKC active sor form of the or-type protein kinase C. Thus, PMA interferes inactive active FIG. 9. Hypothetical model showing posttranslational phos- with phosphorylationor enhances dephosphorylation steps in phorylations of protein kinase C. Protein kinase C is initially the processing of the 74-kDa precursor to the77- and 80-kDa synthesized in an inactive unphosphorylated 74-kDa form and is then protein kinase C forms. The differential degree of influence converted into the 77- and 80-kDa proteinkinase forms by posttrans- of PMA on this posttranslational modification of protein lational modification that involves a t least two phosphorylations. The kinase C may account for the different responses of human first phosphorylation(s) occurs by an unidentified protein kinase C kinase that converts the 74-kDa precursor into the active Ca2+-and breast cancer cells toward growth inhibition by tumor prophospholipid-dependent 77-kDa enzyme. This phosphorylation in- moting phorbol esters (24). SBK CSKll
volves protected phosphorylation siteswhich can only be dephosphorylated by PPlc, PP2Ac, or potato acid phosphatase (PAP) in the presence of PS/PMA. The functional 77-kDa protein kinase C is presumably further regulated by autologous or heterologous phosphorylations involving proteinkinase C, S6 kinase (S6K), casein kinase I1 (CSK II), and PKA. The second phosphorylation(s) probably modulates the activity state of the enzyme (higher affinity for cofactors and substrates) and can be rapidly reversed by cytosolic phosphatases.
Acknowledgments-We thank Dr. P. Parker for the full length bovine brain aPKC cDNA (XbPKC306),Dr. P. Cohen for the purified catalytic subunitsof protein phosphatase 1and 2A, Dr. B. Hemmings for the purified catalytic subunit of CAMP-dependent protein kinase, Dr. S. Ferrari for the purified casein kinase 11, Dr. G. Thomas for the purified S6 kinase, and Dr. T. Meyer for the staurosporine. REFERENCES
1. Nishizuka, Y. (1984) Nature 308,693-698 2. Nishizuka, Y. (1986) Science 233, 305-312 ate the protein kinase C precursor under these conditions.' 3. Berridge, M. J., and Imine, R. F. (1984) Nature 312, 315-321 Moreover, the 74-kDa protein kinaseC form did not undergo 4. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., autophosphorylation and did not accumulate in MDA-MBand Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851 231 cells if protein kinases were inhibited by staurosporine or 5. Anderson, W. B., Estival, A., Tapiovaara, K., and Gopalakrishna, W7. Staurosporine inhibits unspecifically a variety of protein R. (1985) Adu. Cyclic Nucleotide Res. 19, 287-306 kinases including tyrosine-specific protein kinases in a na6. Melloni, E., Pontremoli, S,. Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. nomolar range by competing withATP on their catalytic sites Sci. U. S. A. 82, 6435-6439 (44). These data suggest that the phosphorylation of the 747. Kishimoto, A., Kajikawa, N., Shiota, M., and Nishizuka, Y. (1983) kDa protein kinaseC may occur via a protein kinaseC kinase J . Biol. Chem. 258,1156-1164 that appears to be associated with cellular compartments not 8. Tapley, P. M., and Murray, A. W. (1985) Eur. J . Biochem. 1 5 1 , accessible to theseprotein kinase inhibitors. 419-423 9. Fabbro, D., Regazzi, R., Costa, S. D., Borner, C., and EppenberBy contrast, theimmunocomplexed 77-kDa protein kinase ger, U. (1986) Biochem. Biophys. Res. Commun. 135,65-73 C could be in vitro phosphorylated to an80-kDa species either by purified protein kinase C, S6 kinase, or casein kinase I1 10. Parker, P. J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, S., Stabel, S., Waterfield, M. D., and Ullrich, A. (1986) Science although these phosphorylations were not dependent on PS/ 233,853-859 PMA. Autologous or heterologous phosphorylations are 11. Coussens, L., Parker, P. J., Rhee, L., Yang-Feng, T. L., Chen, E., known to positively or negatively regulate kinase activities Waterfield, M. D., Francke, U., and Ullrich, A. (1986) Science 233,839-866 (52-57). Autologous phosphorylation of protein kinase C (au12. Nishizuka, Y. (1988) Nature 334, 661-665 tophosphorylation) is known to increase its affinity for calY., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and cium, phorbol esters, and protein substrates (17, 58). In con- 13. Ono, Nishizuka, Y., (1988) J. Biol. Chem. 263,6927-6932 trast, the effects of heterologous phosphorylation on protein 14. Fry, M. J., Gebhardt, A., Parker, P. J., and Foulkes, J. G. (1985) kinase C activity and phorbol ester binding have not been EMBO J . 4,3173-3178 studied. It is conceivable, however, that the various protein 15. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S., and Nishizuka, Y. (1982) J. Biol. C k m . 257, 13341-13348 kinases tested phosphorylate the 77-kDa protein kinase C a t different sites. For example, casein kinase I1 is unique in its 16. Parker, P. J., Mitchell, F., Stabel, S., Marais, R., Ullrich, A., and Goris, J. (1987) Adu. Prot. Phosphatases 4,363-373 requirement foracidic domains carboxyl-terminalto thephos- 17. Huang, K.-P., Chan, K.-F. J., Singh, T. J., Nakabayashi, H., and phorylation sites (28). Furthermore, the phosphorylation of Huang, F. L. (1986) J. Biol. Chem. 261, 12134-12140 protein kinase C by the catalytic subunit of PKA does not 18. Woodgett, J. R., and Hunter,T. (1987) J. Biol. Chem. 262,4836affect the M, of the 77-kDa protein kinase C. Thus, only the 4843 comparison of phosphopeptides of i n vivo labeled protein 19. Woodgett, J. R., and Hunter, T. (1987) Mol. Cell Biol. 7,85-96 kinase C with those obtained by phosphorylation of purified 20. Young, S., Parker, P. J., Ullrich, A., andStabel, S. (1987) Biochem. J . 2 4 4 , 775-779 protein kinase C in vitro by various protein kinases in con21. Ohno, S., Akita, Y., Konno, Y., Shinobu, I., and Suzuki, K. (1988) junction with the characterization of its catalytic state will Cell 53, 731-741 enable usto determine the exact phosphorylationsites within 22. Roos, W., Fabbro, D., Kung, W., Costa, S. D., and Eppenberger, the protein kinase C molecule as well as the effects of heterU. (1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 991-995 ologous and autologous phosphorylations on theactivity state 23. Borner, C., Wyss, R., Regazzi, R., Eppenberger, U,. and Fabbro, D. (1987) Int. J . Cancer 40, 344-348 of the enzyme. Taken together the present data demonstrate that protein 24. Borner, C., Eppenberger, U., Wyss, R., and Fabbro, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,2110-2114 kinase C is initiallysynthesized as anon-phosphorylated, 25. Regazzi, R., Fabbro, D., Costa, S. D., Borner, C., and Eppenberinactive 74-kDa precursor. The first phosphorylation(s) ocger, U. (1986) Znt. J. Cancer 37, 731-737 curs by a so far unidentified protein kinase that phosphoryl- 26. Cohen, P., Alemany, S., Hemmings, B. A., Resink, T. J., Stralfors, P., and Tung, H.Y. L. (1988) Methods Enzymol. 159,391-408 ates the 74-kDa precursor into the active 77-kDa protein
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44. Meyer, T., Regenass, U., Fabbro, D., Alteris, E., Rosel, J., Muller, M., Caravatti, G., and Matter, A. (1989) Int. J. Cancer 43, 851-856 45. Hoshijima, M., Kikuchi, A., Tanimoto, T., Kaibuchi, K., and Takai, Y . (1986) Cancer Res. 46, 3000-3004 46. Huang, K.-P., and Huang, F. L. (1986) Biochem. Biophys. Res. Commun. 139,320-326 47. Lee, M.-H., and Bell, R. M. (1986) J . Biol. Chem. 2 6 1 , 1486714870 48. Makowske, M., Birnbaum, M. J., Ballester, R., and Rosen, 0. M. (1986) J . Bid. Chem. 2 6 1 , 13389-13392 49. Knopf, J. L., Lee, M.-H., Sultzman, L. A., Kriz, R. W., Loomis, C. R., Hewick, R. M., and Bell, R. M. (1986) Cell 46, 491-502 50. Ono, Y., Kikkawa, U., Ogita, K., Fujii, T., Kurokawa, T., Asaoka, Y., Sekiguchi, K., Ase, K., Igarashi, K., and Nishizuka, Y. (1987) Science 2 3 6 , 1116-1120 51. House, C., and Kemp, B. E. (1987) Science 238, 1726-1728 52. Cohen, P. (1982) Nature 296,613-620 53. Hunter, T. (1987) Cell 50,823-829 54. Friedman, B., Frackelton, A. R., Ross, A. H., Connors, J. M., Fujiki, H., Sugimura, T., andRosner, M. R. (1984) Proc. Natl. Acad. Sci. U. S. A . 81,3034-3038 55. Jove, R., Kornbluth, S., and Hanafusa, H. (1987) Cell 50, 937943 56. Walsh, D. A., Perkins, J. P., and Krebs, E. G . (1968) J. Biol. Chem. 243,3763-3765 57. Rosen, 0. M., Herrera, R., Olowe, Y., Petruzzelli, L. M., and Cobb, M. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,3237-3240 58. Mochly-Rosen, D., and Koshland,D. E., Jr. (1987) J. Biol. Chem. 262, 2291-2297
