f Vol. 188, No. 3, 1992 November
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIQNS Pages
16, 1992
1084-1089
ACTIVATORS OF PROTEINKINASECINDUCEp34cdc2 HISTONE HlKINASESTIMULATION IN SWISS3T3FIBROBLASTS Noriko Takuwa, Wei Zhou, Mamoru Kumada and Yoh Takuwa
Departments of Physiology and Cardiovascular Biology, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan
Received
October
1,
1992
SUMMARY: Phorbol-12,13-dibutyrate and 1,2-dioctanoylglycerol, activators of protein kinase C (PKC) that stimulate DNA synthesis in serum-deprived Swiss 3T3 fibroblasts, induce histone Hl kinase activity associated with anti-cdc2 immunoprecipitates after a lag period of 15h, a time point close lo Gl/S boundary of the cell cycle in these cells. Downregulation of PKC does not affect the basal cdc2 kinase activity, but potently inhibits both phorbol dibutyrate- and dioctanoylglycerol-induced cdc2 kinase activation. Phorbol dibutyrate induces a dramatic increase in the p34cdc2 protein level as well as the appearance of p35-~36 forms of cdc2 on Western blot. In PKCdownregulated cells, the p34 form of cdc2 remains elevated but p35-p36 forms do not appear upon phorbol dibutyrate stimulation. These results demonstrate that PKC activation leads to cdc2 kinase activation in mitogenically responsive Swiss 3T3 cells, and strongly suggest that both expression of p34cdc2 protein and its posttranslational modification(s) are involved in this process. Western blot analysis of PKC isozymes suggests that either PKCa, PKC6 or PKC& may be involved in p34cdc2 kinase activation and mitogenesis. 0 1992Academic Press,Inc.
Cdc2 protein kinase was first isolated as a key molecule in yeast cell cycle progression involved at both Gl to S and G2 to M phase transitions (1,2). Subsequent studies in higher eukaryotes have identified p34cdc2 as a catalytic subunit of M phase promoting factor (MPF) that play central roles in mitosis (3,4). More recently, implication of p34cdc2 in the initiation of DNA synthesis has also been suggested for higher eukaryotes including mammalian cells (57). Indeed, activation of p34cdc2 histone Hl kinase is not confined to M phase but is also observed in S phase as well in serum-stimulated mammalian cells (8,9). Accumulating evidences indicate that the cell cycle-dependent regulation of ~34~~~~ activity is achieved through both expression and posttranslational modifications. Both mRNA and the protein level of p34cdc2 are reported to increase in late Gl through S phase in serum-stimulated fibroblasts and mitogen-stimulated
lymphocytes
(10,ll).
It is also known that p34cdc2
forms
multisubunit complex with regulatory proteins including cell cycle-specific cyclins, and that both ~34~~~~ and cyclin undergo cell cycle-dependent phosphorylation/dephosphorylation 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
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(10,12-14). It is not known, however, how the signals evoked by serum growth factors or other mitogens lead to expression and activation of ~34~~~ kinase. In the present study, we examined whether activation of protein kinase C (PKC) has any effect on p34cdc2 kinase activity in Swiss 3T3 fibroblasts, a well characterized model system in which activators of PKC induce a synchronous increase in DNA synthesis even in the absence of other growth factor supplement, in a manner dependent on the presence of cellular PKC (15-18). MATERIALS AND METHODS Swiss 3T3 fibroblasts (3T3K) were maintained at subconfluent state in Dulbecco’s modified minimal essential medium (DMEM) supplemented with 10% fetal bovine serum as described (19). Before each experiment cells were made confluent and quiescent by incubating in DMEM containing 0.2% bovine serum albumin (FractionV , Sigma)(DMEM/BSA) for 24h (18). Cells were then incubated in fresh DMEM/BSA in the presence or absence of phorbol12,13-dibutyrate (Sigma) or 1,2dioctanoylglycerol (Sigma) for indicated periods of time. Measurement of p34cdc2 histone Hl kinase activity was performed as described (3,9). In brief, cells were rinsed with Ca2+, Mg2+-free Dulbecco’s phosphate-buffered saline (PBS(-)) and lyzed in the lysis buffer containing 50mM Tris-HCl @H&O), 120mM NaCl, 0.5% NP-40, 100mM NaF, 1mM Na,VO,, O.l%SDS, 2mM EGTA, 8Opg/ml each of leupeptin and aprotinin, and 0.6mM phenylmethylsulfonyl fluoride (PMSF) at 4 “C for 20min. Cell lysate was centrifuged at 10,OOOxgfor Smin at 4°C and the supematant was recovered. Protein concentration was determined, and 75pg protein from each sample was subjected to immunoprecipitation by a rabbit polyclonal antibody (IgG fraction) that was raised against the C terminus peptide of ~34~~~ which is specific for mouse and human ~34~~~ (3,20,21). The immune complex recovered on protein A-Sepharose beads(Sigma) was washed and then the associated histone Hl kinase activity was measured in vitro, in the reaction mixture (40~1) containing 0.4mg/ml of histone Hl (Boehringer-Mannheim), 60pM of ATP, 10mM MgCl2, 1mM dithiothreitol, 50mM Tris-HCl (pH 7.4) and 2pCi of [ y-32P] ATP, at 25 “C for 1Omin. The reaction was terminated by adding 4 x electrophoresis sample buffer (18), and the sample was analyzed on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE), followed by autoradiography using Fuji BAS 2000 Bio-Image Analyzer (Fuji Photo Film Co., Ltd). The radioactivity incorporated into histone Hl was measured (PSL unit). For analysis of cdc2 protein on Western blot, 15Opg of cellular protein was immunoprecipitated with the anti-p34dc2 C terminus peptide antibody as described above and solubilized in 2 x electrophoresis sample buffer. After separation on 12.5% SDS-PAGE, proteins were transferred to Immobilon P membrane (Millipore), and probed with the anti~34~~2 C terminus antibody, followed by alkaline phosphatase-conjugated mouse anti-rabbit IgG antibody (Zymed). For measurement of PKC activity and Western blot analysis of PKC isozymes, cells were washed twice with PBS(-) and lyzed in an ice-cold buffer containing 2OmM Tris/I-ICl (pH7.5) 250mM sucrose, 0.5mM EGTA, 1OmM 2-mercaptoethanol, 1mM PMSF, 6Opg/ml each of leupeptin and aprotinin, and 0.3% Triton X -100. Cell lysates were spun at 100,000 x for 60min at 4 “C and the supernatants were applied to DE-52 columns equilibrated in Buf Ber A (20mM Tris/HCl (pH7.5). 0.5mM EGTA, 0.5mM EDTA, 1mM dithiothreitol and 10% glycerol). PKC was eluted with Buffer A containing 400mM NaCl. Both Caz+dependent and Ca2+-independent PKC activities were measured for control and phorbol dibutyrate-pretreated cells(22), using a synthetic peptide substrate (Amersham). For Western blot analysis of PKC isozymes, trichloroacetic acid-precipitable proteins of DE-52 eluates were solubilized in SDSsample buffer, analyzed on 8% SDS-PAGE, and proteins transferred to Immobilon P membrane. PKC isozymes were detected by employing mouse monoclonal antibodies MC3a, MC2a and MCla (Seikagaku Kogyo) that are specific for PKCa,+ and -y, respectively, and rabbit polyclonal antibodies raised against PKCG, -a, and -5 peptides (BRL). 1085
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RESULTS AND DISCUSSION As shown in Fig.1, addition of phorbol-12,13-dibutyrate
(10e7M) to quiescent Swiss 3T3
cells results in a time- and dose-dependent increase in the histone Hl kinase activity associated with an anti-p34cdc2 immunoprecipitate. The immunoprecipitate by the antibody that was preincubated with the antigen peptide or by pre-immune serum did not show any histone Hl kinase activity. The increase in the ~34~~~ kinase activity is detectable 15h after addition of phorbol dibutyrate, a time point that is close to Gl/S boundary of the cell cycle in these cells (data not shown)(15), and becomes greater with time for up to 21h, when the maximal value of 8.4-fold over the basal control level is observed (Figs.18 and bJ. The ~34~~~ kinase activity observed in phorbol dibutyrate-stimulated cells (6.9-fold) is even greater than that observed in cells stimulated with 10% fetal bovine serum (3.7-fold over the control value) at 24h. The effect of phorbol dibutyrate is dose-dependent with a half maximal and maximal effect obtained at approximately 10m8 and 10m7M, respectively (Figs.la and cJ, which is comparable to the dose-response curve for DNA synthesis (17). It is well established that prolonged treatment of Swiss 3T3 cells with a high concentration of phorbol dibutyrate results in downregulation
of cellular protein kinase C and potently
attenuates mitogenic effects of active phorbol esters (18,22). Indeed, in Swiss 3T3 cells pretreated with 1PM phorbol dibutyrate for 24h, PKC activity of either Ca2+-dependent or Ca2+-independent fraction was reduced to less than 10% of that observed in control cells (data not shown). In these cells the basal level of p34 cdc2 histone Hl kinase activity does not change significantly, however, the phorbol dibutyrate-induced increase in the ~34~~~2 kinase activity is markedly reduced (Fig.5 left). 1,2-Dioctanoylglycerol (90PM), an analogue of natural PKC activator 1,2diacylglycerol,
also induces p34 c&2 kinase activity, although to a
Figure 1. Effect of phorbol-12,13-dibutyrate on p34cdc2 histone Hl kinaseactivity in Swiss 3T3 fibroblasts.(aJCells were incubatedwith either phorbol dibutyrate (PDBu) (10-g- 10e7M) or 10% fetal bovine serum (s) for indicated periods of time (h) and then histone Hl kinase activity associatedwith anti-p34dc2 immunoprecipitateswas measured.m indicates the position of histone Hl. Bars at left.molecular massin KDa. h.,c. Time course @) and doseresponsecurve (Q for PDBu-inducedp34cdc2 histoneHl-kinase activation. Data representthe mean of duplicatedeterminationsfrom a typical experiment. 1086
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CONTROL ) PKC-D
Figure 2. p34cdc2 H’1stone Hl kinase activity induced by either phorbol-12,13-dibutyrate (PDBu) or 1,2-dioctanoylglycerol (Q&$ is inhibited in PKC-downregulated(PKC-D) Swiss 3T3 cells.
lesser extent as compared to phorbol dibutyrate. The effect of dioctanoylglycerol
is also
inhibited in PKC-downregulated cells (Fig.5 rieht). These results indicate that the effects of phorbol dibutyrate and dioctanoylglycerol on p34cdc2 kinase activation are both mediated through PKC activation, just as in the case with DNA synthesis (18). To explore the mechanism underlying the PKCdependent ~34~~~~ kinase activation, we examined whether PKC activation induces ~34~~~ protein expression. As shown in Fig.3, the protein level of p34cdc2 is extremely low in serum-deprived quiescent 3T3 fibroblasts. Phorbol dibutyrate induces a dramatic increase in p34cdc2 protein, and also the appearance of higher molecular weight forms with apparent molecular mass p35-~36. Both p34 and p35-p36 forms appear to be cdc2 protein since all of them disappear in Western blots probed with antibody preincubated with the antigen peptide of p34 cdc2 C terminus sequence. In other types of cells p35-p36 was found to represent hyperphosphorylated
forms of p34cdc2 (23). In
PKC-downregulated cells, the basal content of p34cdc2 protein remains at the elevated level (Fig.3), which only minimally increases in response to phorbol dibutyrate.
In these cells,
phorbol dibutyrate fails to induce the appearance of p35-p36 forms. These results indicate that phorbol dibutyrate induces p34 c&2 kinase activation through p34cdc2 protein expression and, possibly, posttranslational modification(s) PKC-dependent
including phosphorylation of p34cdc2, through
manner. Results in Fig.2 also indicate that p34 form of cdc2 in PKC-
downregulated cells is in its inactive state as a protein kinase. In an attempt to identify the PKC isozyme(s) responsible for p34cdc2 kinase activation, we studied and compared the amount of PKCa,+, control vs. PKC-downregulated
-y,-6,-E
and -5 isoforms on Western blot in
Swiss 3T3 cells (Fig.4). In serum-deprived control Swiss
3T3 cells the existence of PKCa,-6,-E and -c isoforms is observed. Under our experimental conditions specific bands corresponding to PKCg and -y isoforms are not detected. In phorbol dibutyrate-pretreated, PKC activitydownregulated
cells, PKCG and -E have nearly completely
disappeared, and the amount of PKCa has strongly been reduced. In sharp contrast to PKCG, -& and -a isoforms, the area of the major band specifically reactive for anti-PKq antibody (arrowhead in Fig.4, right) barely changes in “PKC-downregulated” cells, although those of the minor bands with relatively higher molecular weights (asterisk) (which also disappear when 1087
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/“i $
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a
YE-* -
3
0
C-,a,
04
“& =
94 -
COMMUNICATIONS
6 P
-
E P
+- UQi-
-.
5 P
-
P
4
Figure 3. Western blot analysis of p34cdc2 in phorbol dibutyrate (PDBu)-stimulated and unstimulated (-) Swiss 3T3 fibroblasts. Note that ~35~36 forms do not appear in PKCdownregulated @KC-D), PDBu-stimulatedcells. Figure 4. Differential effects of phorbol dibutyrate pretreatment on downregulation of PKC isoforms. Confluent Swiss 3T3 fibroblasts were incubated for 24h with either 1uM phorbol dibutyrate @ or vehicle (-) in the absenceof serum. DE-52 column eluates of total cell lysates were analyzed by Western blotting. Immunoblots probed with antibodies against PKCa, PKCG, PKCE and PKC< are shown. Arrowhead indicates the position of respective PKC isoforms. * Seetext for detail.
anti-PKC< antibody is preincubated with the antigen peptide) are markedly reduced. The exact nature of these minor bands reactive for anti-PKC< antibody is not known at present. Whether they represent posttranslationally
modified forms of PKCf or rather the result of antibody
crossreaction with PKCa and/or -Qremains to be seen. Thus, either PKCa (a Ca2+-dependent PKC), or PKCG and/ or PKCE (Caz+-independent PKCs), may be resposible for p34cdc2 kinase activation and mitogenesis in Swiss 3T3 fibroblasts. In addition, the possibility remains to be evaluated that particular form(s) of PKq
contribute(s) to this process.
It has been shown in serum-stimulated human fibroblasts and Swiss 3T3 cells that mRNA and protein levels of p34cdc2 start to increase in late Gl phase, and p34cdc2 becomes phosphorylated just before the onset of S phase (10). The present study demonstrates that PKC activation, which is not considered to be the major signal transduction mechanism stimulated by serum growth factors, induces similar effects as serum with respect to activation of p34cdc2 protein kinase, as well as DNA synthesis (15-18). Thus, it is likely that more than one mitogenic signalling pathways converge at the level of p34cdc2 protein expression and/or activation. With regard to this point, we have observed that stimulation of Swiss 3T3 cells with either phorbol dibutyrate or bombesin and insulin-like growth factor I, the combinations that induce a dramatic synergism on DNA synthcsis(l5,24), results in a synergistic increase in p34cdc2 kinase activation (N.Takuwa et al., unpublished observation). However, we have also observed that the extent of p34cdc2 kinase activation does not always correlates quantitatively with the extent of DNA synthesis : [sH]thymidine incorporation into DNA induced by 10% fetal bovine serum is twice as much as that induced by lo-TM of phorbol dibutyrate, whereas their relative potency with respect to p34cdc2 kinase activation is rather opposite (see above). It would be of interest to test whether activation of other cyclindependent kinases such as cdk2 kinase(25) is also induced by PKC activation in Swiss 3T3 cells. 1088
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It has been recognized that in certain types of cells, including vascular smooth muscle cells, PKC activation during the late Gl phase of the cell cycle rather inhibits growth factor-induced DNA synthesis(26). The molecular mechanism underlying this negative growth regulation by PKC has not been fully understood thus far. Very recently, we have observed in human umbilical vein endothelial cells that PKC activation results in potent inhibition of DNA synthesis and p34cdc2 kinase activation (W.Zhou et al., manuscript in preparation). Thus, PKC activation exerts quite opposite effects on p34cdc2 kinase activation depending on the type of cells, the timing of action during Gl phase and possibly the PKC isozyme involved. Further studies should unveil the molecular basis for PKC-p34cdc2 axis signal transduction in cell growth regulation. ACKNOWLEDGMENTS : This work was supported by grants from the Ministry of Science and Education in Japan and the Tsumura Foundation for Cardiovascular Research. We thank Mrs.M.Sakagami-Imai and Mr.E. Kishimoto for excellent secretarial and technical assistance. REFERENCES 1. Nurse,P. and Thuriaux,P. (1980) Genetics 96,627-637. 2. Nurse,P. and Bissett,Y. (1981) Nature 292558-560. 3. Draetta,G. and Beach,D. (1988) Cell 54,17-26. 4. Labbe,J.C., Picard,A., Peaucellier,G., Cavadore,J.C., Nurse,P. and Doree,M. (1989) Cell 57,253-263. 5. Blow,J.J. and Nurse,P. (1990) Cell 62,855~862. 6. D’Urso,G., Marraccino,R.L., Marshak,D.R. and Roberts,J.M. (1990) Science 250,786791. 7. Dutta,A. and Stillman,B. (1992) EMBO J. 11,2189-2199. 8. Howe,P.H., Draetta,G. and Leof,E.B. (1991) Mol.Cell. Biol. 11,1185-1194. 9. Takuwa,N., Zhou,W., Kumada,M. and Takuwa,Y. (1992) J.Biol.Chem. in press. lO.Lee,M.G., Norbury,C.J., Spurr,N.K. and Nurse,P. (1988) Nature 333,676-679. ll.Furukawa,Y., Piwnica-Worms,H., Emst,T.J., Kanakura,Y. and Griffin,J.D. (1990) Science 250,805~808. 12.Morla,A.O., Draetta,G., Beach,D. and Wang,J.Y.J. (1989) Cell 58,193-203. 13.Pines,J. and Hunter,T. (1989) Cell 58,833-846. llt.Gautier,J. and Maller,J.L. (1991) EMBO J. 10,172-182. lS.Dicker,P. and Rozengurt,E. (1980) Nature 287,607-612. 16.Rozengurt,E., Rodriguez-Pena,A., Coombs,M. and Sinnett-Smith,J. (1984) Proc.Natl.Acad.Sci. U.S.A. 81,5748-5752. 17.Takuwa,N., Takuwa,Y. and Rasmussen,H. (1987) Biochem. J. 243647-653. 18.Takuwa,N., Iwamoto,A., Kumada,M. Yamashita,K. and Takuwa,Y. (1991) J.Biol.Chem. 266,1403-1409. lS).Todaro,G. and Green,H. (1963) J.Cell Biol. 17,299-313. 20.Lee,M.G. and Nurse,P. (1987) Nature 327,31-35. 21.CisekL.J. and Corden,J.L. (1989) Nature 339,676-684. 22.Rodriguez-Pena,A. and Rozengurt,E. (1984) Biochem.Biophys.Res.Commun. 120,10531059. 23.Thomas,N.S.B. (1989) J.Biol.Chem. 264,13697-13700. 24.RozenguQE. and Sinnett-Smith,J. (1983) Proc.Natl. Acad.Sci.U.S.A. 80,2936-2940. 25.Rosenblatt,J., Gu,Y and MorganD.0. (1992) Proc.Nat1.Acad.Sci.U.S.A. 89,2824-2828. 26.Huang.,C.-L. and Ives,H.E. (1987) Nature 329,849-850.
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ACTIVATORS OF PROTEINKINASECINDUCEp34cdc2 HISTONE HlKINASESTIMULATION IN SWISS3T3FIBROBLASTS Noriko Takuwa, Wei Zhou, Mamoru Kumada and Yoh Takuwa
Departments of Physiology and Cardiovascular Biology, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan
Received
October
1,
1992
SUMMARY: Phorbol-12,13-dibutyrate and 1,2-dioctanoylglycerol, activators of protein kinase C (PKC) that stimulate DNA synthesis in serum-deprived Swiss 3T3 fibroblasts, induce histone Hl kinase activity associated with anti-cdc2 immunoprecipitates after a lag period of 15h, a time point close lo Gl/S boundary of the cell cycle in these cells. Downregulation of PKC does not affect the basal cdc2 kinase activity, but potently inhibits both phorbol dibutyrate- and dioctanoylglycerol-induced cdc2 kinase activation. Phorbol dibutyrate induces a dramatic increase in the p34cdc2 protein level as well as the appearance of p35-~36 forms of cdc2 on Western blot. In PKCdownregulated cells, the p34 form of cdc2 remains elevated but p35-p36 forms do not appear upon phorbol dibutyrate stimulation. These results demonstrate that PKC activation leads to cdc2 kinase activation in mitogenically responsive Swiss 3T3 cells, and strongly suggest that both expression of p34cdc2 protein and its posttranslational modification(s) are involved in this process. Western blot analysis of PKC isozymes suggests that either PKCa, PKC6 or PKC& may be involved in p34cdc2 kinase activation and mitogenesis. 0 1992Academic Press,Inc.
Cdc2 protein kinase was first isolated as a key molecule in yeast cell cycle progression involved at both Gl to S and G2 to M phase transitions (1,2). Subsequent studies in higher eukaryotes have identified p34cdc2 as a catalytic subunit of M phase promoting factor (MPF) that play central roles in mitosis (3,4). More recently, implication of p34cdc2 in the initiation of DNA synthesis has also been suggested for higher eukaryotes including mammalian cells (57). Indeed, activation of p34cdc2 histone Hl kinase is not confined to M phase but is also observed in S phase as well in serum-stimulated mammalian cells (8,9). Accumulating evidences indicate that the cell cycle-dependent regulation of ~34~~~~ activity is achieved through both expression and posttranslational modifications. Both mRNA and the protein level of p34cdc2 are reported to increase in late Gl through S phase in serum-stimulated fibroblasts and mitogen-stimulated
lymphocytes
(10,ll).
It is also known that p34cdc2
forms
multisubunit complex with regulatory proteins including cell cycle-specific cyclins, and that both ~34~~~~ and cyclin undergo cell cycle-dependent phosphorylation/dephosphorylation 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Inc. reserved.
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(10,12-14). It is not known, however, how the signals evoked by serum growth factors or other mitogens lead to expression and activation of ~34~~~ kinase. In the present study, we examined whether activation of protein kinase C (PKC) has any effect on p34cdc2 kinase activity in Swiss 3T3 fibroblasts, a well characterized model system in which activators of PKC induce a synchronous increase in DNA synthesis even in the absence of other growth factor supplement, in a manner dependent on the presence of cellular PKC (15-18). MATERIALS AND METHODS Swiss 3T3 fibroblasts (3T3K) were maintained at subconfluent state in Dulbecco’s modified minimal essential medium (DMEM) supplemented with 10% fetal bovine serum as described (19). Before each experiment cells were made confluent and quiescent by incubating in DMEM containing 0.2% bovine serum albumin (FractionV , Sigma)(DMEM/BSA) for 24h (18). Cells were then incubated in fresh DMEM/BSA in the presence or absence of phorbol12,13-dibutyrate (Sigma) or 1,2dioctanoylglycerol (Sigma) for indicated periods of time. Measurement of p34cdc2 histone Hl kinase activity was performed as described (3,9). In brief, cells were rinsed with Ca2+, Mg2+-free Dulbecco’s phosphate-buffered saline (PBS(-)) and lyzed in the lysis buffer containing 50mM Tris-HCl @H&O), 120mM NaCl, 0.5% NP-40, 100mM NaF, 1mM Na,VO,, O.l%SDS, 2mM EGTA, 8Opg/ml each of leupeptin and aprotinin, and 0.6mM phenylmethylsulfonyl fluoride (PMSF) at 4 “C for 20min. Cell lysate was centrifuged at 10,OOOxgfor Smin at 4°C and the supematant was recovered. Protein concentration was determined, and 75pg protein from each sample was subjected to immunoprecipitation by a rabbit polyclonal antibody (IgG fraction) that was raised against the C terminus peptide of ~34~~~ which is specific for mouse and human ~34~~~ (3,20,21). The immune complex recovered on protein A-Sepharose beads(Sigma) was washed and then the associated histone Hl kinase activity was measured in vitro, in the reaction mixture (40~1) containing 0.4mg/ml of histone Hl (Boehringer-Mannheim), 60pM of ATP, 10mM MgCl2, 1mM dithiothreitol, 50mM Tris-HCl (pH 7.4) and 2pCi of [ y-32P] ATP, at 25 “C for 1Omin. The reaction was terminated by adding 4 x electrophoresis sample buffer (18), and the sample was analyzed on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE), followed by autoradiography using Fuji BAS 2000 Bio-Image Analyzer (Fuji Photo Film Co., Ltd). The radioactivity incorporated into histone Hl was measured (PSL unit). For analysis of cdc2 protein on Western blot, 15Opg of cellular protein was immunoprecipitated with the anti-p34dc2 C terminus peptide antibody as described above and solubilized in 2 x electrophoresis sample buffer. After separation on 12.5% SDS-PAGE, proteins were transferred to Immobilon P membrane (Millipore), and probed with the anti~34~~2 C terminus antibody, followed by alkaline phosphatase-conjugated mouse anti-rabbit IgG antibody (Zymed). For measurement of PKC activity and Western blot analysis of PKC isozymes, cells were washed twice with PBS(-) and lyzed in an ice-cold buffer containing 2OmM Tris/I-ICl (pH7.5) 250mM sucrose, 0.5mM EGTA, 1OmM 2-mercaptoethanol, 1mM PMSF, 6Opg/ml each of leupeptin and aprotinin, and 0.3% Triton X -100. Cell lysates were spun at 100,000 x for 60min at 4 “C and the supernatants were applied to DE-52 columns equilibrated in Buf Ber A (20mM Tris/HCl (pH7.5). 0.5mM EGTA, 0.5mM EDTA, 1mM dithiothreitol and 10% glycerol). PKC was eluted with Buffer A containing 400mM NaCl. Both Caz+dependent and Ca2+-independent PKC activities were measured for control and phorbol dibutyrate-pretreated cells(22), using a synthetic peptide substrate (Amersham). For Western blot analysis of PKC isozymes, trichloroacetic acid-precipitable proteins of DE-52 eluates were solubilized in SDSsample buffer, analyzed on 8% SDS-PAGE, and proteins transferred to Immobilon P membrane. PKC isozymes were detected by employing mouse monoclonal antibodies MC3a, MC2a and MCla (Seikagaku Kogyo) that are specific for PKCa,+ and -y, respectively, and rabbit polyclonal antibodies raised against PKCG, -a, and -5 peptides (BRL). 1085
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RESULTS AND DISCUSSION As shown in Fig.1, addition of phorbol-12,13-dibutyrate
(10e7M) to quiescent Swiss 3T3
cells results in a time- and dose-dependent increase in the histone Hl kinase activity associated with an anti-p34cdc2 immunoprecipitate. The immunoprecipitate by the antibody that was preincubated with the antigen peptide or by pre-immune serum did not show any histone Hl kinase activity. The increase in the ~34~~~ kinase activity is detectable 15h after addition of phorbol dibutyrate, a time point that is close to Gl/S boundary of the cell cycle in these cells (data not shown)(15), and becomes greater with time for up to 21h, when the maximal value of 8.4-fold over the basal control level is observed (Figs.18 and bJ. The ~34~~~ kinase activity observed in phorbol dibutyrate-stimulated cells (6.9-fold) is even greater than that observed in cells stimulated with 10% fetal bovine serum (3.7-fold over the control value) at 24h. The effect of phorbol dibutyrate is dose-dependent with a half maximal and maximal effect obtained at approximately 10m8 and 10m7M, respectively (Figs.la and cJ, which is comparable to the dose-response curve for DNA synthesis (17). It is well established that prolonged treatment of Swiss 3T3 cells with a high concentration of phorbol dibutyrate results in downregulation
of cellular protein kinase C and potently
attenuates mitogenic effects of active phorbol esters (18,22). Indeed, in Swiss 3T3 cells pretreated with 1PM phorbol dibutyrate for 24h, PKC activity of either Ca2+-dependent or Ca2+-independent fraction was reduced to less than 10% of that observed in control cells (data not shown). In these cells the basal level of p34 cdc2 histone Hl kinase activity does not change significantly, however, the phorbol dibutyrate-induced increase in the ~34~~~2 kinase activity is markedly reduced (Fig.5 left). 1,2-Dioctanoylglycerol (90PM), an analogue of natural PKC activator 1,2diacylglycerol,
also induces p34 c&2 kinase activity, although to a
Figure 1. Effect of phorbol-12,13-dibutyrate on p34cdc2 histone Hl kinaseactivity in Swiss 3T3 fibroblasts.(aJCells were incubatedwith either phorbol dibutyrate (PDBu) (10-g- 10e7M) or 10% fetal bovine serum (s) for indicated periods of time (h) and then histone Hl kinase activity associatedwith anti-p34dc2 immunoprecipitateswas measured.m indicates the position of histone Hl. Bars at left.molecular massin KDa. h.,c. Time course @) and doseresponsecurve (Q for PDBu-inducedp34cdc2 histoneHl-kinase activation. Data representthe mean of duplicatedeterminationsfrom a typical experiment. 1086
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CONTROL ) PKC-D
Figure 2. p34cdc2 H’1stone Hl kinase activity induced by either phorbol-12,13-dibutyrate (PDBu) or 1,2-dioctanoylglycerol (Q&$ is inhibited in PKC-downregulated(PKC-D) Swiss 3T3 cells.
lesser extent as compared to phorbol dibutyrate. The effect of dioctanoylglycerol
is also
inhibited in PKC-downregulated cells (Fig.5 rieht). These results indicate that the effects of phorbol dibutyrate and dioctanoylglycerol on p34cdc2 kinase activation are both mediated through PKC activation, just as in the case with DNA synthesis (18). To explore the mechanism underlying the PKCdependent ~34~~~~ kinase activation, we examined whether PKC activation induces ~34~~~ protein expression. As shown in Fig.3, the protein level of p34cdc2 is extremely low in serum-deprived quiescent 3T3 fibroblasts. Phorbol dibutyrate induces a dramatic increase in p34cdc2 protein, and also the appearance of higher molecular weight forms with apparent molecular mass p35-~36. Both p34 and p35-p36 forms appear to be cdc2 protein since all of them disappear in Western blots probed with antibody preincubated with the antigen peptide of p34 cdc2 C terminus sequence. In other types of cells p35-p36 was found to represent hyperphosphorylated
forms of p34cdc2 (23). In
PKC-downregulated cells, the basal content of p34cdc2 protein remains at the elevated level (Fig.3), which only minimally increases in response to phorbol dibutyrate.
In these cells,
phorbol dibutyrate fails to induce the appearance of p35-p36 forms. These results indicate that phorbol dibutyrate induces p34 c&2 kinase activation through p34cdc2 protein expression and, possibly, posttranslational modification(s) PKC-dependent
including phosphorylation of p34cdc2, through
manner. Results in Fig.2 also indicate that p34 form of cdc2 in PKC-
downregulated cells is in its inactive state as a protein kinase. In an attempt to identify the PKC isozyme(s) responsible for p34cdc2 kinase activation, we studied and compared the amount of PKCa,+, control vs. PKC-downregulated
-y,-6,-E
and -5 isoforms on Western blot in
Swiss 3T3 cells (Fig.4). In serum-deprived control Swiss
3T3 cells the existence of PKCa,-6,-E and -c isoforms is observed. Under our experimental conditions specific bands corresponding to PKCg and -y isoforms are not detected. In phorbol dibutyrate-pretreated, PKC activitydownregulated
cells, PKCG and -E have nearly completely
disappeared, and the amount of PKCa has strongly been reduced. In sharp contrast to PKCG, -& and -a isoforms, the area of the major band specifically reactive for anti-PKq antibody (arrowhead in Fig.4, right) barely changes in “PKC-downregulated” cells, although those of the minor bands with relatively higher molecular weights (asterisk) (which also disappear when 1087
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Figure 3. Western blot analysis of p34cdc2 in phorbol dibutyrate (PDBu)-stimulated and unstimulated (-) Swiss 3T3 fibroblasts. Note that ~35~36 forms do not appear in PKCdownregulated @KC-D), PDBu-stimulatedcells. Figure 4. Differential effects of phorbol dibutyrate pretreatment on downregulation of PKC isoforms. Confluent Swiss 3T3 fibroblasts were incubated for 24h with either 1uM phorbol dibutyrate @ or vehicle (-) in the absenceof serum. DE-52 column eluates of total cell lysates were analyzed by Western blotting. Immunoblots probed with antibodies against PKCa, PKCG, PKCE and PKC< are shown. Arrowhead indicates the position of respective PKC isoforms. * Seetext for detail.
anti-PKC< antibody is preincubated with the antigen peptide) are markedly reduced. The exact nature of these minor bands reactive for anti-PKC< antibody is not known at present. Whether they represent posttranslationally
modified forms of PKCf or rather the result of antibody
crossreaction with PKCa and/or -Qremains to be seen. Thus, either PKCa (a Ca2+-dependent PKC), or PKCG and/ or PKCE (Caz+-independent PKCs), may be resposible for p34cdc2 kinase activation and mitogenesis in Swiss 3T3 fibroblasts. In addition, the possibility remains to be evaluated that particular form(s) of PKq
contribute(s) to this process.
It has been shown in serum-stimulated human fibroblasts and Swiss 3T3 cells that mRNA and protein levels of p34cdc2 start to increase in late Gl phase, and p34cdc2 becomes phosphorylated just before the onset of S phase (10). The present study demonstrates that PKC activation, which is not considered to be the major signal transduction mechanism stimulated by serum growth factors, induces similar effects as serum with respect to activation of p34cdc2 protein kinase, as well as DNA synthesis (15-18). Thus, it is likely that more than one mitogenic signalling pathways converge at the level of p34cdc2 protein expression and/or activation. With regard to this point, we have observed that stimulation of Swiss 3T3 cells with either phorbol dibutyrate or bombesin and insulin-like growth factor I, the combinations that induce a dramatic synergism on DNA synthcsis(l5,24), results in a synergistic increase in p34cdc2 kinase activation (N.Takuwa et al., unpublished observation). However, we have also observed that the extent of p34cdc2 kinase activation does not always correlates quantitatively with the extent of DNA synthesis : [sH]thymidine incorporation into DNA induced by 10% fetal bovine serum is twice as much as that induced by lo-TM of phorbol dibutyrate, whereas their relative potency with respect to p34cdc2 kinase activation is rather opposite (see above). It would be of interest to test whether activation of other cyclindependent kinases such as cdk2 kinase(25) is also induced by PKC activation in Swiss 3T3 cells. 1088
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It has been recognized that in certain types of cells, including vascular smooth muscle cells, PKC activation during the late Gl phase of the cell cycle rather inhibits growth factor-induced DNA synthesis(26). The molecular mechanism underlying this negative growth regulation by PKC has not been fully understood thus far. Very recently, we have observed in human umbilical vein endothelial cells that PKC activation results in potent inhibition of DNA synthesis and p34cdc2 kinase activation (W.Zhou et al., manuscript in preparation). Thus, PKC activation exerts quite opposite effects on p34cdc2 kinase activation depending on the type of cells, the timing of action during Gl phase and possibly the PKC isozyme involved. Further studies should unveil the molecular basis for PKC-p34cdc2 axis signal transduction in cell growth regulation. ACKNOWLEDGMENTS : This work was supported by grants from the Ministry of Science and Education in Japan and the Tsumura Foundation for Cardiovascular Research. We thank Mrs.M.Sakagami-Imai and Mr.E. Kishimoto for excellent secretarial and technical assistance. REFERENCES 1. Nurse,P. and Thuriaux,P. (1980) Genetics 96,627-637. 2. Nurse,P. and Bissett,Y. (1981) Nature 292558-560. 3. Draetta,G. and Beach,D. (1988) Cell 54,17-26. 4. Labbe,J.C., Picard,A., Peaucellier,G., Cavadore,J.C., Nurse,P. and Doree,M. (1989) Cell 57,253-263. 5. Blow,J.J. and Nurse,P. (1990) Cell 62,855~862. 6. D’Urso,G., Marraccino,R.L., Marshak,D.R. and Roberts,J.M. (1990) Science 250,786791. 7. Dutta,A. and Stillman,B. (1992) EMBO J. 11,2189-2199. 8. Howe,P.H., Draetta,G. and Leof,E.B. (1991) Mol.Cell. Biol. 11,1185-1194. 9. Takuwa,N., Zhou,W., Kumada,M. and Takuwa,Y. (1992) J.Biol.Chem. in press. lO.Lee,M.G., Norbury,C.J., Spurr,N.K. and Nurse,P. (1988) Nature 333,676-679. ll.Furukawa,Y., Piwnica-Worms,H., Emst,T.J., Kanakura,Y. and Griffin,J.D. (1990) Science 250,805~808. 12.Morla,A.O., Draetta,G., Beach,D. and Wang,J.Y.J. (1989) Cell 58,193-203. 13.Pines,J. and Hunter,T. (1989) Cell 58,833-846. llt.Gautier,J. and Maller,J.L. (1991) EMBO J. 10,172-182. lS.Dicker,P. and Rozengurt,E. (1980) Nature 287,607-612. 16.Rozengurt,E., Rodriguez-Pena,A., Coombs,M. and Sinnett-Smith,J. (1984) Proc.Natl.Acad.Sci. U.S.A. 81,5748-5752. 17.Takuwa,N., Takuwa,Y. and Rasmussen,H. (1987) Biochem. J. 243647-653. 18.Takuwa,N., Iwamoto,A., Kumada,M. Yamashita,K. and Takuwa,Y. (1991) J.Biol.Chem. 266,1403-1409. lS).Todaro,G. and Green,H. (1963) J.Cell Biol. 17,299-313. 20.Lee,M.G. and Nurse,P. (1987) Nature 327,31-35. 21.CisekL.J. and Corden,J.L. (1989) Nature 339,676-684. 22.Rodriguez-Pena,A. and Rozengurt,E. (1984) Biochem.Biophys.Res.Commun. 120,10531059. 23.Thomas,N.S.B. (1989) J.Biol.Chem. 264,13697-13700. 24.RozenguQE. and Sinnett-Smith,J. (1983) Proc.Natl. Acad.Sci.U.S.A. 80,2936-2940. 25.Rosenblatt,J., Gu,Y and MorganD.0. (1992) Proc.Nat1.Acad.Sci.U.S.A. 89,2824-2828. 26.Huang.,C.-L. and Ives,H.E. (1987) Nature 329,849-850.
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