Roles of Specific Isoforms of Protein Kinase C in the Transcriptional Control of Cyclin D1 and Related Genes
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m302016200
ISSN1083-351X
AutoresJae‐Won Soh, I. Bernard Weinstein,
Tópico(s)Cell death mechanisms and regulation
ResumoAlthough protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (ΔNPS and CAT), and dominant negative mutants of PKCs α, β1, β2, γ, δ, ϵ, η, ζ, and ι. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-α and PKC-ϵ were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-δ inhibited its activity. Transient transfection studies with a series of 5′-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-α. Deletion of the AP-1 enhancer element located at position –954 upstream from the initiation site abolished PKC-α-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-α and PKC-ϵ. The constitutively active mutants of PKC-α and PKC-ϵ also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-α or PKC-ϵ displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-α or PKC-ϵ in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation. Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (ΔNPS and CAT), and dominant negative mutants of PKCs α, β1, β2, γ, δ, ϵ, η, ζ, and ι. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-α and PKC-ϵ were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-δ inhibited its activity. Transient transfection studies with a series of 5′-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-α. Deletion of the AP-1 enhancer element located at position –954 upstream from the initiation site abolished PKC-α-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-α and PKC-ϵ. The constitutively active mutants of PKC-α and PKC-ϵ also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-α or PKC-ϵ displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-α or PKC-ϵ in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation. Protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; JNK, c-Jun N-terminal kinase; SRF; serum response factor; SRE; serum response element; TCF, ternary complex factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; CAT, catalytic domains; DAG, diacylglycerol; WT, wild type; DN, dominant negative; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; IP, immunoprecipitation; GST, glutathione S-transferase; MARCKS, myristoylated alanine-rich C kinase substrate; ΔNPS, N-terminal pseudosubstrate deleted mutant.1The abbreviations used are: PKC, protein kinase C; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; JNK, c-Jun N-terminal kinase; SRF; serum response factor; SRE; serum response element; TCF, ternary complex factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; CAT, catalytic domains; DAG, diacylglycerol; WT, wild type; DN, dominant negative; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; IP, immunoprecipitation; GST, glutathione S-transferase; MARCKS, myristoylated alanine-rich C kinase substrate; ΔNPS, N-terminal pseudosubstrate deleted mutant. is a multigene family that encodes at least 11 distinct isoforms of lipid-regulated serine/threonine kinases (1Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar, 2Basu A. Pharmacol. Ther. 1993; 59: 257-280Crossref PubMed Scopus (206) Google Scholar). Specific isoforms play pivotal roles in several signal transduction pathways that regulate cellular growth, transformation, and differentiation (3Blobe G.C. Obeid L.M. Hannun Y.A. Cancer Metastasis Rev. 1994; 13: 411-431Crossref PubMed Scopus (259) Google Scholar, 4Grunicke H.H. Uberall F. Semin. Cancer Biol. 1992; 3: 351-360PubMed Google Scholar). The isoforms are classified into three groups, based on their structure and cofactor requirement: (i) classic PKCs (α, βI, βII, and γ), which are activated by diacylglycerol (DAG) or calcium, (ii) novel PKCs (δ, ϵ, η, θ, and μ), which are activated by DAG but not by calcium, and (iii) atypical PKCs (ζ and ι), which are not responsive to either DAG or calcium. Each of these isoforms contains an N-terminal regulatory domain and a C-terminal catalytic kinase domain. The regulatory domains contain a pseudosubstrate domain, an autoinhibitory domain with substrate-like sequences that maintain the enzyme in an inactive state presumably by interacting with the substrate binding site in the catalytic domain. PKC activators like DAG, phorbol esters, and calcium are thought to relieve this intramolecular inhibition, resulting in a conformational change that liberates the substrate binding domain from the pseudosubstrate domain, thereby activating the enzyme. In previous studies we obtained evidence that in NIH3T3 fibroblasts PKC-α and PKC-ϵ can enhance the activities of at least three signaling pathways that converge on the serum response element (SRE): c-Raf-MEK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF (5Soh J.W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (246) Google Scholar). The SRE is a transcriptional control element that plays an important role in the transcription of c-fos and other genes involved in cell proliferation. These findings suggest that specific isoforms of PKC integrate complex networks of signal transduction pathways that control gene expression. Cyclin D1 plays a critical role in the progression of mammalian cells through the G1 phase of the cell cycle. Amplification and/or overexpression of the cyclin D1 gene is often seen in several types of human cancer (6Jiang W. Zhang Y.J. Kahn S.M. Hollstein M.C. Santella R.M. Lu S.H. Harris C.C. Montesano R. Weinstein I.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9026-9030Crossref PubMed Scopus (379) Google Scholar). The cyclin D1 promoter is one of the major targets for several growth stimulatory signaling pathways (7Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar, 8Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1891) Google Scholar). Therefore, in the present study we examined the possible roles of specific isoforms of PKC in the transcriptional control of cyclin D1, using serum-starved NIH3T3 mouse fibroblasts as a model system. We present evidence that, among the nine isoforms of PKC we examined, constitutively active mutants of PKC-α and PKC-ϵ were the most potent activators of the cyclin D1 promoter. We found that the AP-1 enhancer element in the cyclin D1 promoter is required for activation of the cyclin D1 promoter by PKC-α and PKC-ϵ, because activation of the cyclin D1 promoter by PKC-α or PKC-ϵ was abolished by either deletion of the AP-1 site or expression of a dominant negative c-Jun. Constitutively active mutants of PKC-α and PKC-ϵ also activated the promoters for c-fos, c-jun, and cyclin E and when stably expressed in NIH3T3 cells stimulated cell growth. Thus, these findings provide evidence that in murine fibroblasts PKC-α and PKC-ϵ play important roles in enhancing cell cycle progression and cell proliferation. Plasmid Construction—The expression vector pHACE (5Soh J.W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (246) Google Scholar) was used to generate plasmids that encode WT or PKC mutants with a C-terminal HA tag (see Fig. 1A and Table I). pHACE-PKC-WT expression plasmids were generated by ligating full-length open reading frames of different PKC isoforms into pHACE digested with EcoRI. pHACE-PKC-DN expression plasmids were generated by ligating full-length open reading frames of PKC isoforms with a dominant negative (DN) (K→ R or K→ M) point mutation at the ATP binding site into pHACE digested with EcoRI. pHACE-PKC-ΔNPS expression plasmids were generated by ligating cDNA fragments encoding pseudosubstrate deletion (ΔNPS) mutants of PKC isoforms into pHACE digested with EcoRI. pHACE-PKC-CAT expression plasmids were generated by ligating cDNA fragments encoding only the catalytic domains (CAT) of PKC isoforms into pHACE digested with EcoRI. All of the cDNA fragments of these PKC mutants were generated by PCR and were analyzed to confirm their sequences, using an automated DNA sequencer (Applied Biosystems). The expression vectors encoding the WT and mutant forms of PKC-α, δ, ϵ, or ζ have been described previously (5Soh J.W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (246) Google Scholar). The cDNA for rat PKC-β1 was described previously (9Housey G.M. Johnson M.D. Hsiao W.L. O'Brian C.A. Murphy J.P. Kirschmeier P. Weinstein I.B. Cell. 1988; 52: 343-354Abstract Full Text PDF PubMed Scopus (421) Google Scholar). The cDNA for mouse PKC-β2 was a gift from Dr. C. L. Ashendel (10Tang Y.M. Ashendel C.L. Nucleic Acids Res. 1990; 18: 5310Crossref PubMed Scopus (14) Google Scholar). The cDNA for mouse PKC-γ was a gift from Dr. R. M. Bell (11Knopf J.L. Lee M.H. Sultzman L.A. Kriz R.W. Loomis C.R. Hewick R.M. Bell R.M. Cell. 1986; 46: 491-502Abstract Full Text PDF PubMed Scopus (454) Google Scholar). The cDNA for mouse PKC-η was a gift from Dr. S. Ohno (12Osada S. Mizuno K. Saido T.C. Akita Y. Suzuki K. Kuroki T. Ohno S. J. Biol. Chem. 1990; 265: 22434-22440Abstract Full Text PDF PubMed Google Scholar). The cDNA for human PKC-ι was a gift from Dr. T. Biden (13Selbie L.A. Schmitz-Peiffer C. Sheng Y. Biden T.J. J. Biol. Chem. 1993; 268: 24296-24302Abstract Full Text PDF PubMed Google Scholar). The cyclin D1 promoter-luciferase plasmids and pRSV-c-Jun-N138 were gifts from Dr. R. G. Pestell (7Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar), and the c-fos promoter-luciferase and c-jun promoter-luciferase plasmids were gifts from Dr. R. Prywes (14Han T.H. Lamph W.W. Prywes R. Mol. Cell. Biol. 1992; 12: 4472-4477Crossref PubMed Scopus (90) Google Scholar). The cyclin E promoter-luciferase plasmid was a gift from Dr. R. A. Weinberg (15Geng Y. Eaton E.N. Picon M. Roberts J.M. Lundberg A.S. Gifford A. Sardet C. Weinberg R.A. Oncogene. 1996; 12: 1173-1180PubMed Google Scholar). The pJH-v-src plasmid was a gift from Dr. J. T. Parsons (16Reynolds A.B. Roesel D.J. Kanner S.B. Parsons J.T. Mol. Cell. Biol. 1989; 9: 629-638Crossref PubMed Scopus (286) Google Scholar).Table ICoding sequences of the PKC mutantsWTDNΔNPSCATPKC-α2-6722-672 (K368R)30-672326-672PKC-β12-6712-671 (K371R)30-671329-671PKC-β22-6732-673 (K371R)30-673329-673PKC-γ2-6972-697 (K380R)29-697338-697PKC-δ2-6742-674 (K376R)152-674334-674PKC-ϵ2-7372-737 (K437R)164-737395-737PKC-η2-6832-683 (K384R)166-683342-683PKC-ζ2-5922-592(K281M)124-592239-592PKC-ι2-5872-587 (K273M)125-587232-587 Open table in a new tab Cell Cultures, Transfection, and Reporter Assays—NIH3T3 mouse fibroblasts were grown in Dulbecco's minimal essential medium (DMEM) containing 10% calf serum. For reporter assays, triplicate samples of 1 × 105 cells in 35-mm plates were transfected using Lipofectin (Invitrogen) with 1 μg of the reporter plasmid, 0.05–5 μg of various expression vectors, and 1 μg of the control plasmid pCMV-β-gal. The pcDNA3 plasmid DNA was added to the transfections to achieve the same total amount of plasmid DNA per transfection. Twenty-four hours after transfection, cell extracts were prepared and luciferase assays were done using the Luciferase Assay System (Promega). Luciferase activities were normalized with respect to parallel β-galactosidase activities, to correct for differences in transfection efficiency. β-Galactosidase assays were performed using the β-Galactosidase Enzyme Assay System (Promega). Western Blot Analysis—NIH3T3 cells were grown in DMEM containing 10% calf serum, and COS-7 cells were grown in DMEM containing 10% fetal bovine serum. With both cell types, 2 × 105 cells in 60-mm plates were transfected using Lipofectin (Invitrogen) with 5 μg of the indicated expression vectors or the control vector pcDNA3. Six hours after transfection, the cells were fed with DMEM containing 10% fetal bovine serum and incubated overnight. The cells were then trypsinized and transferred to 10-cm plates and grown for 24 h before protein extraction. Cellular proteins were extracted by cell lysis in radioimmune precipitation assay buffer (50 mm Tris HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mm EDTA, 2 mm EGTA, 1 mm dithiothreitol) that contained protease inhibitors (10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.1 mm phenylmethylsulfonyl fluoride) and phosphatase inhibitors (1 mm NaF, 0.1 mm Na3VO4, 10 mm β-glycerophosphate). 50 μg of total cell extract protein was subjected to SDS-PAGE. Proteins were then transferred to an Immobilon-P (Millipore) membrane at 60 V for 3 h at 4 °C. The membranes were subsequently blocked with 5% dry milk in TBS-T (20 mm Tris-HCl, pH 7.6, 137 mm NaCl, 0.05% Tween 20) and then probed with the indicated antibody. The immunoblots were visualized with the Enhanced Chemiluminescence (ECL) Western blotting system (Amersham Biosciences). The anti-HA antibody (Covance), anti-cyclin D1 antibody (BD Pharmingen), and anti-cyclin E antibody (BD Pharmingen) were used at a 1:1000 dilution. PKC Kinase Assay—COS-7 cells were transfected with the indicated expression vectors or the control vector pcDNA3, as described earlier (5Soh J.W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (246) Google Scholar), and cellular proteins were extracted by cell lysis in PKC extraction buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 0.1% Tween 20, 1 mm EDTA, 2.5 mm EGTA, 10% glycerol) that contained protease inhibitors (10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.1 mm phenylmethylsulfonyl fluoride) and phosphatase inhibitors (1 mm NaF, 0.1 mm Na3VO4, 10 mm β-glycerophosphate). HA-tagged PKC proteins were immunoprecipitated from 500 μg of cell extract protein using 3 μg of the anti-HA antibody and 30 μl of protein G-Sepharose, after a 3-h incubation at 4 °C. The immunoprecipitates were washed twice with PKC extraction buffer and then twice with IP kinase buffer (50 mm HEPES, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 2.5 mm EGTA, 1 mm NaF, 0.1 mm Na3VO4, 10 mm β-glycerophosphate) and resuspended in 20 μl of IP kinase buffer. The kinase assay was initiated by adding 40 μl of IP kinase buffer containing 10 μg of a GST-MARCKS substrate and 5 μCi of [γ-32P]ATP. The reactions were performed at 30 °C for 30 min. The reactions were terminated by adding SDS sample buffer and boiled for 5 min. The reaction products were then analyzed by SDS-PAGE and autoradiography. Recombinant GST-MARCKS proteins were expressed in Escherichia coli strain BL21(DE3)/LysS and purified to homogeneity using glutathione S-Sepharose beads (Amersham Biosciences). The experiments were repeated three times and gave similar results. Generation and Characterization of NIH3T3 Cell Lines That Stably Express the ΔNPS Mutants of PKC-α and PKC-ϵ—NIH3T3 cells were transfected with the control vector pcDNA3, pHACE-PKC-α-ΔNPS, or pHACE-PKC-ϵ-ΔNPS, using Lipofectin (Invitrogen). Twenty-four hours after transfection, the cells were transferred to DMEM containing 10% calf serum and neomycin (600 μg/ml, Invitrogen) to select for cells that stably expressed the transfected plasmids. Neomycin-resistant clones were pooled and passaged in DMEM containing 10% calf serum and neomycin (200 μg/ml). For growth curve analysis, cells were plated in triplicate at a density of 2 × 104 cells per well in 6-well (35 mm) plates with 2 ml of DMEM medium containing 10% calf serum. The cells were refed with fresh medium every 3 days. The number of cells per well was counted using a Coulter counter, every day for the subsequent 7 days. Generation of Constitutively Active and Dominant Negative Mutants of Specific Isoforms of PKC—The presence of multiple PKC isoforms in mammalian cells and the paucity of low molecular weight isoform-specific inhibitors of PKC, or a comprehensive series of isoform-specific PKC mutants, have made it difficult to determine the specific roles of individual isoforms in cell cycle progression and cell proliferation. Therefore, as described under “Experimental Procedures” (Fig. 1A and Table I), we developed a series of expression vectors that encode HA-tagged wild type (WT), dominant negative (DN), constitutively active pseudosubstrate deleted (ΔNPS) and constitutively active catalytic domain fragments (CAT) of PKCs α, β1, β2, γ, δ, ϵ, η, ζ, and ι. Some of the mutants of PKCs α, δ, ϵ, and ζ were developed and described in one of our previous publications (5Soh J.W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (246) Google Scholar). This series of expression vectors was transfected into COS-7 cells to characterize the proteins encoded by each of these constructs. Western blot analysis (Fig. 1B) indicated that all but one of these constructs (PKC-δ-ΔNPS) expressed significant amounts of the related HA-tagged proteins and that all of the major bands for these proteins were of the expected sizes. PKC-δ-ΔNPS was expressed at a much lower level than the other proteins (Fig. 1B), perhaps due to its instability or toxicity (as discussed below). It was also of interest to examine the in vitro kinase activities of these proteins. Therefore, immunoprecipitates of the same COS-7 cell extracts were prepared using an anti-HA antibody, and these immunoprecipitates were added to an in vitro kinase assay that contained a GST-MARCKS-(96–184) fusion protein (5Soh J.W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (246) Google Scholar) as the substrate. Western blot analysis showed that equal amounts of HA-tagged PKC proteins were immunoprecipitated (data not shown). We found that all of the PKC-WT, PKC-ΔNPS, and PKC-CAT constructs but none of the PKC-DN constructs displayed kinase activities with this substrate (Fig. 1C). Most of the ΔNPS mutants had slightly higher kinase activities than the corresponding WT proteins, and the CAT mutants had much higher kinase activities than the corresponding WT or ΔNPS proteins, except for PKC-ι-ΔNPS, which had higher kinase activity than PKC-ι-CAT. The fact that for most of the isoforms the CAT mutants had higher activity than the ΔNPS mutants suggests that the N-terminal regulatory domains of PKCs may contain kinase inhibitory sequences in addition to the pseudosubstrate domain. We found that the PKC-δ-ΔNPS mutant had a relatively high level of kinase activity (Fig. 1C) even though this protein was only expressed at a low level (Fig. 1B). Activation of Cyclin D1 Promoter by Specific Isoforms of PKC—The ability of specific isoforms of PKC to activate the cyclin D1 promoter, in the absence of exogenous growth factors, was studied by using transient transfection reporter assays. NIH3T3 mouse fibroblasts were transfected with the control plasmid, PKC-WT, PKC-ΔNPS, or PKC-CAT constructs together with the –1745CD1-luciferase reporter plasmid, which contains the full-length cyclin D1 promoter. The cells were then serum-starved for 24 h and assayed for luciferase activity (Fig. 2A). Among the nine PKC-WT constructs tested, only PKC-ϵ-WT was able to cause a statistically significant activation of the cyclin D1 promoter (about 3-fold). However, when we transfected the PKC-ΔNPS constructs, which lack the pseudosubstrate sequences, the PKC-ΔNPS mutants of PKC-α and -ϵ caused significant activation of the cyclin D1 promoter (6- to 7-fold) and the PKC-ΔNPS mutants of PKCs β1, β2, γ, η, ζ, and ι caused moderate activation of the cyclin D1 promoter (2- to 4-fold). When we transfected the constitutively active PKC-CAT constructs, the CAT mutants of PKCs α, β1, β2, γ, ϵ, η, ζ, and ι caused significant activation of the cyclin D1 promoter (3-to 12-fold). Of these constructs, PKC-α-CAT and PKC-ϵ-CAT were the most potent, because they caused about a 12-fold activation of the cyclin D1 promoter. In contrast to the stimulation seen with other CAT mutants, the PKC-δ-CAT mutant inhibited cyclin D1 promoter activity (by about 40%), which is consistent with evidence that PKC-δ can inhibit cell growth and induce apoptosis (17Mishima K. Ohno S. Shitara N. Yamaoka K. Suzuki K. Biochem. Biophys. Res. Commun. 1994; 201: 363-372Crossref PubMed Scopus (35) Google Scholar, 18Li L. Lorenzo P.S. Bogi K. Blumberg P.M. Yuspa S.H. Mol. Cell. Biol. 1999; 19: 8547-8558Crossref PubMed Google Scholar). Down-regulation of endogenous cyclin D1 expression by PKC-δ has been reported in rat fat pad epididymal endothelial cells and rat smooth muscle cells (19Ashton A.W. Watanabe G. Albanese C. Harrington E.O. Ware J.A. Pestell R.G. J. Biol. Chem. 1999; 274: 20805-20811Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 20Page K. Li J. Corbit K.C. Rumilla K.M. Soh J.W. Weinstein I.B. Albanese C. Pestell R.G. Rosner M.R. Hershenson M.B. Am. J. Respir. Cell Mol. Biol. 2002; 27: 204-213Crossref PubMed Scopus (36) Google Scholar, 21Fukumoto S. Nishizawa Y. Hosoi M. Koyama H. Yamakawa K. Ohno S. Morii H. J. Biol. Chem. 1997; 272: 13816-13822Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The v-src expression vector was used as a positive control and showed strong activation of the cyclin D1 promoter, as described previously (22Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines 3rd, G.K. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In general the CAT mutants gave higher activity than the corresponding ΔNPS mutants (Fig. 2A). These results are consistent with our in vitro kinase assays (Fig. 1C) and provide further evidence that there are inhibitory sequences in the N-terminal regions of these isoforms of PKC in addition to the pseudosubstrate region. The fact that the CAT mutants for these nine isoforms of PKC differed considerably in their activities (Fig. 2A) provides evidence that even though they lack the N-terminal regulatory domain they retain specificity. This is especially evident with PKC-ϵ, because both the WT and CAT constructs of this isoform had high activity and with PKC-δ, because both the WT and CAT constructs of this isoform had no or actually an inhibitory effect on cyclin D1 promoter activity (Fig. 2A). Furthermore, the relatively high activities of the CAT mutants of PKC-α and PKC-ϵ are not simply a result of their expression at higher levels than the other CAT mutants (Fig. 1B). We found that the ΔNPS and CAT mutants of PKC-α and PKC-ϵ also activate the cyclin D1 promoter in human epithelial cells, including HeLa, MCF-7, and SW480 cells. 2J.-W. Soh and I. B. Weinstein, unpublished data. Therefore, these findings are not confined to mouse fibroblasts. To confirm the roles of PKC isoforms in the transcriptional control of cyclin D1, we examined the effects of dominant negative mutants (DN) of the same PKC isoforms on serum-induced cyclin D1 promoter activity. NIH3T3 mouse fibroblasts were transfected with either the control plasmid or PKC-DN constructs together with the cyclin D1-luciferase reporter plasmid. The cells were then serum-starved for 24 h, treated with or without 20% serum for 24 h to induce cyclin D1 promoter activity, and assayed for luciferase activity. Fig. 2B shows that activation of the cyclin D1 promoter by serum was strongly inhibited by the DN mutants of PKC-α and PKC-ϵ (by about 80%), and partially by the DN mutants of PKCs β1, β2, γ, η, ζ, or ι (by 10–30%). The DN mutants of PKC-δ did not cause significant inhibition of the activation of the cyclin D1 promoter by serum, which is consistent with our finding that the activated mutants of PKC-δ did not activate the cyclin D1 promoter (Fig. 2A). Taken together, these experiments provide evidence that PKC-α and PKC-ϵ are the two major PKC isoforms among the nine PKC isoforms examined in our studies that activate signal transduction pathways that lead to activation of the cyclin D1 promoter. Involvement of AP-1 in Activation of the Cyclin D1 Promoter by Specific Isoforms of PKC—To map the region of the cyclin D1 promoter required for activation by specific isoforms of PKC, a series of cyclin D1 promoter truncation mutants (Fig. 3A) were transfected in the presence or absence of the constitutively active mutant of PKC-α (PKC-α-ΔNPS). The cyclin D1 promoter mutants, which lack the AP-1, SP1, and CRE transcriptional response elements, were also used to map the response elements required for activation by specific isoforms of PKC. As shown in Fig. 3B, transient transfection studies with a series of 5′-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-α. Deletion of the AP-1 enhancer element located at the –954 position upstream from the initiation site completely abolished PKC-α-dependent activation of cyclin D1 expression. However, deletion of the SP1 or CRE enhancer elements did not have any effect on PKC-α-dependent activation of the cyclin D1 promoter. Similar results were obtained with the constitutively active mutant of PKC-ϵ (PKC-ϵ-ΔNPS) (data not shown). To confirm the important role of the AP-1 site in activation of the cyclin D1 promoter by PKC-α, an increasing amount (0, 0.5, 1, or 2 μg) of a plasmid containing a dominant negative mutant of c-Jun (c-Jun-N138), which lacks the N-terminal transcriptional activation domain (Δ2–138) (7Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar, 23Baichwal V.R. Tjian R. Cell. 1990; 63: 815-825Abstract Full Text PDF PubMed Scopus (142) Google Scholar), was transfected together with the activated mutant of PKC-α (PKC-α-ΔNPS) and the –1745CD1-luciferase reporter plasmid. The dominant negative mutant of c-Jun (c-Jun-N138) inhibited activation of cyclin D1 promoter by PKC-α-ΔNPS in a dose-dependent manner (Fig. 3C). The dominant negative mutant of c-Jun (c-Jun-N138) also inhibited activation of the cyclin D1 promoter by PKC-ϵ-ΔNPS in a dose-dependent manner (data not shown). Taken together with the studies described in Fig. 3B, these results indicate that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-α and PKC-ϵ in these cells. Activation of c-fos, c-jun, and Cyclin E Promoters by Specific Isoforms of PKC—We also examined the effects of these isoforms of PKC on the expression of other genes involved in cell proliferation and cell cycle progression using transient transfection assays with promoter-luciferase constructs of c-fos, c-jun, and cyclin E. c-fos and c-jun are immediate early response genes and are targets of several growth factors and oncogenes. Cyclin E is an important regulator of the G1/S transition of the cell cycle, together with cyclin D1. These studies focused on PKC-α and PKC-ϵ, because, as described above, they were the most potent activators of the cyclin D1 promoter. NIH3T3 cells were transfected with either the c-fos, c-jun, or cyclin E promoter reporter plasmid together with either the control plasmid or PKC-α-WT, PKC-α-ΔNPS, PKC-α-CAT, PKC-α-DN, PKC-ϵ-WT, PKC-ϵ-ΔNPS, PKC-ϵ-CAT, or PKC-ϵ-DN constructs. Again, the v-src expression vector was used as a positive control. Twenty-four hours after growing the transfected cells in serum-free medium, cell extracts were prepared and assayed for luciferase activity. We found that the ΔNPS and CAT mutants of both PKC-α and PKC-ϵ
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