Identification of Serine 643 of Protein Kinase C-δ as an Important Autophosphorylation Site for Its Enzymatic Activity
1997; Elsevier BV; Volume: 272; Issue: 39 Linguagem: Inglês
10.1074/jbc.272.39.24550
ISSN1083-351X
AutoresWeiqun Li, Jiachang Zhang, Donald P. Bottaro, Wei Li, Jacalyn H. Pierce,
Tópico(s)Enzyme Structure and Function
ResumoTo investigate the role of serine/threonine autophosphorylation of protein kinase C-δ (PKC-δ), we mutated serine 643 of PKC-δ to an alanine residue (PKC-δS643A). Two different expression vectors containing PKC-δS643A mutant cDNAs were transfected and expressed in 32D myeloid progenitor cells.In vitro autophosphorylation assays demonstrated 65–83% reduction in autophosphorylation of PKC-δS643A in comparison to wild type PKC-δ (PKC-δWT). The enzymatic activity of PKC-δS643A mutant as measured by phosphorylating the PKC-δ pseudosubstrate region-derived substrate was also reduced more than 70% in comparison to that of PKC-δWT. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that at least one phosphopeptide was absent in PKC-δS643A when compared with PKC-δWT, further substantiating that serine 643 is phosphorylated in vivo. Localization and 12-O-tetradecanoylphorbol-13-acetate-dependent translocation and tyrosine phosphorylation of PKC-δS643A were not altered in comparison to PKC-δWT, indicating that mutagenesis did not affect the structural integrity of the mutant protein. 12-O-Tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells overexpressing PKC-δS643A mutant protein was impaired in comparison to that of PKC-δWT transfectant. Taken together, our results demonstrate that serine 643 of PKC-δ is a major autophosphorylation site, and phosphorylation of this site plays an important role in controlling its enzymatic activity and biological function. To investigate the role of serine/threonine autophosphorylation of protein kinase C-δ (PKC-δ), we mutated serine 643 of PKC-δ to an alanine residue (PKC-δS643A). Two different expression vectors containing PKC-δS643A mutant cDNAs were transfected and expressed in 32D myeloid progenitor cells.In vitro autophosphorylation assays demonstrated 65–83% reduction in autophosphorylation of PKC-δS643A in comparison to wild type PKC-δ (PKC-δWT). The enzymatic activity of PKC-δS643A mutant as measured by phosphorylating the PKC-δ pseudosubstrate region-derived substrate was also reduced more than 70% in comparison to that of PKC-δWT. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that at least one phosphopeptide was absent in PKC-δS643A when compared with PKC-δWT, further substantiating that serine 643 is phosphorylated in vivo. Localization and 12-O-tetradecanoylphorbol-13-acetate-dependent translocation and tyrosine phosphorylation of PKC-δS643A were not altered in comparison to PKC-δWT, indicating that mutagenesis did not affect the structural integrity of the mutant protein. 12-O-Tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells overexpressing PKC-δS643A mutant protein was impaired in comparison to that of PKC-δWT transfectant. Taken together, our results demonstrate that serine 643 of PKC-δ is a major autophosphorylation site, and phosphorylation of this site plays an important role in controlling its enzymatic activity and biological function. Protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; WT, wild type; TPA, 12-O-tetradecanoylphorbol-13-acetate; HA, hemagglutinin; PDGF-βR, platelet-derived growth factor β receptor; PAGE, polyacrylamide gel electrophoresis; anti-Tyr(P), anti-phosphotyrosine; mAb, monoclonal antibody.is composed of a family of serine/threonine kinases. To date, 11 different PKC isoenzymes have been identified that are divided into three different subgroups, conventional PKCs (cPKCs), novel PKCs (nPKCs), and atypical PKCs (1Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1469) Google Scholar, 2Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4226) Google Scholar, 3Ohno S. Akita Y. Hata A. Osada S.-I. Kubo K. Konno Y. Akimoto K. Mizuno K. Saido T. Kuroki T. Suzuki K. Adv. Enzyme Regul. 1991; 31: 287-303Crossref PubMed Scopus (110) Google Scholar). PKCs have been defined as important signaling molecules in cell growth, differentiation, secretion of hormones and neurotransmitters, and cellular transformation (2Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4226) Google Scholar). PKC-δ belongs to nPKC subgroup and is ubiquitously expressed in many tissues and cell lines (4Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar). We have focused our efforts on understanding the role of PKC-δ in various signaling transduction pathways. Overexpression of wild type of PKC-δ (PKC-δWT) in 32D myeloid progenitor cells led to monocytic differentiation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment (5Mischak H. Pierce J.H. Goodnight J. Kazanietz M.G. Blumberg P.M. Mushinski J.F. J. Biol. Chem. 1993; 268: 20110-20115Abstract Full Text PDF PubMed Google Scholar), suggesting a causal role for PKC-δ in hematopoietic cell differentiation. An ATP binding mutant of PKC-δ (PKC-δK376R) was generated by site-directed mutagenesis and was demonstrated to lack autophosphorylation capacity in vitro completely (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Moreover, the PKC-δK376R mutant competitively inhibited PKC-δWT phosphorylation of an exogenous substrate in vitro. Recently, our group and several others (7Denning M.F. Dlugosz A.A. Howett M.K. Yuspa S.H. J. Biol. Chem. 1993; 268: 26079-26081Abstract Full Text PDF PubMed Google Scholar, 8Denning M.F. Dlugosz A. Threadgill D.W. Magnuson T. Yuspa S.H. J. Biol. Chem. 1996; 271: 5325-5331Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 9Gschwendt M. Kielbassa K. Kittstein W. Marks F. FEBS Lett. 1994; 347: 85-89Crossref PubMed Scopus (98) Google Scholar, 10Li W. Mischak H. Yu J.-C. Wang L.-M. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar, 11Smith H. Chang E.-Y. Szallasi Z. Blumberg P.M. Rivera J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9112-9116Crossref PubMed Scopus (69) Google Scholar, 12Soltoff S.P. Toker A. J. Biol. Chem. 1995; 270: 13490-13495Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) observed tyrosine phosphorylation of PKC-δ in vivo in response to its activation by various agonists. PKC-δ was also demonstrated to be an important substrate in the platelet-derived growth factor β receptor (PDGF-βR) pathway (13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar). It was phosphorylated by the activated PDGF-βR in vivo and in vitro on tyrosine residue(s) (10Li W. Mischak H. Yu J.-C. Wang L.-M. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar, 13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar). The relevance of PKC-δ in mediating c-sis/PDGF-B transformation of NIH 3T3 cells was recently elucidated (14Li W. Michieli P. Alimandi M. Lorenzi M.V. Wu Y. Wang L.-H. Heidaran M.A. Pierce J.H. Oncogene. 1996; 13: 731-737PubMed Google Scholar). In this study, expression of the PKC-δK376R mutant led to dramatic inhibition of c-sis-induced NIH 3T3 cell transformation. These results demonstrate that PKC-δ plays a physiological role in a signaling pathway leading to malignant transformation of fibroblasts induced by sis oncogene. Serine/threonine phosphorylation of PKC in vivo was first observed approximately 10 years ago (15Borner C. Filipuzzi I. Wartmann M. Eppenberger U. Fabbro D. J. Biol. Chem. 1989; 264: 13902-13909Abstract Full Text PDF PubMed Google Scholar, 16Huang K.-P. Chan K.-F.J. Singh T.J. Nakabayashi H. Huang F.L. J. Biol. Chem. 1986; 261: 12134-12140Abstract Full Text PDF PubMed Google Scholar, 17Mitchell F.E. Marais R.M. Parker P.J. Biochem. J. 1989; 261: 131-136Crossref PubMed Scopus (54) Google Scholar, 18Mochly-Rosen D. Koshland Jr., D.E. J. Biol. Chem. 1987; 262: 2291-2297Abstract Full Text PDF PubMed Google Scholar, 19Molina C.A. Ashendel C.L. Cancer Res. 1991; 51: 4624-4630PubMed Google Scholar). Several in vivo phosphorylation sites have been mapped utilizing different methods (20Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 21Flint A.J. Paladini R.D. Koshland D.E. Science. 1990; 249: 408-411Crossref PubMed Scopus (108) Google Scholar, 22Tsutakawa S. Medzihradszky K.F. Flint A.J. Burlingame A.L. Koshland Jr., D.E. J. Biol. Chem. 1995; 270: 26807-26812Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Based on studies performed on cPKCs (20Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 23Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 24Bornancin F. Parker P.J. J. Biol. Chem. 1997; 272: 3544-3549Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 25Cazaubon S.M. Parker P.J. J. Biol. Chem. 1993; 268: 17559-17563Abstract Full Text PDF PubMed Google Scholar, 26Dutil E.M. Keranen L.M. DePaoli-Roach A.A. Newton A.C. J. Biol. Chem. 1994; 269: 29359-29362Abstract Full Text PDF PubMed Google Scholar, 27Orr J.W. Newton A.C. J. Biol. Chem. 1994; 269: 27715-27718Abstract Full Text PDF PubMed Google Scholar), it is generally believed that PKC is first synthesized as an immature precursor protein that does not show any catalytic activity. Phosphorylation of PKC on the "activation loop," which corresponds to threonines 497 and 500 of PKC-α (23Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and βII (26Dutil E.M. Keranen L.M. DePaoli-Roach A.A. Newton A.C. J. Biol. Chem. 1994; 269: 29359-29362Abstract Full Text PDF PubMed Google Scholar), respectively, by an unidentified PKC kinase then renders PKC catalytic domain competent. However, transphosphorylation of PKC on its activation loop does not alter the mobility of the protein as observed by SDS-polyacrylamide gel electrophoresis (PAGE). Subsequent autophosphorylation on threonine 641 of PKC-βII results in the first upward shift of the mobility of the protein. This event is followed by a second autophosphorylation on serine 660 of PKC-βII which further shifts the protein to the mature 80-kDa form. Generation of diacylglycerol through different mechanisms recruits PKC to the membrane where the pseudosubstrate region-mediated autoinhibition of the catalytic domain is released. The enzyme is then able to phosphorylate substrates and transmit the downstream signals. How the mature enzyme returns to the cytosol after activation remains unclear. This may be regulated by serine/threonine phosphatase activity (1Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1469) Google Scholar). Autophosphorylation of PKC has been observed both in vivoand in vitro (15Borner C. Filipuzzi I. Wartmann M. Eppenberger U. Fabbro D. J. Biol. Chem. 1989; 264: 13902-13909Abstract Full Text PDF PubMed Google Scholar, 16Huang K.-P. Chan K.-F.J. Singh T.J. Nakabayashi H. Huang F.L. J. Biol. Chem. 1986; 261: 12134-12140Abstract Full Text PDF PubMed Google Scholar, 17Mitchell F.E. Marais R.M. Parker P.J. Biochem. J. 1989; 261: 131-136Crossref PubMed Scopus (54) Google Scholar, 18Mochly-Rosen D. Koshland Jr., D.E. J. Biol. Chem. 1987; 262: 2291-2297Abstract Full Text PDF PubMed Google Scholar, 19Molina C.A. Ashendel C.L. Cancer Res. 1991; 51: 4624-4630PubMed Google Scholar). It is thought that autophosphorylation of PKC enhances its binding to phorbol ester and reduces the K m for its substrates in vitro (16Huang K.-P. Chan K.-F.J. Singh T.J. Nakabayashi H. Huang F.L. J. Biol. Chem. 1986; 261: 12134-12140Abstract Full Text PDF PubMed Google Scholar, 18Mochly-Rosen D. Koshland Jr., D.E. J. Biol. Chem. 1987; 262: 2291-2297Abstract Full Text PDF PubMed Google Scholar). Several in vivo autophosphorylation sites for different PKC isoenzymes have been mapped (20Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 21Flint A.J. Paladini R.D. Koshland D.E. Science. 1990; 249: 408-411Crossref PubMed Scopus (108) Google Scholar, 22Tsutakawa S. Medzihradszky K.F. Flint A.J. Burlingame A.L. Koshland Jr., D.E. J. Biol. Chem. 1995; 270: 26807-26812Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Recently, conserved threonine autophosphorylation sites on two cPKCs (PKC-α and PKC-βI) were characterized by site-directed mutagenesis (23Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 28Zhang J. Wang L. Petrin J. Bishop W.R. Bond R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6130-6134Crossref PubMed Scopus (33) Google Scholar, 29Zhang J. Wang L. Schwartz J. Bond R.W. Bishop W.R. J. Biol. Chem. 1994; 269: 19578-19584Abstract Full Text PDF PubMed Google Scholar). Mutation of threonine 638 to alanine in the PKC-α molecule did not dramatically affect its enzymatic activity (23Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In striking contrast, mutation of this conserved site (threonine 642 to alanine) in PKC-βI completely abolished its enzymatic activity and in vivophosphorylation (29Zhang J. Wang L. Schwartz J. Bond R.W. Bishop W.R. J. Biol. Chem. 1994; 269: 19578-19584Abstract Full Text PDF PubMed Google Scholar). Since PKC-δ belongs to the nPKC subfamily and a serine residue rather than a threonine residue exists at this conserved position (see Fig. 1), we have attempted to elucidate whether PKC-δ is phosphorylated on this conserved site and, if so, whether this phosphorylation would influence PKC-δ function. Our results indicate that serine 643 is a major PKC-δ autophosphorylation site, and phosphorylation of this site significantly affects its enzymatic activity. The Bio-Rad Muta-gene Phagemid in vitro mutagenesis kit (version 2) was used for the site-directed mutagenesis. The oligonucleotide 5′-GAATGAGAAACCTCAGCTTGCATTCAG-3′ was used as a mutant primer in the in vitro mutagenesis reaction where the serine residue at amino acid 643 of murine PKC-δ was changed to alanine (underlined in the sequence). The successful mutation of this site generated a new BsmI restriction site that was used to screen all the reaction products. The mutation was confirmed by DNA sequencing. The PKC-δS643A mutant cDNA was subcloned into pCEV-HA (three hemagglutinin epitope repeats, neo selection) and pLTR (two HA epitope repeats, gpt selection) vectors, generating pCEV-δS643A-HA and pLTR-δS643A-HA, respectively. The generation of these two vectors and subcloning of PKC-δWT cDNA into these vectors have been previously described (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The 32D cells were transfected with different cDNA expression vectors using the electroporation procedure described previously (5Mischak H. Pierce J.H. Goodnight J. Kazanietz M.G. Blumberg P.M. Mushinski J.F. J. Biol. Chem. 1993; 268: 20110-20115Abstract Full Text PDF PubMed Google Scholar). 32D cells and transfectants were cultured in RPMI 1640 medium with 10% fetal calf serum and 5% WEHI-3B conditioned medium as a source of murine interleukin-3. These procedures have been described previously (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar,10Li W. Mischak H. Yu J.-C. Wang L.-M. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar, 13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar, 30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Briefly, the 32D transfectants were serum-starved for 2 h and left untreated or stimulated with 100 ng/ml TPA (Sigma) for 10 min. The cell pellets were lysed in Triton X-100 containing lysis buffer (13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar) and clarified by centrifugation. For immunoprecipitation, equal amounts of proteins (1–5 mg per sample) were incubated with polyclonal anti-PKC-δ serum (5 μl per sample, Calbiochem) together with 40 μl of protein G-coupled Sepharose (Pharmacia Biotech, Inc.) or with anti-HA monoclonal antibody (mAb; 4 μg per sample, Boehringer Mannheim) together with 25 μl of protein A-Sepharose beads (Pierce). Anti-phosphotyrosine (anti-Tyr(P), 2 μg/ml, Upstate Biotechnology) and anti-PKC-δ (1:1000) were utilized for immunoblot analysis. The enhanced chemiluminescence system (Amersham Corp.) was used to visualize proteins, and the densities of the bands from SDS-PAGE and autoradiography were quantified by using a densitometer (Molecular Dynamics). The method for the subcellular fractionation has been described before (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar). The in vitro autophosphorylation assay utilizing anti-HA antibody for immunoprecipitation was performed by following a previously described protocol (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Briefly, cell lysates were immunoprecipitated with anti-HA antibody as described above. Washed immunoprecipitates were incubated on ice for 30 min with 50 μl of autophosphorylation buffer that contained 20 mm Tris-HCl, pH 7.5, 5 mmmagnesium acetate, 50 μg/ml phosphatidylserine (Sigma), 100 ng/ml TPA, 10 μg/ml leupeptin, 1 mmNa3VO4, 1 μm ATP (Boehringer Mannheim), and 5 μCi of [γ-32P]ATP (3000 Ci/mmol, Amersham Corp.). The reaction was stopped by washing twice with Triton X-100 containing lysis buffer, and denatured proteins were separated by SDS-PAGE. The dried gel was autoradiographed. Both in vivo labeling and subsequent two-dimensional phosphopeptide analysis have been described previously (30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Briefly, serum-starved 32D transfectants were labeled with [32P]orthophosphate (1 mCi/ml; NEN Life Science Products) for 3 h and were stimulated with TPA (100 ng/ml) for 10 min. Cell lysates were immunoprecipitated with anti-HA mAb, and immunoprecipitates were resolved by SDS-PAGE. Radiolabeled PKC-δWT-HA and PKC-δS643A-HA bands were excised from the gel and exhaustively digested with trypsin (tosylphenylalanyl chloromethyl ketone-treated). The resulting phosphopeptides were resolved by thin layer electrophoresis, pH 8.9, followed by ascending chromatography, pH 1.9. Dried plates were autoradiographed for 1 week. DE52 ion exchange chromatography to enrich PKC from the cell lysates and the subsequent measurement of PKC activity utilizing PKC-δ pseudosubstrate region-derived peptide as a substrate have been described previously (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar, 30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Direct measurement of PKC-δ activity on PKC-δ substrate utilizing anti-HA immunoprecipitates as the kinase sources was also employed. Briefly, the equal amounts of protein (6 mg per sample) from the various PKC-δ transfectants were immunoprecipitated with anti-HA antibody (4 μg per sample). Washed immunoprecipitates were incubated at room temperature with 40 μl of reaction buffer that contained 10 μm PKC-δ substrate derived from PKC-δ pseudosubstrate region (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), 20 mm Tris-HCl, pH 7.5, 1 mm CaCl2, 10 μm magnesium acetate, 1 μm TPA, 50 μg/ml phosphatidylserine (Sigma), 30 μm ATP, and 30 μCi of [γ-32P]ATP for 20 min. The reaction tube was centrifuged, and 20 μl of the supernatant was spotted on phosphocellulase disk sheets (Life Technologies, Inc.). The sheets were washed twice with 1% phosphoric acid and twice with distilled water, and samples were analyzed by liquid scintillation. The nonspecific catalytic activity was measured in the same reaction buffer except that TPA and phosphatidylserine were omitted from the reaction. The specific PKC-δ activity was calculated by subtracting the nonspecific catalytic activity from the total catalytic activity and expressed as counts per min (cpm). 32D cells or 32D transfectants were untreated or exposed to TPA (100 ng/ml) overnight. Cells were incubated with fluorescein isothiocyanate-conjugated anti-Mac-1 (CalTag) or anti-FcγII/III receptor (anti-FcγII/IIIR, Pharmigen) as described previously (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The cells were subjected to flow cytometry using a Becton-Dickinson FACScan. In an attempt to define which amino acids within PKC-δ are autophosphorylation sites and determine whether mutation of one of these sites would affect PKC-δ enzymatic activity, we chose to mutate serine 643 to alanine by site-directed mutagenesis. This putative autophosphorylation site is conserved in other PKC sequences, including PKC-α, PKC-βI, and PKC-βII (Fig.1). In vivo phosphopeptide mapping or site-directed mutagenesis of the corresponding threonine sites within PKC-α, PKC-βI, and PKC-βII revealed that these residues were all phosphorylated in vivo (20Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 21Flint A.J. Paladini R.D. Koshland D.E. Science. 1990; 249: 408-411Crossref PubMed Scopus (108) Google Scholar, 22Tsutakawa S. Medzihradszky K.F. Flint A.J. Burlingame A.L. Koshland Jr., D.E. J. Biol. Chem. 1995; 270: 26807-26812Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 23Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 28Zhang J. Wang L. Petrin J. Bishop W.R. Bond R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6130-6134Crossref PubMed Scopus (33) Google Scholar, 29Zhang J. Wang L. Schwartz J. Bond R.W. Bishop W.R. J. Biol. Chem. 1994; 269: 19578-19584Abstract Full Text PDF PubMed Google Scholar). The mutant cDNA, designated PKC-δS643A, was inserted into the pCEV-HA (3 × HA repeats) vector, generating pCEV-δS643A-HA, or into pLTR-HA vector (2 × HA repeats), generating pLTR-δS643A-HA. PKC-δWT cDNA was previously inserted into these same vectors and designated pCEV-δWT-HA and pLTR-δWT-HA, respectively (30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 32D cells were transfected with expression vectors containing the various cDNA constructs, and drug-resistant 32D transfectants were subjected to immunoprecipitation and immunoblot analysis to detect PKC-δS643A and PKC-δWT expression. As shown in Fig.2, immunoprecipitation with anti-PKC-δ serum followed by immunoblot analysis with the anti-HA mAb detected both pLTR-δWT-HA and pCEV-δWT-HA proteins with mobilities of 80 and 90 kDa, respectively. The mobilities of PKC-δWT proteins expressed in these two vectors were identical to those reported in our previous study (30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Endogenous PKC-δ expression in 32D cells was not detected, since the anti-HA mAb was utilized for immunoprecipitation. The levels of PKC-δS643A expression in cells transfected with pLTR-HA and pCEV-HA vectors were 2.8- and 1.8-fold higher than those of PKC-δWT in the corresponding vectors, respectively (Fig. 2). We performed in vitroautophosphorylation assays utilizing the anti-HA mAb for immunoprecipitation. As shown in Fig.3 A, autophosphorylation of pLTR-δS643A-HA protein was reduced by 54% when compared with that of the pLTR-δWT-HA molecule. Autophosphorylation of the pCEV-δS643A-HA protein was decreased by 37% when compared with that of pCEV-δWT-HA (Fig. 3 B). Autophosphorylation of endogenous PKC-δ from parental 32D cells was not detected since the anti-HA mAb would not recognize endogenous PKC-δ. By normalizing protein expression levels of PKC-δS643A in comparison to those of PKC-δWT in the various transfectants (see Fig. 2), an 83% reduction in pLTR-δS643A-HA autophosphorylation and a 65% reduction in pCEV-δS643A-HA autophosphorylation were observed. These results strongly suggest that serine 643 of PKC-δ is a major autophosphorylation site, and mutation of this site dramatically reduces autophosphorylation. To confirm that serine 643 is an in vivo phosphorylation site, two-dimensional tryptic phosphopeptide analysis was performed. As shown in Fig. 4 A, tryptic digestion of in vivo labeled PKC-δWT-HA from the TPA-treated transfectant resulted in the detection of approximately 20 distinct phosphopeptides. The phosphopeptide pattern generated from PKC-δWT-HA is consistent to that generated in a previous study (30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), assuring that this assay is very reproducible. Although most of PKC-δWT-HA phosphopeptides were also detected from tryptic digestion of in vivo labeled PKC-δS643A-HA after TPA treatment of 32D/pLTR-δS643A-HA transfectant, two phosphopeptides (peptides 5 and 14) were absent from PKC-δS643A-HA sample. The reduced intensity of peptide 5 in a mixture experiment, where equal amounts of PKC-δWT-HA and PKC-δS643A-HA samples were mixed before performing two-dimensional phosphopeptide analysis, confirmed that peptide 5 was missing in PKC-δS643A-HA (compare peptide 5 in Fig. 4, Aand C). Since the PKC-δWT-HA sample migrated slightly slower than the others in chromatography, only a tail of peptide 14 can be observed (Fig. 4 A). This peptide was not detected in PKC-δS643A-HA sample (Fig. 4 B). Therefore, whether the intensity of peptide 14 detected in the mixture experiment was reduced (Fig. 4 C) is difficult to judge. In addition, the intensity of peptide 11 was greatly reduced in the PKC-δS643A-HA sample when compared with PKC-δWT-HA, and intermediate intensity was observed in the mixture experiment (Fig. 4 C). On the other hand, phosphopeptide 19 may be absent in PKC-δWT-HA. Taken together, the results of two-dimensional phosphopeptide analysis clearly indicate that the absence or reduction in intensity of phosphopeptides 5, 14, and 11 may account for the reduced autophosphorylation of PKC-δS643Ain vitro (see Fig. 3). The enzymatic activity of PKC-δS643A expressed in pLTR-HA system was measured utilizing two separate procedures. In the first assay, the activities were measured utilizing anti-HA immunoprecipitates as the kinase sources. This method has been recently used in other PKC studies to measure PKC activity (8Denning M.F. Dlugosz A. Threadgill D.W. Magnuson T. Yuspa S.H. J. Biol. Chem. 1996; 271: 5325-5331Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar,31Ueda Y. Hirai S. Osada S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). As shown in Table I, the immunoprecipitates derived from pLTR-δWT-HA and pLTR-δS643A-HA mutant transfectants displayed similar nonspecific catalytic activities when they were incubated with the PKC-δ pseudosubstrate region-derived peptide in the absence of TPA and phosphatidylserine, two important cofactors required for specific PKC activation in vitro. However, the specific PKC-δ catalytic activity of pLTR-δS643A-HA mutant was reduced by 54% when compared with that of pLTR-δWT-HA protein.Table IThe PKC-δS643A mutant protein expressed in 32D cells possesses reduced enzymatic activity as measured by utilizing anti-HA immunoprecipitates as PKC-δ kinase sourcesCell linesTotal catalytic activityNonspecific activityPKC-δ activity32D/pLTR-δWT-HA1,401,383246,7421,154,64132D/pLTR-δS643A-HA747,810214,401533,409 (54%)1-aThe % inhibition of enzymatic activity was determined by subtracting the PKC-δ activity of the pLTR-δS643A-HA transfectant from the pLTR-δWT-HA transfectant and dividing the difference by the activity of the pLTR-δWT-HA transfectant.The method for measuring PKC-δ activity by using anti-HA immunoprecipitates as PKC-δ sources was described under "Experimental Procedures." Only one sample from each lysate was utilized for anti-HA immunoprecipitation and the subsequent activity assay. Thus, no standard deviation was available. PKC-δ specific activity was obtained by subtracting nonspecific activity from total catalytic activity. The activity is presented as cpm.1-a The % inhibition of enzymatic activity was determined by subtracting the PKC-δ activity of the pLTR-δS643A-HA transfectant from the pLTR-δWT-HA transfectant and dividing the difference by the activity of the pLTR-δWT-HA transfectant. Open table in a new tab The method for measuring PKC-δ activity by using anti-HA immunoprecipitates as PKC-δ sources was described under "Experimental Procedures." Only one sample from each lysate was utilized for anti-HA immunoprecipitation and the subsequent activity assay. Thus, no standard deviation was available. PKC-δ specific activity was obtained by subtracting nonspecific activity from total catalytic activity. The activity is presented as cpm. In another PKC activity assay, DE52 ion exchange chromatography was utilized to enrich PKC-δ proteins before performing the kinase assay (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). pLTR-δWT-HA overexpression resulted in a 14-fold increase in the enzymatic activity compared with that of endogenous PKC-δ (TableII). The increased activity observed in the pLTR-δWT-HA transfectant correlated with the levels of overexpressed PKC-δ protein (data not shown). Expression of pLTR-δS643A-HA reduced its specific catalytic activity by 39% compared with that of pLTR-δWT-HA. By normalizing the protein expression level of pLTR-δS643A-HA in comparison to that of pLTR-δWT-HA, a 78–84% reduction in pLTR-δS643A-HA enzymatic activity was calculated from the results of these two assays (see Fig.2). In summary, these results indicate that PKC-δ serine 643 is not only important for autophosphorylation but also for transphosphorylation of its in vitro substrate.Table IIThe PKC-δS643A mutant protein expressed in 32D cells possesses reduced enzymatic activity as measured by utilizing DE52 column eluates as PKC-δ kinase sourcesEluates of DE52 columnTotal catalytic activityNonspecific activityPKC-δ activity32D14,060 ± 5036,438 ± 432 7,62232D/pLTR-δWT-HA130,123 ± 8,32620,051 ± 264110,07232D/pLTR-δS643A-HA79,106 ± 2,78611,708 ± 332 67,398 (39%)2-aThe % inhibition of enzymatic activity was determined by subtracting the PKC-δ activity of the pLTR-δS643A-HA transfectant from the pLTR-δWT-HA transfectant and dividing the difference by the activity of the pLTR-δWT-HA transfectant.The method for PKC enrichment by DE52 ion exchange chromatography and the subsequent activity assay has been described previously (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). PKC-δ specific activity was obtained by subtracting nonspecific activity from total catalytic activity. The results represent the mean value of three individual samples. The activity is presented as cpm.2-a The % inhibition of enzymatic activity was determined by subtracting the PKC-δ activity of the pLTR-δS643A-HA transfectant from the pLTR-δWT-HA transfectant and dividing the difference by the activity of the pLTR-δWT-HA transfectant. Open table in a new tab The method for PKC enrichment by DE52 ion exchange chromatography and the subsequent activity assay has been described previously (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). PKC-δ specific activity was obtained by subtracting nonspecific activity from total catalytic activity. The results represent the mean value of three individual samples. The activity is presented as cpm. Recent work on the PKC-αT638A mutant suggested that mutation of threonine 638 rendered the enzyme very sensitive to heat treatment (23Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Thus, we were interested in determining whether there were any changes in the heat sensitivity of PKC-δS643A mutant in comparison to PKC-δWT. As shown in Fig. 5, pLTR-δWT-HA, pLTR-δS643A-HA, and endogenous PKC activities remained very stable even after a 30-min period of preincubation at 25 °C. Surprisingly, both pLTR-δWT-HA and pLTR-δS643A-HA mutant activities were slightly increased after the preincubation period. This result suggests that phosphorylation of PKC-δ on serine 643 does not affect the heat stability of the enzyme, even though the enzymatic activity and autophosphorylation of PKC-δS643A are greatly reduced in comparison to PKC-δWT. PKC-δ normally resides in the cytosol (S100) of the cell. In response to stimulation by TPA, a portion translocates to the membrane fraction (P100) (13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar). Our previous data demonstrated that PKC-δ was tyrosine-phosphorylated in vivo in response to TPA stimulation, and tyrosine-phosphorylated PKC-δ could be detected only in the membrane fraction (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 10Li W. Mischak H. Yu J.-C. Wang L.-M. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar, 13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar, 30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Thus, we investigated whether mutation of serine 643 would affect localization, translocation, or tyrosine phosphorylation of the enzyme. As shown in Fig. 6 A, the pLTR-δS643A-HA mutant as well as pLTR-δWT-HA proteins resided in the cytosol in resting cells after cell fractionation and immunoprecipitation with anti-HA mAb followed by anti-PKC-δ immunoblot analysis. Stimulation with TPA for 10 min caused translocation of a similar portion of both pLTR-δWT-HA and pLTR-δS643A-HA mutant proteins to the membrane fraction (Fig. 6 A, lanes 4 and 6). Reblotting the membrane with anti-Tyr(P) mAb showed that both pLTR-δWT-HA and pLTR-δS643A-HA mutant proteins were tyrosine-phosphorylated in TPA-stimulated samples (Fig. 6 B, lanes 4 and 6). As previously demonstrated (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 13Li W. Yu J.-C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar), tyrosine phosphorylation was observed only in the membrane fraction. Taken together, the results indicate that autophosphorylation of PKC-δ on serine 643 does not affect localization, translocation, or tyrosine phosphorylation of the enzyme. TPA treatment of 32D cells overexpressing PKC-δWT was able to mediate monocytic differentiation, as judged by changes in morphology, cell adhesion, nonspecific esterase staining, and cell surface differentiation marker expression (5Mischak H. Pierce J.H. Goodnight J. Kazanietz M.G. Blumberg P.M. Mushinski J.F. J. Biol. Chem. 1993; 268: 20110-20115Abstract Full Text PDF PubMed Google Scholar, 6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Since mutation of PKC-δ on serine 643 reduced its autophosphorylation and its enzymatic activity, we tested whether PKC-δ-mediated monocytic differentiation would be affected. Treatment of the pLTR-δS643A-HA mutant transfectant with TPA overnight resulted in reduced cell adhesion and less morphological changes indicative of the macrophage phenotype as analyzed by Wright-Giemsa staining when compared with the pLTR-δWT-HA transfectant (data not shown). Flow cytometric analysis was utilized to detect cell surface differentiation marker expression. As seen in Fig. 7, stimulation of pLTR-δWT-HA transfectant with TPA overnight resulted in increased expression of Mac-1 (Fig. 7 A) and FcγII/IIIR (Fig. 7 B). TPA treatment of the pLTR-δS643A-HA mutant transfectant resulted in reduced increases in marker expression in comparison to the pLTR-δWT-HA transfectant (Fig. 7, A andB). However, the TPA-induced increase in marker expression observed for the pLTR-δS643A-HA mutant transfectant was still greater than that for the parental 32D cells, indicating that the remaining kinase activity provided by the pLTR-δS643A-HA mutant was able to partially mediate the differentiation process. These results suggest that serine autophosphorylation on amino acid 643 plays an important role in PKC-δ-mediated monocytic differentiation of 32D myeloid progenitor cells. In the present study, we have demonstrated that serine 643 of PKC-δ is a major autophosphorylation site in vitro and autophosphorylation of PKC-δ on this site is required for its full enzymatic activity. TPA-induced monocytic differentiation of 32D cells overexpressing PKC-δS643A is reduced in comparison to the PKC-δWT transfectant, suggesting that the mutant protein is less efficient at activating key substrate(s) which affect the differentiation process. The effects of site-directed mutagenesis of PKC-α and PKC-βI at similarly conserved sites were recently reported (23Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 29Zhang J. Wang L. Schwartz J. Bond R.W. Bishop W.R. J. Biol. Chem. 1994; 269: 19578-19584Abstract Full Text PDF PubMed Google Scholar). Although noin vitro autophosphorylation data were presented in either study, transphosphorylation of the histone substrate in vitro by PKC-αT638A mutant was reduced by 26% (23Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In contrast, the PKC-βIT642A mutant completely abolished in vivo phosphorylation and enzymatic activity (29Zhang J. Wang L. Schwartz J. Bond R.W. Bishop W.R. J. Biol. Chem. 1994; 269: 19578-19584Abstract Full Text PDF PubMed Google Scholar). Whether mutagenesis of PKC-βI affected the general conformation of the protein remains to be determined. This was suggested by the inability to label in vivo the PKC-βIT642A mutant protein with [32P]orthophosphate. Although an ATP binding mutant of PKC-δ (PKC-δK376R) generated in our laboratory was completely devoid of autophosphorylation capacity (6Li W. Yu J.-C. Shin D.-Y. Pierce J.H. J. Biol. Chem. 1995; 270: 8311-8318Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), it could still be labeledin vivo by [32P]orthophosphate. 2W. Li, unpublished observations.Two-dimensional phosphopeptide mapping of the PKC-δK376R mutant revealed that at least two autophosphorylation sites were absent when compared with PKC-δWT, indicating that other sites in addition to serine 643 must contribute to PKC-δ autophosphorylation.2Moreover, the present results provide evidence that autophosphorylation of the PKC-δS643A mutant is not completely abolished (see Fig. 3). PKC-δS643A mutant could be labeled in vivo to a similar extent as PKC-δWT (see Fig. 4). Based on recent mapping and site-directed mutagenesis results involving PKC-α at serine 657 (24Bornancin F. Parker P.J. J. Biol. Chem. 1997; 272: 3544-3549Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) and PKC-βII at serine 660 (20Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), we predict that the corresponding serine 662 of PKC-δ may be an additional autophosphorylation site. Generation of a serine 643 to alanine mutant of PKC-δ did not affect the translocation of PKC-δ from the cytosol to the membrane in response to TPA stimulation, nor did it affect its tyrosine phosphorylation in vivo. These data indicate that site-directed mutagenesis did not alter the general conformation of the molecule. This is also suggested by the similar two-dimensional phosphopeptide pattern observed for both PKC-δWT and PKC-δS643A (see Fig. 4, A and B). Translocation of PKC from the cytosol to the membrane is dependent on the binding of phorbol ester or endogenously produced diacylglycerol to the regulatory domain of PKC (1Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1469) Google Scholar). Tyrosine phosphorylation of PKC-δ has also been mapped at the N terminus of PKC-δ (30Li W. Li W. Chen X.-H. Kelley C.A. Alimandi M. Zhang J. Chen Q. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1996; 271: 26404-26409Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Therefore, it was not surprising that mutation of serine 643 did not affect these events since this mutation resides in the C terminus of the molecule. Although phosphorylation has been implicated to be important for PKC localization, expression of PKC-δS643A did not alter the localization of the molecule. This can be best explained by the finding that in vitroautophosphorylation was diminished by only 65–83% in the mutant (see Fig. 3). Thus, alternative autophosphorylation sites may compensate and allow the mutant protein to normally regulate localization through phosphorylation and dephosphorylation dynamics. In summary, our results demonstrate that serine 643 is a major autophosphorylation site of PKC-δ. Autophosphorylation of PKC-δ on this site is indispensable for its full enzymatic activity but is not required or sufficient for determining the localization, translocation, or tyrosine phosphorylation of PKC-δ. Mapping the remaining autophosphorylation site(s) within PKC-δ should make it feasible to determine the complete role of autophosphorylation and its effects on the various aspects of PKC-δ function. We are grateful to Nelson Ellmore for excellent technical assistance.
Referência(s)