Artigo Acesso aberto Revisado por pares

Activation of PKC δ in the Rat Corpus Luteum during Pregnancy

1999; Elsevier BV; Volume: 274; Issue: 52 Linguagem: Inglês

10.1074/jbc.274.52.37499

ISSN

1083-351X

Autores

Carl A. Peters, Evelyn T. Maizels, Mary Hunzicker-Dunn,

Tópico(s)

Estrogen and related hormone effects

Resumo

Maintenance of pregnancy in the rat requires the corpus luteum. At a time when rat placental lactogens (rPLs) are required to support progesterone production by the corpus luteum and when relaxin expression is initiated, expression of a specific protein kinase C (PKC) isoform, PKC δ, is dramatically increased. We therefore assessed whether prolactin (PRL) receptor activation promotes activation of PKC δ in a luteinized granulosa cell model. We also assessed the activation status of PKC δ in corpora lutea obtained when the corpus luteum is exposed to chronically high concentrations of rPLs. The activity of PKC δ was assessed by two means: an immune complex (IC) assay and Western blotting with a phospho-epitope-specific antibody that detects PKC δ phosphorylated on serine 662. PKC δ activation in the IC kinase assay was determined by the ability of immunoprecipitated PKC δ to phosphorylate the PKC δ-preferential substrate small heat shock protein (HSP-27). Treatment of luteinized rat granulosa cells with phorbol myristate acetate, a known activator of PKC, promoted a 7-fold increase in HSP-27 phosphorylation by PKC δ. Similarly, immunoreactivity with the phospho-epitope-specific PKC δ antibody was increased in extracts prepared from luteinized granulosa cells treated with phorbol myristate acetate or followingin vitro activation of recombinant PKC δ. Using these assays, we assessed whether PRL receptor agonists were capable of activating PKC δ in luteinized granulosa cells. PRL receptor agonists induced translocation PKC δ from the cytosolic to the Triton-soluble membrane fraction and increased PKC δ activity assessed by both IC kinase assay and Western blotting with phospho-epitope-specific PKC δ antibody. Analysis of PKC δ activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC δ antibody revealed that PKC δ activity was increased throughout the second half of pregnancy. These results demonstrate that PRL receptor activation promotes the acute activation of PKC δ in luteinized rat granulosa cells. At a time when the rat is exposed to chronically high concentrations of rPLs, PKC δ is increasingly expressed and active. Maintenance of pregnancy in the rat requires the corpus luteum. At a time when rat placental lactogens (rPLs) are required to support progesterone production by the corpus luteum and when relaxin expression is initiated, expression of a specific protein kinase C (PKC) isoform, PKC δ, is dramatically increased. We therefore assessed whether prolactin (PRL) receptor activation promotes activation of PKC δ in a luteinized granulosa cell model. We also assessed the activation status of PKC δ in corpora lutea obtained when the corpus luteum is exposed to chronically high concentrations of rPLs. The activity of PKC δ was assessed by two means: an immune complex (IC) assay and Western blotting with a phospho-epitope-specific antibody that detects PKC δ phosphorylated on serine 662. PKC δ activation in the IC kinase assay was determined by the ability of immunoprecipitated PKC δ to phosphorylate the PKC δ-preferential substrate small heat shock protein (HSP-27). Treatment of luteinized rat granulosa cells with phorbol myristate acetate, a known activator of PKC, promoted a 7-fold increase in HSP-27 phosphorylation by PKC δ. Similarly, immunoreactivity with the phospho-epitope-specific PKC δ antibody was increased in extracts prepared from luteinized granulosa cells treated with phorbol myristate acetate or followingin vitro activation of recombinant PKC δ. Using these assays, we assessed whether PRL receptor agonists were capable of activating PKC δ in luteinized granulosa cells. PRL receptor agonists induced translocation PKC δ from the cytosolic to the Triton-soluble membrane fraction and increased PKC δ activity assessed by both IC kinase assay and Western blotting with phospho-epitope-specific PKC δ antibody. Analysis of PKC δ activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC δ antibody revealed that PKC δ activity was increased throughout the second half of pregnancy. These results demonstrate that PRL receptor activation promotes the acute activation of PKC δ in luteinized rat granulosa cells. At a time when the rat is exposed to chronically high concentrations of rPLs, PKC δ is increasingly expressed and active. protein kinase C phosphatidylserine diacylglycerol prolactin phorbol myristate acetate estrogen rat placental lactogen Dulbecco's modified Eagle's medium/Ham's F-12 Triton-soluble immune complex 27-kDa heat shock protein 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate 1-phosphatidylinositol 3-kinase signal transducer and activator of transcription The corpus luteum is a transient endocrine gland of the ovary formed following ovulation by the differentiation of granulosa and thecal cells (1Gibori G. Khan I. Warshaw M.L. McLean M.P. Puryear T.K. Nelson S. Durkee T.J. Azhar S. Steinschneider A. Rao M.C. Recent Prog. Horm. Res. 1988; 44: 377-424PubMed Google Scholar). In the rat, the corpus luteum is the sole source of the progesterone that is necessary to maintain pregnancy to term and is thus necessary throughout pregnancy (1Gibori G. Khan I. Warshaw M.L. McLean M.P. Puryear T.K. Nelson S. Durkee T.J. Azhar S. Steinschneider A. Rao M.C. Recent Prog. Horm. Res. 1988; 44: 377-424PubMed Google Scholar). It is therefore of great interest to assess the signal transduction pathways employed within the corpus luteum that are involved in the regulation of its function. PKC1 is a family of serine/threonine kinases that has been implicated in the regulation of numerous signaling pathways (2Mochly-Roesn D. Kauvar L.M. Adv. Pharmacol. 1998; 44: 91-145Crossref PubMed Scopus (89) Google Scholar, 3Buckley A.R. Crowe P.D. Russell D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8649-8653Crossref PubMed Scopus (154) Google Scholar). The PKC family consists of 10 different isoforms that have been grouped into three categories based on the structural and functional differences among family members (4Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1359) Google Scholar). Conventional isoforms α, βI, βII, and γ isoforms are activated by PS, DAG, and Ca2+. Novel isoforms do not require Ca2+ for kinase activity and are represented by the δ, ε, η, and θ isoforms. The atypical isoforms ζ and ι require only PS for activation. As the number of PKC isoforms has increased, so has the expectation that distinct PKC isoforms will have distinct functions within a cell. This has been, to some extent, borne out by the specific roles of PKC isoforms in mitogenesis (5Gschwendt M. Eur. J. Biochem. 1999; 259: 555-564Crossref PubMed Scopus (230) Google Scholar, 6Acs P. Wang Q.J. Bogi K. Marquez A.M. Lorenzo P.S. Biro T. Szallasi Z. Mushinski J.F. Blumberg P.M. J. Biol. Chem. 1997; 272: 28793-28799Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), gene expression (5Gschwendt M. Eur. J. Biochem. 1999; 259: 555-564Crossref PubMed Scopus (230) Google Scholar, 7Hata A. Akita Y. Suzuki K. Ohno S. J. Biol. Chem. 1993; 268: 9122-9129Abstract Full Text PDF PubMed Google Scholar, 8Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar), and secretion (9Taylor M.J. Clark C.L. Biol. Reprod. 1988; 39: 743-750Crossref PubMed Scopus (8) Google Scholar, 10Kiley S.C. Parker P.J. Fabbro D. Jaken S. Mol. Endocrinol. 1992; 6: 120-131PubMed Google Scholar, 11Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar, 12Billiard J. Koh D. Babcock D.F. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12192-12197Crossref PubMed Scopus (50) Google Scholar). The ability of a distinct PKC isoform to regulate discrete biological functions is likely due to three factors: (a) the requirements for activation of a PKC isoform as determined by that isoform's structure (4Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1359) Google Scholar); (b) the localization of different PKC isoforms to distinct subcellular locales, thus limiting access of a particular PKC isoform to relevant substrates (2Mochly-Roesn D. Kauvar L.M. Adv. Pharmacol. 1998; 44: 91-145Crossref PubMed Scopus (89) Google Scholar); and (c) the substrate specificity of PKC isoforms (13Kolch W. Heidecker G. Kochs G. Hummel R. Vahldi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-4252Crossref PubMed Scopus (1159) Google Scholar, 14Kielbassa K. Müller H.-J. Meyer H.E. Marks F. Gschwendt M. J. Biol. Chem. 1995; 270: 6156-6162Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 15Municio M.M. Lozano J. Sanchez P. Moscat J. Diaz-Meco M.T. J. Biol. Chem. 1995; 270: 15884-15891Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 16Nishikawa K. Toker A. Johannes F.-J. Songyang Z. Cantley L.C. J. Biol. Chem. 1997; 272: 952-960Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 17Maizels E.T. Peters C.A. Kline M. Cutler R.E. Shanmugam M. Hunzicker-Dunn M. Biochem. J. 1998; 332: 703-712Crossref PubMed Scopus (101) Google Scholar). The ovary of the rat has been found to express the same subset of PKC isoforms throughout all the stages of development that have been analyzed (18Cutler R.E. Maizels E.T. Brooks E.J. Mizuno K. Ohno S. Hunzicker-Dunn M. Biochim. Biophys. Acta. 1993; 1179: 260-270Crossref PubMed Scopus (27) Google Scholar). These are the conventional isoforms α, βI, and βII, the novel isoforms δ and ε, and the atypical isoform ζ. The δ isoform can be distinguished from the other isoforms by the striking increase of both PKC δ mRNA and protein levels in corpora lutea in the second half of pregnancy (19Cutler R.E. Maizels E.T. Hunzicker-Dunn M. Endocrinology. 1994; 135: 1669-1678Crossref PubMed Scopus (39) Google Scholar). The rat corpus luteum is maintained in the second half of pregnancy by the combined actions of intraluteal E2 and PRL-like hormones such as the placenta-derived rPL-1 (1Gibori G. Khan I. Warshaw M.L. McLean M.P. Puryear T.K. Nelson S. Durkee T.J. Azhar S. Steinschneider A. Rao M.C. Recent Prog. Horm. Res. 1988; 44: 377-424PubMed Google Scholar, 20Keyes P.L. Possley R.M. Brabec R.K. Biol. Reprod. 1987; 37: 699-707Crossref PubMed Scopus (7) Google Scholar). We have found that rPL-1 treatment of luteinized granulosa cells induces phosphorylation of Stat 3 on both tyrosine 705 and serine 727 and induction of relaxin mRNA expression, 2C. A. Peters, E. T. Maizels, M. C. Robertson, R. P. Shiu, M. S. Soloff, and M. Hunzicker-Dunn, submitted for publication. a major product of the rat corpus luteum in the second half of pregnancy (21Sherwood O.D. Downing S.J. Guico-Lamm M.L. O'Day-Bowman M.B. Fields P.A. Oxf. Rev. Reprod. Biol. 1993; 15: 143-189PubMed Google Scholar). Both Stat 3 serine phosphorylation and induction of relaxin expression by rPL-1 were abrogated by the PKC δ inhibitor rottlerin.2 Based on the ability of the PKC δ inhibitor rottlerin to block rPL-1-induced Stat 3 serine phosphorylation and relaxin mRNA expression, we now seek direct evidence (a) that PRL receptor activation by rPL-1 activates PKC δ in a luteinized granulosa cell model and (b) that PKC δ is active in anin vivo setting in the corpus luteum of pregnancy, coincident with high rPLs in serum of rats. Our results show that signaling through the PRL receptor promotes acute activation of PKC δ in rat luteinized granulosa cells and that the PKC δ in corpora lutea obtained when rPLs are elevated is active. These results thus implicate the PRL signaling pathway in the activation of PKC δ in corpora lutea of pregnancy. The following materials were purchased: [γ-32P]ATP (specific activity 3000 Ci/mmol) from NEN Life Science Products; SDS-polyacrylamide gel electrophoresis reagents from Bio-Rad; protein standards from Diversified Biotech (Boston, MA); recombinant HSP-27 from Stressgen Biotechnology (Victoria, British Columbia, Canada); Hybond C-extra nitrocellulose and ECL reagents from Amersham Pharmacia Biotech; GF109203X from Alexis (San Diego, CA); purified recombinant PKC δ from Pan Vera (Madison, WI); PKC δ-specific monoclonal antibody directed to the N terminus of PKC δ from Transduction Labs (lot 2, released April 1995) (Lexington, KY). M-4 (PKC α) monoclonal antibody was obtained from K. Leach (The Upjohn Company), and PKC δ serine 662 phospho-epitope-specific antibody was a gift from New England Biolabs (Beverly, MA). All other PKC-specific antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); 4G-10 (anti-phosphotyrosine) monoclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). All other biochemical reagents were purchased from Sigma. Final concentrations are indicated throughout. Rats were obtained at 21 days of age from Charles River Laboratories (Portage, MI) and were maintained in accordance with "Guidelines for the Care and Use of Experimental Animals" by protocols approved by the Northwestern University Animal Care and Use Committee. Follicles were collected from 30-day-old rats that had been administered a low dose of human chorionic gonadotrophin (0.15 IU) given subcutaneously twice daily for 2 days. On the following day, a high dose of human chorionic gonadotrophin (10 IU) was given to rats via tail vein injection, and ovaries were isolated 7 h later (22Richards J.S. Hedin L. Caston L. Endocrinology. 1986; 118: 1660-1668Crossref PubMed Scopus (60) Google Scholar, 23Hickey G.J. Krasnow J.S. Beattie W.G. Richards J.S. Mol. Endocrinol. 1990; 4: 3-12Crossref PubMed Scopus (255) Google Scholar). Cells were harvested by mechanical dispersion and put into culture by modifications of the method of Bley et al. (24Bley M.A. Simon J.C. Saragueta P.E. Baranao J.L. Biol. Reprod. 1991; 44: 880-888Crossref PubMed Scopus (26) Google Scholar) as described by Carr et al. (25Carr D.W. DeManno D.A. Atwood A. Hunzicker-Dunn M. Scott J.D. J. Biol Chem. 1993; 268: 20729-20732Abstract Full Text PDF PubMed Google Scholar). The medium used for all procedures was DMEM/F-12 without phenol red and with 15 mmHEPES, 3.15 g/liter glucose, 1% charcoal-stripped fetal bovine serum, 100 IU penicillin G, and 100 μg/ml streptomycin. Following sequential incubations at 37 °C in 6 mm EGTA in DMEM/F-12 and 0.5m sucrose in DMEM/F-12, ovaries were returned to DMEM/F-12. Granulosa cells were released into the medium from all follicles using 30-gauge needles and gentle pressure. Cells were pelleted at 100 × g for 15 min, counted using trypan blue, and plated at a density of approximately 1 × 106 cells/ml on plastic dishes. Cells were cultured in humidified atmosphere at 37 °C, 5% CO2 with 10 nm estradiol-17β (in ethanol, final concentration 0.5%). The medium was changed every 3 days. Pregnant rats were obtained from Charles River Laboratories. On the appropriate day of pregnancy rats were sacrificed, ovaries were dissected, corpora lutea were removed, and protein was collected from the corpora lutea as specified within. Subcellular fractions of cell or tissue extracts were prepared by homogenization in protease/phosphatase inhibitor-enriched homogenization buffer (10 mm potassium phosphate, pH 7.0, 1 mm EDTA, 5 mm EGTA, 10 mmMgCl2, 2 mm dithiothreitol, 1 mmsodium vanadate, 80 mm β-glycerophosphate, 100 μg/ml pepstatin A, 10 μg/ml leupeptin, 40 μg/ml phenylmethylsulfonyl fluoride) followed by centrifugation at 105,000 × gfor 70 min. The soluble fraction was removed and the pellet resuspended in the same buffer adjusted to 0.1% Triton X-100 and incubated with stirring for 60 min followed by centrifugation at 105,000 ×g for 30 min. Alternatively, clarified cell lysates were prepared by homogenization in a lysis buffer (10 mmpotassium phosphate, pH 7.0, 1 mm EDTA, 5 mmEGTA, 10 mm MgCl2, 2 mmdithiothreitol, 1 mm sodium vanadate, 50 mmβ-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, 0.1% deoxycholic acid) followed by centrifugation at 20,000 × g for 20 min. Samples were denatured by adding 3× stop (3% SDS, 150 mm Tris-HCl, 2.4 mm EDTA, 3% β-mercaptoethanol, 30% glycerol, and 0.5% bromphenol blue) followed by heating for 5 min at 100 °C. Protein concentrations in both fractions were determined (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard. Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to membranes for Western blot analysis. Western blot analysis was performed using the ECL detection system (Amersham Pharmacia Biotech) following the protocol provided by the manufacturer. Where appropriate, membranes were stripped of antibodies according to the protocol provided with the ECL detection system. Densitometric quantitation was performed by image analysis using a Bio-Rad Molecular Analyst or BioImage Intelligent Quantifier software. Cells were cultured for 9 days with E2 and subsequently treated with 10 nm PMA or ethanol vehicle for 10 min or with 5 μg/ml rPL-1 for 5 min. Clarified cell lysates or subcellular fractions of cell extracts were prepared, and PKC δ or control immunoprecipitations were performed on samples containing 500 μg of total protein. Antibody-antigen complexes were precipitated by further incubation with an anti-mouse Ig antibody, where applicable, and protein A-conjugated Sepharose or with protein A/G-conjugated agarose alone. Pelleted proteins were washed with low salt (10 mm Tris-HCl, pH 7.2, 150 mm NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mm sodium vanadate, and 40 μg/ml phenylmethylsulfonyl fluoride) and high salt (10 mm Tris-HCl, pH 7.2, 1 mm NaCl, 0.1% Nonidet-P40, 1 mm sodium vanadate, and 40 μg/ml phenylmethylsulfonyl fluoride) radioimmunoprecipitation assay (RIPA) buffer. Immunoprecipitates were resuspended in 50 μl of TE (10 mm Tris, pH 7.5, 0.1 mm EGTA). Precipitated proteins were subjected to an in vitro kinase assay in a final volume of 110 μl (containing 45 μmα-glycerophosphate (pH 7.0), 9 mm MgCl2, 0.9 mm dithiothreitol, 4.5 μm ATP, 5 μCi of [γ-32P]ATP, and 5 μg of exogenous substrate). Where indicated, the PKC inhibitor GF109203X (bisindolylmaleimide) was added at a final concentration of 5 μm. Incubations were typically for 5 or 10 min (unless otherwise indicated) at 37 °C, and reactions were terminated by adding 50 μl of 3× stop and heat denaturation. Proteins in the samples were separated by SDS-polyacrylamide gel electrophoresis, and the top half of the gel, containing PKC δ, was transferred to a membrane and subjected to Western blotting while the bottom half of the gel, containing exogenous substrate, was dried and exposed to film to detect incorporation of labeled phosphate. Alternatively, the entire gel was transferred, and phosphorylation was detected by exposure to film followed by Western blotting. A similar procedure was employed to analyze PKC δ activation during pregnancy. Pregnant rats were sacrificed on the indicated day of pregnancy, and ovaries were isolated. Corpora lutea were isolated and homogenized as described above for use in the IC kinase assay. Where indicated, kinase assay included lipids (PS (45 μg/ml) and 1,2-diolein (1.6 μg/ml)). Reactions were conducted as described above with 3.5 nm recombinant PKC δ replacing immunoprecipitated PKC δ. To begin to assess the activation of PKC isoforms during pregnancy, we employed the fact that translocation to a membrane fraction is widely recognized as an index of activation of PKC for many isoforms (2Mochly-Roesn D. Kauvar L.M. Adv. Pharmacol. 1998; 44: 91-145Crossref PubMed Scopus (89) Google Scholar). Subcellular fractions of corpora lutea from days 11, 18, and 21 of pregnancy were prepared and the cytosol and T.S. (membrane) fractions analyzed by Western blot analysis. Results depicted in Fig. 1 show that all PKC isoforms expressed are partially active at some time during the second half of pregnancy based on their presence in the T.S. fraction. PKCs α and ε are both detected in the T.S. fraction on days 11 and 21 of pregnancy and to a reduced extent on day 18 of pregnancy. In contrast, PKC βII is detected in the T.S. fraction predominately on day 18. PKC ζ is detected in the T.S. fraction only on day 21 of pregnancy. PKC δ exhibits the previously described increase in expression (19Cutler R.E. Maizels E.T. Hunzicker-Dunn M. Endocrinology. 1994; 135: 1669-1678Crossref PubMed Scopus (39) Google Scholar), and increased amounts of PKC δ are detected in the T.S. fraction as pregnancy progresses to term. Because luteal PKC δ exhibits an increase in expression during pregnancy (19Cutler R.E. Maizels E.T. Hunzicker-Dunn M. Endocrinology. 1994; 135: 1669-1678Crossref PubMed Scopus (39) Google Scholar) and appears to be partly activated throughout the second half of pregnancy, we sought to analyze PKC δ activity more closely. To this end we employed an assay that involves the immunoprecipitation of PKC δ. The kinase activity of the precipitated PKC δ is then assessed by its ability to phosphorylatein vitro HSP-27, a PKC δ preferential substrate (17Maizels E.T. Peters C.A. Kline M. Cutler R.E. Shanmugam M. Hunzicker-Dunn M. Biochem. J. 1998; 332: 703-712Crossref PubMed Scopus (101) Google Scholar). This assay is conducted in the absence of exogenous activators so that the kinase activity that is measured reflects that which was attained in the cell or tissue. Fig. 2 A shows the results of an IC kinase assay performed on samples from luteinized granulosa cells. Cells were treated with either vehicle or 10 nm PMA for 10 min. The results of PKC δ immune precipitations from cytosol and T.S. fractions reveal that PMA promotes both the translocation of PKC δ from the cytosol to the T.S. fraction and the partial down-regulation of PKC δ (Fig. 2 A, top panel; compare amount of PKC δ in lane 1 with that in lanes 3and 4). PMA also induced the tyrosine phosphorylation of the PKC δ translocated into the T.S. fraction (Fig. 2 A, second panel). Tyrosine phosphorylation of PKC δ has been observed by several groups to be a consequence of PKC δ activation, especially in response to PMA, but the function of PKC δ tyrosine phosphorylation is not yet fully understood (5Gschwendt M. Eur. J. Biochem. 1999; 259: 555-564Crossref PubMed Scopus (230) Google Scholar). The autophosphorylation of PKC δ on serine/threonine residues during the in vitro kinase assay (Fig. 2 A, third panel) mirrors the amount of PKC δ immunoprecipitated in each lane. Phosphorylation of the exogenous substrate HSP-27 by immunoprecipitated PKC δ from vehicle and PMA-treated cells is shown in the bottom panel of Fig. 2 A. Although PKC δ exhibits activity in the cytosolic fraction of control cells (lane 1), phosphorylation of HSP-27 by PKC δ is clearly enhanced in the T.S. fraction of PMA-treated cells consistent with PKC δ translocation to this fraction (lane 4). PMA-stimulated activation of PKC δ is most clearly appreciated when the amount of phosphorylated HSP-27 is assessed relative to the amount of PKC δ that is immunoprecipitated (Fig. 2 B). Results of this analysis show that PMA-dependent PKC δ activation is readily detected by this IC kinase assay. We further evaluated the characteristics of the in vitro PKC δ IC kinase assay. Cells were treated with 10 nm PMA for 10 min and then homogenized in a membrane extracting buffer. PKC δ was immunoprecipitated, and the IC kinase assay reaction was performed for 1–10 min. The upper panel of Fig. 3 A is a PKC δ Western blot that shows that equivalent amounts of PKC δ were immunoprecipitated. The lower panel shows that HSP-27 phosphorylation increases with time of incubation. When PKC δ antibody is omitted, PKC δ is not immunoprecipitated and HSP-27 is not phosphorylated (Fig. 3 B). HSP-27 phosphorylation by immunoprecipitated PKC δ is nearly undetectable when the in vitro reaction is performed in the presence of the PKC inhibitor GF109203X (27Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudett V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 28Wilkinson S.E. Parker P.J. Nixon J.S. Biochem. J. 1993; 294: 335-337Crossref PubMed Scopus (495) Google Scholar) (Fig. 3 C). Taken together, these results show that the PKC δ IC kinase assay detects authentic activation of PKC δ attained in PMA-treated luteinized granulosa cells. Based on our evidence that the IC kinase assay readily detects active PKC δ and utilizing this assay, we sought to analyze the activity of PKC δ in corpora lutea obtained during the second half of pregnancy. PKC δ was immunoprecipitated from corpora lutea collected from days 11, 18, and 21 of pregnancy and homogenized in a membrane extracting buffer. The amount of PKC δ immunoprecipitated from these days of pregnancy (Fig. 4 A, top panel) correlates with the increase in PKC δ expression previously observed (19Cutler R.E. Maizels E.T. Hunzicker-Dunn M. Endocrinology. 1994; 135: 1669-1678Crossref PubMed Scopus (39) Google Scholar). Tyrosine phosphorylation of PKC δ is also observed, particularly on day 21 of pregnancy. Consistent with the translocation analysis shown in Fig. 1, HSP-27 phosphorylation in the IC kinase assay is detected in each of the luteal samples and increases as pregnancy progresses (Fig. 4 A, bottom panel). These results suggest that PKC δ is indeed active throughout the second half of pregnancy, as predicted from the results presented in Fig. 1. PKC δ exhibits similar activity in corpora lutea obtained on days 18 and 21. To determine whether this level of PKC δ activity reflects maximal activation of PKC δ, we assessed the activity of PKC δ in an IC kinase assay upon addition of the PKC activators PS and DAG. Results show that PKC δ immunoprecipitated from corpora lutea on day 18 of pregnancy can be further activated in vitro when PS and DAG are added to thein vitro reaction (Fig. 4 B). Autophosphorylation of PKC δ on serine 643 is reported to be important for the regulation of PKC δ activity (29Li W. Zhang J. Bottaro D.P. Li W. Pierce J.H. J. Biol. Chem. 1997; 272: 24550-24555Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, mutation of this serine to an alanine did not abolish PKC δ autophosphorylation or activity (29Li W. Zhang J. Bottaro D.P. Li W. Pierce J.H. J. Biol. Chem. 1997; 272: 24550-24555Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 30Stempka L. Schnozler M. Radke S. Rincke G. Marks F. Gschwendt M. J. Biol. Chem. 1999; 274: 8886-8892Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Serine 662 of PKC δ has also been hypothesized as a site of autophosphorylation because of corresponding autophosphorylation sites on PKC α (serine 657) and PKC βII (serine 660). Using an epitope-specific antibody that reacts with PKC δ phosphorylated on serine 662, we sought to assess whether serine 662 autophosphorylation occurs coincident with activation of PKC δ. The time-dependent activation in vitro of recombinant PKC δ by PS and DAG is shown (Fig. 5 A). PKC δ exhibits increased histone phosphorylation and autophosphorlyation with time of incubation, as shown in the lower two panels of Fig. 5 A, and a corresponding increase in immunoreactivity as detected with the PKC δ serine 662 phospho-epitope-specific antibody (Fig. 5 A, top panel). A PKC δ Western blot confirms that equivalent amounts of PKC δ are present in each lane (Fig. 5 A, top panel). We also evaluated the ability of the PKC δ serine 662 phospho-epitope-specific antibody to detect PMA-dependent PKC δ activation in luteinized granulosa cells. Luteinized granulosa cells were stimulated with 10 nm PMA or vehicle for 10 min. Results show that the phosphorylation of PKC δ on serine 662 is also increased following PMA-dependent activation of PKC δ in luteinized granulosa cells (Fig. 5 B). PKC δ exhibits some basal activity, based on phospho-epitope-specific antibody immunoreactivity in the absence of PMA treatment, consistent with the results shown in Fig. 2. To further confirm the activation of PKC δ in rat corpora lutea during pregnancy, Western blotting with the serine 662 phospho-epitope-specific antibody was performed on extracts prepared from corpora lutea obtained on day 11, 18, or 21 of pregnancy. The increase in PKC δ expression is again apparent (Fig. 6 , lower panel). Immunoreactivity with the serine 662 phospho-epitope-specific antibody is equivalent on day 18 and 21 of pregnancy, and both are clearly increased compared with the reactivity seen on day 11 of pregnancy (Fig. 6 , upper panel). Thus, the relative activity of PKC δ detected in the corpora lutea of pregnancy by both membrane translocation (Fig. 1) and IC kinase assay (Fig. 4) is mirrored by reactivity with the serine 662 phospho-epitope-specific antibody. During the second half of pregnancy, when we detect activated PKC δ, the rat corpus luteum is maintained exclusively by the combined actions of intraluteal E2, aromatized from androgens provided by the placenta (1Gibori G. Khan I. Warshaw M.L. McLean M.P. Puryear T.K. Nelson S. Durkee T.J. Azhar S. Steinschneider A. Rao M.C. Recent Prog. Horm. Res. 1988; 44: 377-424PubMed Google Scholar), and PRL-like hormones such as the placenta-derived rPL-1 (1Gibori G. Khan I. Warshaw M.L. McLean M.P. Puryear T.K. Nelson S. Durkee T.J. Azhar S. Steinschneider A. Rao M.C. Recent Prog. Horm. Res. 1988; 44: 377-424PubMed Google Scholar, 20Keyes P.L. Possley R.M. Brabec R.K. Biol. Reprod. 1987; 37: 699-707Crossref PubMed Scopus

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