Molecular Mechanism of the Inhibition of Phospholipase C β3 by Protein Kinase C
2000; Elsevier BV; Volume: 275; Issue: 39 Linguagem: Inglês
10.1074/jbc.m004276200
ISSN1083-351X
AutoresCaiping Yue, Chun-Ying Ku, Mingyao Liu, Melvin I. Simon, Barbara M. Sanborn,
Tópico(s)Venomous Animal Envenomation and Studies
ResumoActivation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCβ) pathway. In turn, phosphorylation of PLCβ by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca2+ release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Gαq-coupled (oxytocin and M1 muscarinic) and Gαi-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50–100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCβ3. Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCβ3 was stimulated directly by Gαq or Gβγ in overexpression assays. PKC phosphorylated PLCβ3 at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCβ3 and site-directed mutagenesis identified Ser1105 as the predominant phosphorylation site. Ser1105 is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023–18027). Similar to PKA, the inhibition by PKC of Gαq-stimulated PLCβ3 activity was completely abolished by mutation of Ser1105 to Ala. In contrast, mutation of Ser1105 or Ser26, another putative phosphorylation target, to Ala had no effect on inhibition of Gβγ-stimulated PLCβ3activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Gαq-stimulated PLCβ3 as a result of phosphorylation of Ser1105. Moreover, PKC and PKA both inhibit Gβγ-stimulated activity by mechanisms that do not involve Ser1105. Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCβ) pathway. In turn, phosphorylation of PLCβ by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca2+ release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Gαq-coupled (oxytocin and M1 muscarinic) and Gαi-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50–100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCβ3. Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCβ3 was stimulated directly by Gαq or Gβγ in overexpression assays. PKC phosphorylated PLCβ3 at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCβ3 and site-directed mutagenesis identified Ser1105 as the predominant phosphorylation site. Ser1105 is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023–18027). Similar to PKA, the inhibition by PKC of Gαq-stimulated PLCβ3 activity was completely abolished by mutation of Ser1105 to Ala. In contrast, mutation of Ser1105 or Ser26, another putative phosphorylation target, to Ala had no effect on inhibition of Gβγ-stimulated PLCβ3activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Gαq-stimulated PLCβ3 as a result of phosphorylation of Ser1105. Moreover, PKC and PKA both inhibit Gβγ-stimulated activity by mechanisms that do not involve Ser1105. phospholipase C phosphatidylinositide phosphatidylinositol 1,4,5-trisphosphate protein kinase C cAMP-dependent protein kinase formyl-Met-Leu-Phe phorbol 12-myristate 13-acetate thymelea- toxin Dulbecco's modified Eagle's medium phosphate-buffered saline 8-[4-chlorophenythiol]-cAMP 4-morpholineethanesulfonic acid Stimulation of seven transmembrane receptors coupled to the Gαq or Gαi subunits of heterotrimeric G proteins results in activation of PLCβ1 isoforms that hydrolyze phosphatidylinositol 4,5-bisphosphate to generate the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (1Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (925) Google Scholar, 2Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (350) Google Scholar). IP3 binds to a receptor in endoplasmic reticulum and releases intracellular calcium from its stores. Diacylglycerol, alone or in conjunction with elevated intracellular calcium, activates PKC and initiates additional cellular responses (3Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1471) Google Scholar). Currently, four isoforms of mammalian PLCβ have been identified and characterized (4Alvarez R.A. Ghalayini A.J. Xu P Hardcastle A Bhattacharya S Rao P.N. Pettenati M.J. Anderson R.E. Baehr W. Genomics. 1995; 29: 53-61Crossref PubMed Scopus (26) Google Scholar, 5Bahk Y.Y. Lee Y.H. Lee T.G. Seo J Ryu S.H. Suh P.G. J. Biol. Chem. 1994; 269: 8240-8245Abstract Full Text PDF PubMed Google Scholar, 6Lee C.W. Park D.J. Lee K.H. Kim C.G. Rhee S.G. J. Biol. Chem. 1993; 268: 21318-21327Abstract Full Text PDF PubMed Google Scholar, 7Kim M.J. Bahk Y.Y. Min D.S. Lee S.J. Ryu S.H. Suh P.G. Biochem. Biophys. Res. 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Phosphorylation of PLCβ3 or PLCβ2 by PKA inhibits their activity and establishes a mechanism for cross-talk between Gαq- or Gαi-coupled and Gαs-coupled receptors (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,19Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (190) Google Scholar). The inhibition of G protein-coupled receptor-mediated PI turnover or intracellular calcium release by protein kinase C has been reported (20Woodruff M.L. Chaban V.V. Worley C.M. Dirksen E.R. Am. J. Physiol. 1999; 276: L669-L678PubMed Google Scholar, 21Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Cunningham M.L. Filtz T.M. Harden T.K. Mol. Pharmacol. 1999; 56: 265-271Crossref PubMed Scopus (14) Google Scholar, 23Litosch I. Recept. Signal Transduct. 1996; 6: 87-98PubMed Google Scholar, 24Ryu S.H. Kim U.H. Wahl M.I. Brown A.B. Carpenter G. Huang K.P. Rhee S.G. J. Biol. Chem. 1990; 265: 17941-17945Abstract Full Text PDF PubMed Google Scholar, 25Richardson R.M. Ali H. Tomhave E.D. Haribabu B. Snyderman R. J. Biol. Chem. 1995; 270: 27829-27833Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Protein kinase C is comprised of three subfamilies, the conventional (α, β1, β2, and γ), novel (δ, ε, η, μ, and θ), and atypical (ζ and λ) PKCs (3Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1471) Google Scholar). The conventional and novel PKCs are activated subsequent to the stimulation of Gαq- or Gαi-coupled receptors (3Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1471) Google Scholar, 26Asaoka Y. Nakamura S. Yoshida K. Nishizuka Y. Trends Biochem. Sci. 1992; 17: 414-417Abstract Full Text PDF PubMed Scopus (365) Google Scholar). The inhibition of PI turnover by PKC may present a feedback for determining the frequency and amplitude of signals being transmitted. The mechanisms by which PKC inhibits agonist-stimulated PI turnover have not been well defined. PKC can phosphorylate certain G protein-coupled receptors (platelet-activating factor receptor, C5A receptor) and thereby inhibit PI turnover or intracellular calcium release (reviewed in Ref. 27Ali H. Richardson R.M. Haribabu B. Snyderman R. J. Biol. Chem. 1999; 274: 6027-6030Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). PKC also appears to inhibit agonist-stimulated PI turnover at a post-receptor level (25Richardson R.M. Ali H. Tomhave E.D. Haribabu B. Snyderman R. J. Biol. Chem. 1995; 270: 27829-27833Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 28Phillippe M. Saunders T. Basa A. Am. J. Physiol. 1997; 273: E665-E673PubMed Google Scholar). Although phosphorylation of PLCβ1 and PLCβ2by PKC has been reported (23Litosch I. Recept. Signal Transduct. 1996; 6: 87-98PubMed Google Scholar, 24Ryu S.H. Kim U.H. Wahl M.I. Brown A.B. Carpenter G. Huang K.P. Rhee S.G. J. Biol. Chem. 1990; 265: 17941-17945Abstract Full Text PDF PubMed Google Scholar, 29Filtz T.M. Cunningham M.L. Stanig K.J. Paterson A. Harden T.K. Biochem. J. 1999; 338: 257-264Crossref PubMed Scopus (35) Google Scholar, 30Litosch I. Biochem. J. 1997; 326: 701-707Crossref PubMed Scopus (45) Google Scholar), the physiological relevance of these observations has not been demonstrated. PLCβt, a turkey PLCβ isoform with highest homology to PLCβ2, is phosphorylated by conventional PKCs, and its catalytic activity is inhibited (29Filtz T.M. Cunningham M.L. Stanig K.J. Paterson A. Harden T.K. Biochem. J. 1999; 338: 257-264Crossref PubMed Scopus (35) Google Scholar). PLCβ3 is not phosphorylated by PKCαin vitro (23Litosch I. Recept. Signal Transduct. 1996; 6: 87-98PubMed Google Scholar). Nonetheless, a correlation between PLCβ3 phosphorylation and PKC inhibition of receptor-initiated PI turnover has been reported (21Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 31Strassheim D. Law P.Y. Loh H.H. Mol. Pharmacol. 1998; 53: 1047-1053PubMed Google Scholar). To determine the importance of PLCβ3 phosphorylation by PKC, we have identified the phosphorylation site on PLCβ3and investigated which PKC subfamily can catalyze the phosphorylation. We report the identification of Ser1105 as the predominant PKC phosphorylation site, the involvement of conventional PKCs in this phosphorylation, and the convergence of PKC and PKA on phosphorylation and inhibition of PLCβ3 by Gαq. Furthermore, we find that Gβγ-stimulated PLCβ3activity is inhibited by both PKC and PKA by mechanisms that do not involve Ser1105 phosphorylation. Thymeleatoxin (Tx), Gö 6976, PKC catalytic fragment, PKCβ1, and PKCγ were obtained from Calbiochem. H-89 was purchased from Seikagaku America, Inc. (Rockville MD). PMA, CPT-cAMP (8-[4-chlorophenythiol]-cAMP), and other chemicals were purchased from Sigma. Lys C was obtained from Wako Bioproducts (Richmond, VA). Modified sequence grade trypsin, GeneEditor site-directed mutagenesis kit, and the gel drying film were purchased from Promega (Madison, WI). LipofectAMINE, Dulbecco's modified Eagle's medium (DMEM), phosphate-free DMEM, and all other cell culture reagents were obtained from Life Technologies, Inc. [3H]Inositol (22 Ci/mmol) was obtained from American Radiolabeled Chemical Co. (St. Louis, MO), [32P]orthophosphate (5 mCi/ml) and γ-[32P]ATP (3000 Ci/mmol) were from Amersham Pharmacia Biotech. The RBL-2H3 cell line stably expressing fMLP receptor and fMLP were provided by Dr. D. Haviland, University of Texas, Houston. The plasmid encoding PKA catalytic subunit was provided by Dr. G. S. McKnight, Washington University (Seattle, WA). PLCβ3, Gαq, Gβ1, and Gγ2 plasmids were constructed as described elsewhere (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 32Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-52301Crossref PubMed Scopus (173) Google Scholar). Site-directed mutation of Ser26 to Ala was achieved with the mutagenic primer (5′-CGGCGCGGGGCTAAGTTCATCAAATGG-3′) identically as described for the Ser1105 → Ala mutation (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) using GeneEditor. All plasmid sequences were confirmed by DNA sequencing. Construction of baculovirus containing PLCβ3 Ser1105 → Ala(His)6 and purification of the recombinant protein from Sf9 cells were carried out as described for PLCβ3(His)6 (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). For in vivo phosphorylation, COSM6 cells seeded in 6-well plates were transfected with PLCβ3(His)6 plasmid and metabolically labeled with [32P] ortho-phosphate (0.10 mCi) in 0.5 ml of phosphate-free DMEM for 90 min. After PMA (1 μm) treatment for 30 min, cells were lysed in 500 μl of M-PER lysis buffer (Pierce) containing a mixture of protease and phosphatase inhibitors (21Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and centrifuged at 15,000 × g for 5 min at 4 °C. Phosphorylated PLCβ3(His)6was isolated with nickel-nitrilotriacetic acid resin, separated on a 7.5% SDS-polyacrylamide gel, stained with Coomassie Blue, and analyzed by autoradiography. In vitro phosphorylation by PKC was carried out according to protocols provided by the vendor. Briefly, 0.8 μmpurified recombinant PLCβ3(His)6 or PLCβ3Ser1105 → Ala(His)6was incubated with purified constitutively active PKC fragment (0.04 μm) in the presence of 2.5 μCi of [γ-32P]ATP and 100 μm ATP in a total volume of 10 μl of PKC buffer (50 mm MES, pH 6.5, 1.25 mm EGTA, 12.5 mm MgCl2) for the times specified at 30 °C. Equal amounts of PLCβ3(His)6 were also incubated for 40 min with purified PKCβ1 or PKCγ (20 ng) in a total volume of 10 μl of reaction buffer (20 mm HEPES, pH 7.4, 100 μm CaCl2, 10 mmMgCl2, 100 μg/ml phosphatidylserine, 20 μg/ml diacylglycerol, 0.03% Triton X-100). Reactions were terminated by adding 10 μl of 2× SDS sample buffer and boiling for 5 min. Proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue. The phosphorylated bands were localized by autoradiography. The stoichiometry of PLCβ3phosphorylation by PKC was determined at 100 min by filter binding assay as described elsewhere (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). For two-dimensional tryptic peptide mapping and phosphoamino acid analysis, 32P-labeled PLCβ3from in vivo or in vitro phosphorylation reactions was separated by SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Blue, dried between two layers of drying membranes, and exposed to Biomax-MS x-ray film (Eastman Kodak Co.). PLCβ3 bands were cut out and rehydrated in 50 mm ammonium bicarbonate, pH 8 (buffer A), overnight. After peeling off the drying membrane, each gel slice was boiled for 5 min in 100 μl of buffer A containing 5 mm dithiothreitol. The tube was cooled to room temperature, 50 μl of 100 mmiodoacetic acid was added, and the tube was incubated for 30 min in the dark at room temperature. The gel slice was washed again in buffer A and ground with a disposable pestle. The residual Coomassie Blue dye was removed by rinsing the gel slurry with 50 μl of 50% acetonitrile in buffer A. The tube was centrifuged at 15,000 × gfor 5 min, and the supernatant was discarded. The pellet was resuspended in 50 μl of acetonitrile and incubated for 5 min. The tube was centrifuged again, and the pellet was dried in a SpeedVac for 10 min after removal of supernatant. The pellet was resuspended in 75 μl of buffer A, and 2 μg of trypsin was added. The tube was incubated at 37 °C for 5 h before the addition of another 2 μg of trypsin, and the total incubation time was between 18 and 24 h. The liquid containing the digested peptides was recovered and further prepared for two-dimensional peptide mapping with a Hunter thin layer electrophoresis system (C.B.S. Scientific Co., Del Mar, CA) according to the protocol provided by the manufacturer. External markers for each dimension were included in each thin layer plate to facilitate the comparison between samples. For phosphoamino acid analysis, about 100 cpm of total tryptic peptides mixture was used. Peptide sequencing using 32P-labeled PLCβ3(His)6 (150 pmol) recombinant protein purified from Sf9 cells was carried out as described elsewhere (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). HeLa, COSM6, and RBL-2H3 cells were cultured as described for PHM1-41 cells (33Monga M. Ku C.Y. Dodge K.L. Sanborn B.M. Biol. Reprod. 1996; 55: 427-432Crossref PubMed Scopus (58) Google Scholar). HeLa and COSM6 cells (1.8 × 105/well) were seeded in 6-well plates and transfected 16–24 h later as described (34Dodge K.L. Sanborn B.M. Endocrinology. 1998; 139: 2265-2271Crossref PubMed Google Scholar) with M1 receptor (1 μg), Gαq (0.5 μg), Gβ1(0.375 μg), Gγ2 (0.375 μg), and PLCβ3(0.25 μg) as indicated. Empty rcCMV vector was added to bring the total amount of plasmid DNA to 1.25 μg per well. For effects of endogenous PKC on agonist-stimulated PI turnover, near confluent PHM1 and RBL-2H3 cells (12-well plates) and COSM6 and HeLa cells (6-well plates) were treated with 1 μm PMA or 100 ng/ml thymeleatoxin for 30 min in PBS+ (phosphate-buffered saline (PBS) plus 1.2 mm Ca2+, 1.0 mmMg2+, and 1.0 mm glucose) containing 10 mm LiCl prior to stimulation by agonists (100 nm oxytocin, 15 μm carbachol, or 100 nm fMLP) for 30 min. Where indicated, H-89 (10 μm) or Gö 6976 (8 μm) were added to PHM1-41 cells. After 15 min, PMA or CPT-cAMP were added, followed by oxytocin 15 min later. For direct stimulation of PLCβ3 by Gαq or Gβ1γ2, transfected COSM6 cells were first treated with 1 μm PMA for 30 min in PBS+ followed by addition of 20 mm LiCl for 30 min. Cells were lysed, and total IPs were determined as described (19Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (190) Google Scholar). The effect of activation of endogenous PKC on predominantly Gαq-coupled oxytocin receptor-initiated PI turnover (35Ku C.Y. Qian A. Wen Y. Anwer K. Sanborn B.M. Endocrinology. 1995; 136: 1509-1515Crossref PubMed Google Scholar) was studied in PHM1-41 cells, a human myometrial smooth muscle cell line (33Monga M. Ku C.Y. Dodge K.L. Sanborn B.M. Biol. Reprod. 1996; 55: 427-432Crossref PubMed Scopus (58) Google Scholar). Stimulation of PHM1 cells with 100 nm oxytocin significantly increased the production of total IPs. Pretreating cells with 1 μm PMA completely inhibited this increase (Fig. 1 A). The PMA effect was not specific to the oxytocin receptor or to PHM1-41 cells. A similar inhibitory effect of PMA was also evident with Gαq-coupled M1 muscarinic receptor transfected into HeLa (Fig. 1 B) or COSM6 (Fig. 1 C) cells. In addition, PMA also significantly inhibited Gαi-coupled fMLP receptor-initiated PI turnover (36Jiang H. Kuang Y. Wu Y. Smrcka A. Simon M.I. Wu D. J. Biol. Chem. 1996; 271: 13430-13434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) in RBL-2H3 cells (Fig.1 D) in which the only PLCβ form expressed is PLCβ3 (21Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This occurred under conditions where the fMLP receptor has been shown not to be phosphorylated by PKC (37Richardson R.M. Ali H. Pridgen B.C. Haribabu B. Snyderman R. J. Biol. Chem. 1998; 273: 10690-10695Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These observations, together with those previously reported (21Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 31Strassheim D. Law P.Y. Loh H.H. Mol. Pharmacol. 1998; 53: 1047-1053PubMed Google Scholar), establish that the PKC inhibitory effect on G protein-coupled receptor-initiated PI turnover is a general mechanism and that the PKC effect can occur at a post-receptor level. To investigate the potential role of specific PKCs in this process, the effect of Tx, a specific activator of conventional PKCs (38Ryves W.J. Evans A.T. Olivier A.R. Parker P.J. Evans F.J. FEBS Lett. 1991; 288: 5-9Crossref PubMed Scopus (176) Google Scholar), was compared with PMA, which activates both conventional and novel PKCs (38Ryves W.J. Evans A.T. Olivier A.R. Parker P.J. Evans F.J. FEBS Lett. 1991; 288: 5-9Crossref PubMed Scopus (176) Google Scholar), in PHM1-41 and RBL-2H3 cell lines. In both cases, Tx was as effective as PMA in inhibiting oxytocin or fMLP-stimulated PI turnover at the concentration tested (Fig. 1, A and D). In addition, Gö 6976, an inhibitor of conventional PKC (39Qatsha K.A. Rudolph C. Marme D. Schachtele C. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4674-4678Crossref PubMed Scopus (136) Google Scholar), was able to reverse the PMA inhibitory effect by ∼50% at a concentration of 4 μm (data not shown). These data provide evidence that conventional PKCs are capable of inhibiting Gαq- or Gαi-coupled receptor-initiated PI turnover. Because PLCβ3 is present in all four cell lines mentioned above and can be phosphorylated by PKC, at least in RBL-2H3 cells (21Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), it is highly possible that PKC inhibits PI turnover by decreasing PLCβ3 activity. If so, PKC should inhibit the direct stimulation of PLCβ3 by Gαq or Gβγ. COSM6 cells transfected with both PLCβ3 and Gαq plasmids exhibited a marked increase in total [3H]IPs compared with transfection with either plasmid alone (Fig. 2 A). Consistent with the prediction, pretreating cells with PMA nearly abolished Gαq-stimulated PLCβ3 activity. Tx elicited a similar inhibitory effect on Gαq-stimulated PLCβ3 activity (data not shown). Cotransfection of Gβ1γ2 and PLCβ3 into COSM6 cells also resulted in marked increase in PI turnover. This increase was significantly reduced by PMA (Fig. 2 B), but the reduction was not of the magnitude observed for Gαq-stimulated PLCβ3. Thus PKC inhibition of PI turnover occurs at a post-receptor level, and this effect may require the phosphorylation of PLCβ3. PLCβ3 overexpressed in COSM6 cells exhibited significant 32P incorporation under basal conditions. Nonetheless, PMA induced a substantial increase in 32P incorporation into PLCβ3 (Fig.3 A). The phosphorylation of PLCβ3 by PKC was investigated further in vitro. Purified recombinant PLCβ3 was incubated with catalytically active PKC fragments (a rat brain mixture of multiple PKC isoforms, including α, β, and γ) in the presence of [γ-32P]ATP. As shown in Fig. 3 B, PLCβ3 was phosphorylated in a time-dependent manner. A stoichiometry of 0.4 mol of phosphate/PLCβ3 was achieved after incubation with PKC for 100 min under these conditions. In similar experiments, no phosphorylation was seen in the absence of PKC (data not shown). Purified PKCβ1 or PKCγ also phosphorylated PLCβ3 in vitro, whereas no phosphorylation of PLCβ3 was observed in the absence of kinase (Fig. 3 C). As shown by two-dimensional phosphopeptide mapping of in vivo 32P-labeled PLCβ3, trypsin digestion yielded multiple phosphopeptides in the basal state (Fig.4 A). PMA specifically induced phosphorylation on one predominant site (Fig. 4 B, indicated by the arrow). Minor sites increased by PMA were also present (indicated by arrowhead). We cannot exclude the contribution of incomplete digestion by trypsin to this pattern. In vitro, PKC phosphorylated PLCβ3 on one predominant site (Fig. 4 C, arrow). The migration of this peptide relative to the standards was identical to those observed in digests after in vivo phosphorylation. The phosphorylation occurred exclusively on serine residues (Fig.4 D). We utilized in vitro phosphorylated recombinant PLCβ3(His)6 to identify the PKC phosphorylation sites. After isolation by SDS-polyacrylamide gel electrophoresis, 32P-labeled PLCβ3 was digested with Lys C instead of trypsin to achieve more complete cleavage and fewer peptides (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The digestion mixture was separated by reverse-phase high pressure liquid chromatography, and fractions were recovered and counted. Fig.5 A shows the 32P distribution among these fractions. About 8% of 32P was found in the follow-through (fraction −1 to −4) and appeared to be free 32P as judged by phosphopeptide mapping (data not shown). Nearly 60% of the total 32P was recovered in fraction 12. This fraction was subjected to peptide sequencing. Although two peptides were identified in this fraction, nearly 90% of the total 32P was found in the fourth cycle (Fig.5 B). This clearly identified Ser1105 and not Ser1107 in the peptide Arg-His-Asn-Ser1105-Ile-Ser-Glu-Ala-Lys as the amino acid phosphorylated. Furthermore, mutation of Ser1105significantly diminished PLCβ3 phosphorylation by PKC in vitro (Fig. 5 C). This strongly argues that Ser1105 is the predominant site for PKC. Residual weak phosphorylation associated with Ser1105 → Ala mutant PLCβ3 could indicate the presence of other minor sites. Interestingly, Ser1105, unique to PLCβ3 among the PLCβ isoforms, is preferentially phosphorylated by PKA as well (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We have previously shown that phosphorylation by PKA of Ser1105 is required for inhibition of Gαq-stimulated PLCβ3 activity by PKA. The finding that PKC also phosphorylates Ser1105 suggested that the same mechanism was utilized by PKC. To test this hypothesis, the Ser1105 → Ala mutant PLCβ3 was cotransfected with Gαq into COSM6 cells, and the effect of PMA was evaluated. As shown before (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), the Ser1105 → Ala mutant PLCβ3 was as effective as the wild type enzyme in coupling to Gαq (Fig.6). Importantly, PMA inhibited Gαq-stimulated wild type PLCβ3 activity but had no effect on Gαq-stimulated Ser1105 → Ala PLCβ3 activity. These data unequivocally identify phosphorylation of Ser1105 by PKC as responsible for PKC inhibition of Gαq-stimulated PLCβ3activity. We also investigated the effect of mutating Ser1105 on PKC inhibition of Gβγ-stimulated PLCβ3 activity. The Ser1105 → Ala mutant PLCβ3 was as effective as wild type PLCβ3 in coupling to Gβ1γ2 (Fig.7 A). However in contrast to Gαq-stimulated PLCβ3 activity, PKC inhibited Gβ1γ2-stimulated Ser1105 → Ala mutant PLCβ3 activity to the similar degree as it did the wild type PLCβ3. Thus Ser1105 is not absolutely required for PKC inhibition of Gβ1γ2-stimulated PLCβ3activity. The N-terminal region of PLCβ3 appears to contribute to its interaction with Gβγ (40Bell R.M. Burns D.J. J. Biol. Chem. 1991; 266: 4661-4664Abstract Full Text PDF PubMed Google Scholar). We had identified Ser26 in the peptide Arg-Arg-Gly-Ser-Lys as a potential phosphorylation site in this region. However, there was no effect of mutating Ser26 to Ala on PKC inhibition of Gβγ-stimulated PLCβ3 activity (Fig.7 A). In the face of the inability of mutation of Ser1105 and Ser26 to reverse the effect of PKC on Gβγ-stimulated PLCβ3 activity, we examined the effect of mutation of these residues on PKA-mediated inhibition as well. As seen in Fig.7 B, PKA inhibited Gβγ-stimulated PLCβ3activity. Mutation of Ser1105 or Ser26 also had no effect on inhibition by PKA of Gβγ-stimulated PLCβ3 activity. Phosphoryation of Ser1105 by PKC or PKA suppressed Gαq-stimulated PLCβ3 activity. This fact raised the interesting possibility that PKC activation might lead to PKA activation, resulting in indirect phosphorylation of PLCβ3 at the PKA site or vice versa. We addressed this possibility in PHM1-41 cells. As shown in Fig.8, H-89, a specific PKA inhibitor, reversed the inhibition by cAMP but did not affect the inhibition by PMA of oxytocin-stimulated PI turnover. Similarly, Gö 6976, a specific PKC inhibitor, significantly diminished the inhibitory effect of PMA but not of cAMP on oxytocin-stimulated PI turnover. These data indicate that PKC and PKA exert their inhibitory effects independent of each other. We have presented evidence that PKC inhibits Gαq-coupled (oxytocin and M1 muscarinic) and Gαi-coupled receptor (fMLP) receptor-initiated PI turnover in four different cell lines expressing PLCβ3. The response to endogenous PKC activation by PMA differs in order of magnitude between cell lines and the state of the receptor (endogenous or transfected). This variation may reflect differences in relative membrane permeability of PMA and the localization and abundance of the PKC isoforms responsible or the relative contribution of Gαq-coupling to PLCβ3 to PI turnover. We have also demonstrated in cotransfection assays that the PKC inhibitory effect occurred at the G protein-PLCβ3 level, and we have provided direct evidence to support the hypothesis that phosphorylation of PLCβ3 is involved in the PKC inhibitory effect on Gαq-coupled activation. The use of in vitro phosphorylated PLCβ3 for identifying the PKC phosphorylation site is supported by the demonstration that a similar site was phosphorylated by PKC in vivo and in vitro. PKC phosphorylates predominantly one residue, Ser1105, that is also phosphorylated by PKA (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The marked reduction of in vitro phosphorylation of the Ser1105 → Ala PLCβ3 mutant further corroborates this finding. However, the remaining weak phosphorylation associated with this mutant indicates that PKC may phosphorylate other minor sites as well. Mutation of Ser1105 to Ala reversed completely the inhibition of Gαq-stimulated PLCβ3 activity by PKC. This provides conclusive evidence for the direct inhibition of PLCβ3 by PKC, a response identical to that seen previously for PKA (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We also demonstrated that the inhibitory effect of PKC occurs in the absence of PKA inhibition, suggesting that it is not a consequence of indirect PKA activation. The convergence of PKC and PKA on Ser1105 underscores the importance of Ser1105 in the regulation of Gαq-stimulated PLCβ3 activity in diverse cellular processes and suggests possible redundancy for the inhibition of PLCβ3 activity by these two kinases. In addition, these data also argue that the effect of PKC or PKA targets PLCβ3 and not G protein or proteins involved in the production of substrate phosphatidylinositol 4,5-bisphosphate, as mutation of Ser1105 can completely reverse the inhibition by PKC or PKA of Gαq-stimulated PLCβ3 activity. In marked contrast, Ser1105 does not appear to be critical for inhibition of Gβγ-stimulated PLCβ3 activity by either PKC or PKA. Ser26 was also not required, although the N-terminal region of PLCβ3 appears to contribute to its interaction with Gβγ (40Bell R.M. Burns D.J. J. Biol. Chem. 1991; 266: 4661-4664Abstract Full Text PDF PubMed Google Scholar). At present the mechanism for the inhibition of Gβγ-stimulated PLCβ3 activity by PKC or PKA remains unknown. It is unlikely that Gβ1γ2 is the direct target for the inhibitory effects of PKA or PKC as these proteins are not phosphorylated by PKC or PKA in vitro. 2C. Yue and B. M. Sanborn, unpublished observations. Identification of PKA or PKC minor phosphorylation sites may help to solve this question. Alternatively, the mechanism may involve phosphorylation of other molecules indirectly involved in the coupling (12Yue C. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The effects of a conventional PKC-specific activator and an inhibitor indicate that conventional PKCs are capable of phosphorylating PLCβ3. This conclusion is supported by in vitro phosphorylation of PLCβ3 by the constitutively active PKC fragment and by purified PKCβ1 and PKCγ. The wide distribution of conventional PKCs (26Asaoka Y. Nakamura S. Yoshida K. Nishizuka Y. Trends Biochem. Sci. 1992; 17: 414-417Abstract Full Text PDF PubMed Scopus (365) Google Scholar) and PLCβ3 (2Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (350) Google Scholar) in tissues correlates well with the generality of the PKC inhibitory effect on receptor-initiated PI turnover. We conclude that conventional PKCs phosphorylate PLCβ3and inhibit Gαq- and Gβγ-stimulated PLCβ3 activity. PKC and PKA act similarly in that they inhibit Gαq-stimulated PLCβ3 as a result of phosphorylation of Ser1105. Moreover, PKA and PKC both inhibit Gβγ-stimulated activity by mechanisms that do not involve Ser1105. We thank Dr. S. McKnight and Dr. D. Haviland for providing valuable experimental materials.
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