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J. Biol. Chem., Vol. 275, Issue 39, 30220-30225, September 29, 2000

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Molecular Mechanism of the Inhibition of Phospholipase C ₃ by Protein Kinase C^*

Caiping Yue, Chun-Ying Ku, Mingyao Liu^§, Melvin I. Simon^¶, and Barbara M. Sanborn

From the  Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225, the ^§ Department of Medical Biochemistry and Genetics, Center for Cancer Biology and Nutrition, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, and the ^¶ Department of Biology, California Institute of Technology, Pasadena, California 91125

Received for publication, May 18, 2000, and in revised form, June 23, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLC) pathway. In turn, phosphorylation of PLC 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 G_q-coupled (oxytocin and M1 muscarinic) and G_i-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLC₃. 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 PLC₃ was stimulated directly by G_q or G in overexpression assays. PKC phosphorylated PLC₃ at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLC₃ 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 G_q-stimulated PLC₃ 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 G-stimulated PLC₃ activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit G_q-stimulated PLC₃ as a result of phosphorylation of Ser¹¹⁰⁵. Moreover, PKC and PKA both inhibit G-stimulated activity by mechanisms that do not involve Ser¹¹⁰⁵.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulation of seven transmembrane receptors coupled to the G_q or G_i subunits of heterotrimeric G proteins results in activation of PLC¹ isoforms that hydrolyze phosphatidylinositol 4,5-bisphosphate to generate the second messengers inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (1, 2). 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 (3). Currently, four isoforms of mammalian PLC have been identified and characterized (4-10). Significantly, PLC₃ is ubiquitously expressed and activated by all known PLC activators (G_q, G, and calcium) (2). Regulation of PLC₃ may be of great importance in many cellular processes (11-15). Insufficient expression of PLC₃ has been correlated with increased sensitivity to tumor formation (15, 16), whereas overexpression of PLC₃ seems to suppress tumor growth (17). PLC₃ knockout mice exhibit altered response to µ-opioids (11) or early embryonic lethality (18).

Phosphorylation appears to play an important role in regulating the activity of PLC isoforms. Phosphorylation of PLC₃ or PLC₂ by PKA inhibits their activity and establishes a mechanism for cross-talk between G_q- or G_i-coupled and G_s-coupled receptors (12, 19). The inhibition of G protein-coupled receptor-mediated PI turnover or intracellular calcium release by protein kinase C has been reported (20-25). Protein kinase C is comprised of three subfamilies, the conventional (, ₁, ₂, and ), novel (, , , µ, and ), and atypical ( and ) PKCs (3). The conventional and novel PKCs are activated subsequent to the stimulation of G_q- or G_i-coupled receptors (3, 26). 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. 27). PKC also appears to inhibit agonist-stimulated PI turnover at a post-receptor level (25, 28). Although phosphorylation of PLC₁ and PLC₂ by PKC has been reported (23, 24, 29, 30), the physiological relevance of these observations has not been demonstrated. PLC_t, a turkey PLC isoform with highest homology to PLC₂, is phosphorylated by conventional PKCs, and its catalytic activity is inhibited (29). PLC₃ is not phosphorylated by PKC in vitro (23). Nonetheless, a correlation between PLC₃ phosphorylation and PKC inhibition of receptor-initiated PI turnover has been reported (21, 31).

To determine the importance of PLC₃ phosphorylation by PKC, we have identified the phosphorylation site on PLC₃ and 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 PLC₃ by G_q. Furthermore, we find that G-stimulated PLC₃ activity is inhibited by both PKC and PKA by mechanisms that do not involve Ser¹¹⁰⁵ phosphorylation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Thymeleatoxin (Tx), Gö 6976, PKC catalytic fragment, PKC₁, and PKC 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 -[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).

Cloning, Site-directed Mutagenesis, and Protein Purification-- PLC₃, G_q, G₁, and G₂ plasmids were constructed as described elsewhere (12, 32). Site-directed mutation of Ser²⁶ to Ala was achieved with the mutagenic primer (5'-CGGCGCGGGGCTAAGTTCATCAAATGG-3') identically as described for the Ser¹¹⁰⁵  Ala mutation (12) using GeneEditor. All plasmid sequences were confirmed by DNA sequencing. Construction of baculovirus containing PLC₃ Ser¹¹⁰⁵  Ala(His)₆ and purification of the recombinant protein from Sf9 cells were carried out as described for PLC₃ (His)₆ (12).

In Vivo and in Vitro ³²P Labeling and Isolation of PLC₃-- For in vivo phosphorylation, COSM6 cells seeded in 6-well plates were transfected with PLC₃(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 (21) and centrifuged at 15,000 × g for 5 min at 4 °C. Phosphorylated PLC₃(His)₆ was 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 µM purified recombinant PLC₃(His)₆ or PLC₃Ser¹¹⁰⁵  Ala(His)₆ was incubated with purified constitutively active PKC fragment (0.04 µM) in the presence of 2.5 µCi of [-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 PLC₃(His)₆ were also incubated for 40 min with purified PKC₁ or PKC (20 ng) in a total volume of 10 µl of reaction buffer (20 mM HEPES, pH 7.4, 100 µM CaCl₂, 10 mM MgCl₂, 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 PLC₃ phosphorylation by PKC was determined at 100 min by filter binding assay as described elsewhere (12).

Phosphoamino Acid Analysis, Peptide Mapping, and Sequencing-- For two-dimensional tryptic peptide mapping and phosphoamino acid analysis, ³²P-labeled PLC₃ from 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.). PLC₃ 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 mM iodoacetic 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 × g for 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 PLC₃(His)₆ (150 pmol) recombinant protein purified from Sf9 cells was carried out as described elsewhere (12).

Cell Culture, Transfection, and PI Turnover-- HeLa, COSM6, and RBL-2H3 cells were cultured as described for PHM1-41 cells (33). HeLa and COSM6 cells (1.8 × 10⁵/well) were seeded in 6-well plates and transfected 16-24 h later as described (34) with M1 receptor (1 µg), G_q (0.5 µg), G₁ (0.375 µg), G₂ (0.375 µg), and PLC₃ (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 mM Mg²⁺, 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 PLC₃ by G_q or G₁₂, 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 (19).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PKC Inhibits Oxytocin, M1 Muscarinic, and fMLP Receptor-initiated PI Turnover-- The effect of activation of endogenous PKC on predominantly G_q-coupled oxytocin receptor-initiated PI turnover (35) was studied in PHM1-41 cells, a human myometrial smooth muscle cell line (33). 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. 1A). 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 G_q-coupled M1 muscarinic receptor transfected into HeLa (Fig. 1B) or COSM6 (Fig. 1C) cells. In addition, PMA also significantly inhibited G_i-coupled fMLP receptor-initiated PI turnover (36) in RBL-2H3 cells (Fig. 1D) in which the only PLC form expressed is PLC₃ (21). This occurred under conditions where the fMLP receptor has been shown not to be phosphorylated by PKC (37). These observations, together with those previously reported (21, 31), 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.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Prior treatment with PMA or Tx inhibits oxytocin (OT), carbachol, or fMLP-stimulated total IP production in PHM1-41 (A), HeLa (B), COSM6 (C) or RBL-2H3 (D) cells, respectively. HeLa and COSM6 cells were transfected with (M1R) or without (Vector) a plasmid expressing M1 receptor and were stimulated with 15 µM carbachol. Where indicated, PBS was used as control reagent. Data are presented as the means ± S.E. (n = 3) of 1 of 2-4 similar experiments and were analyzed by analysis of variance and Duncan's test. Groups with different letters are different from each other at p < 0.05.

To investigate the potential role of specific PKCs in this process, the effect of Tx, a specific activator of conventional PKCs (38), was compared with PMA, which activates both conventional and novel PKCs (38), 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 (39), 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 G_q- or G_i-coupled receptor-initiated PI turnover.

PKC Inhibits the Direct Stimulation of PLC₃ by G_q and G-- Because PLC₃ is present in all four cell lines mentioned above and can be phosphorylated by PKC, at least in RBL-2H3 cells (21), it is highly possible that PKC inhibits PI turnover by decreasing PLC₃ activity. If so, PKC should inhibit the direct stimulation of PLC₃ by G_q or G. COSM6 cells transfected with both PLC₃ and G_q plasmids exhibited a marked increase in total [³H]IPs compared with transfection with either plasmid alone (Fig. 2A). Consistent with the prediction, pretreating cells with PMA nearly abolished G_q-stimulated PLC₃ activity. Tx elicited a similar inhibitory effect on G_q-stimulated PLC₃ activity (data not shown). Cotransfection of G₁₂ and PLC₃ into COSM6 cells also resulted in marked increase in PI turnover. This increase was significantly reduced by PMA (Fig. 2B), but the reduction was not of the magnitude observed for G_q-stimulated PLC₃. Thus PKC inhibition of PI turnover occurs at a post-receptor level, and this effect may require the phosphorylation of PLC₃.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   PMA inhibits Gq-stimulated PLC3 (A) and G-stimulated PLC3 (B) activity in COSM6 cells transfected with plasmids expressing Gq, G, and PLC3. Data are presented as the means ± S.E. (n = 3) of 1 of 3 similar experiments and were analyzed by analysis of variance and Duncan's test. Groups with different letters are different from each other at p < 0.05.

Phosphorylation of PLC₃ by PKC in Vivo and in Vitro-- PLC₃ overexpressed in COSM6 cells exhibited significant ³²P incorporation under basal conditions. Nonetheless, PMA induced a substantial increase in ³²P incorporation into PLC₃ (Fig. 3A). The phosphorylation of PLC₃ by PKC was investigated further in vitro. Purified recombinant PLC₃ was incubated with catalytically active PKC fragments (a rat brain mixture of multiple PKC isoforms, including , , and ) in the presence of [-³²P]ATP. As shown in Fig. 3B, PLC₃ was phosphorylated in a time-dependent manner. A stoichiometry of 0.4 mol of phosphate/PLC₃ 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 PKC₁ or PKC also phosphorylated PLC₃ in vitro, whereas no phosphorylation of PLC₃ was observed in the absence of kinase (Fig. 3C).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   A, in vivo 32P labeling of PLC3(His)6 isolated from COSM6 cells transfected with PLC3(His)6 plasmid and pretreated with (PMA) or without (control) PMA. B, time-dependent phosphorylation of PLC3(His)6 by PKC in vitro. Reactions were terminated at the times indicated in minutes. Coomassie Blue staining of the respective gels is shown below the autoradiographs. C, autoradiography of PLC3(His)6 after incubation without () or with (+) purified PKC1 and PKC in vitro.

Ser¹¹⁰⁵ Is the Predominant Phosphorylation Site for PKC-- As shown by two-dimensional phosphopeptide mapping of in vivo ³²P-labeled PLC₃, trypsin digestion yielded multiple phosphopeptides in the basal state (Fig. 4A). PMA specifically induced phosphorylation on one predominant site (Fig. 4B, 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.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Two-dimensional tryptic peptide mapping of PLC3(His)6 32P-labeled in vivo (A and B) or in vitro (C). Two markers were applied on each TLC plate as migration controls for each dimension. The black markers on the top of each panel indicate the position of one such marker; others outside of the displayed region were also used in lining up the plates. "O" depicts the sample origin. The predominant PKC-stimulated phosphorylation site is indicated by the arrows (B and C) and the minor sites by the arrowheads. Longer exposure of C revealed some minor sites as well. D, two-dimensional phosphoamino acid analysis of PLC3(His)6 phosphorylated by PKC in vitro. The dotted circles indicate the migration positions of phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) standards.

In vitro, PKC phosphorylated PLC₃ on one predominant site (Fig. 4C, 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. 4D). We utilized in vitro phosphorylated recombinant PLC₃(His)₆ to identify the PKC phosphorylation sites. After isolation by SDS-polyacrylamide gel electrophoresis, ³²P-labeled PLC₃ was digested with Lys C instead of trypsin to achieve more complete cleavage and fewer peptides (12). The digestion mixture was separated by reverse-phase high pressure liquid chromatography, and fractions were recovered and counted. Fig. 5A 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. 5B). 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 Ser¹¹⁰⁵ significantly diminished PLC₃ phosphorylation by PKC in vitro (Fig. 5C). This strongly argues that Ser¹¹⁰⁵ is the predominant site for PKC. Residual weak phosphorylation associated with Ser¹¹⁰⁵  Ala mutant PLC₃ could indicate the presence of other minor sites. Interestingly, Ser¹¹⁰⁵, unique to PLC₃ among the PLC isoforms, is preferentially phosphorylated by PKA as well (12).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   A, 32P distribution among fractions collected after reverse-phase high pressure liquid chromatography separation of Lys C-digested PLC3(His)6 labeled with 32P in vitro. Fraction 12 has ~60% of the total 32P. B, sequence of peptides in fraction 12 and associated 32P. The serine residue with more than 90% of total 32P loaded onto the sequencing membrane is denoted by *. filter represents 32P left on the sequencing membrane after 10 cycles. C, in vitro phosphorylation by PKC (30 min at 30 °C) of recombinant wild type (WT) or Ser1105  Ala mutant (S/A) PLC3(His)6 purified from Sf9 cells. The Coomassie Blue staining (Coomassie) and autoradiography (autorad) of the same gel are shown.

Functional Analysis of Phosphorylation of PLC₃ by PKC Versus PKA-- We have previously shown that phosphorylation by PKA of Ser¹¹⁰⁵ is required for inhibition of G_q-stimulated PLC₃ 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 PLC₃ was cotransfected with G_q into COSM6 cells, and the effect of PMA was evaluated. As shown before (12), the Ser¹¹⁰⁵  Ala mutant PLC₃ was as effective as the wild type enzyme in coupling to G_q (Fig. 6). Importantly, PMA inhibited G_q-stimulated wild type PLC₃ activity but had no effect on G_q-stimulated Ser¹¹⁰⁵  Ala PLC₃ activity. These data unequivocally identify phosphorylation of Ser¹¹⁰⁵ by PKC as responsible for PKC inhibition of G_q-stimulated PLC₃ activity.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Mutation of Ser1105 to Ala (S/A) reversed the inhibition by PKC of Gq-stimulated PLC3 in COSM6 cells transfected with plasmids expressing Gq and PLC3 plasmids. Data are presented as the means ± S.E. (n = 3) of 1 of 3 similar experiments and were analyzed by analysis of variance and Duncan's test. Groups with different letters are different from each other at p < 0.05.

We also investigated the effect of mutating Ser¹¹⁰⁵ on PKC inhibition of G-stimulated PLC₃ activity. The Ser¹¹⁰⁵  Ala mutant PLC₃ was as effective as wild type PLC₃ in coupling to G₁₂ (Fig. 7A). However in contrast to G_q-stimulated PLC₃ activity, PKC inhibited G₁₂-stimulated Ser¹¹⁰⁵  Ala mutant PLC₃ activity to the similar degree as it did the wild type PLC₃. Thus Ser¹¹⁰⁵ is not absolutely required for PKC inhibition of G₁₂-stimulated PLC₃ activity. The N-terminal region of PLC₃ appears to contribute to its interaction with G (40). 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 G-stimulated PLC₃ activity (Fig. 7A).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Inhibition of G-stimulated Ser1105  Ala and Ser26  Ala mutant PLC3 activity by PKC (A) or PKA (B) in COSM6 cells transfected with plasmids expressing G, wild type (WT), Ser1105  Ala or Ser26  Ala mutant PLC3. A, data are presented as the means ± S.E. ( n = 3) of 1 of 3 similar experiments and were analyzed by analysis of variance and Duncan's test. Groups with different letters are different from each other at p < 0.05. B, plasmid encoding the PKA catalytic subunit was cotransfected into COSM6 cells (filled bars), and its expression was induced with 60 µM ZnSO4 for 24 h after transfection. Data represent the mean of duplicate determinations (range denoted by the error bars) in 1 of 2 similar experiments.

In the face of the inability of mutation of Ser¹¹⁰⁵ and Ser²⁶ to reverse the effect of PKC on G-stimulated PLC₃ activity, we examined the effect of mutation of these residues on PKA-mediated inhibition as well. As seen in Fig. 7B, PKA inhibited G-stimulated PLC₃ activity. Mutation of Ser¹¹⁰⁵ or Ser²⁶ also had no effect on inhibition by PKA of G-stimulated PLC₃ activity.

Inhibition of Oxytocin-stimulated Total IP Production in PHM1-41 Cells by PKC or PKA Represents Independent Pathways-- Phosphoryation of Ser¹¹⁰⁵ by PKC or PKA suppressed G_q-stimulated PLC₃ activity. This fact raised the interesting possibility that PKC activation might lead to PKA activation, resulting in indirect phosphorylation of PLC₃ 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.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Inhibition of oxytocin-stimulated total IP production in PHM1-41 cells by PKC or PKA represents independent pathways. Data are presented as the means ± S.E. (n = 3) from 1 of 2 similar experiments and were analyzed by analysis of variance and Duncan's test. Groups with different letters are different from each other at p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have presented evidence that PKC inhibits G_q-coupled (oxytocin and M1 muscarinic) and G_i-coupled receptor (fMLP) receptor-initiated PI turnover in four different cell lines expressing PLC₃. 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 G_q-coupling to PLC₃ to PI turnover. We have also demonstrated in cotransfection assays that the PKC inhibitory effect occurred at the G protein-PLC₃ level, and we have provided direct evidence to support the hypothesis that phosphorylation of PLC₃ is involved in the PKC inhibitory effect on G_q-coupled activation.

The use of in vitro phosphorylated PLC₃ 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 (12). The marked reduction of in vitro phosphorylation of the Ser¹¹⁰⁵  Ala PLC₃ 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 G_q-stimulated PLC₃ activity by PKC. This provides conclusive evidence for the direct inhibition of PLC₃ by PKC, a response identical to that seen previously for PKA (12). 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 G_q-stimulated PLC₃ activity in diverse cellular processes and suggests possible redundancy for the inhibition of PLC₃ activity by these two kinases. In addition, these data also argue that the effect of PKC or PKA targets PLC₃ 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 G_q-stimulated PLC₃ activity.

In marked contrast, Ser¹¹⁰⁵ does not appear to be critical for inhibition of G-stimulated PLC₃ activity by either PKC or PKA. Ser²⁶ was also not required, although the N-terminal region of PLC₃ appears to contribute to its interaction with G (40). At present the mechanism for the inhibition of G-stimulated PLC₃ activity by PKC or PKA remains unknown. It is unlikely that G₁₂ is the direct target for the inhibitory effects of PKA or PKC as these proteins are not phosphorylated by PKC or PKA in vitro.² 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 (12).

The effects of a conventional PKC-specific activator and an inhibitor indicate that conventional PKCs are capable of phosphorylating PLC₃. This conclusion is supported by in vitro phosphorylation of PLC₃ by the constitutively active PKC fragment and by purified PKC₁ and PKC. The wide distribution of conventional PKCs (26) and PLC₃ (2) in tissues correlates well with the generality of the PKC inhibitory effect on receptor-initiated PI turnover.

We conclude that conventional PKCs phosphorylate PLC₃ and inhibit G_q- and G-stimulated PLC₃ activity. PKC and PKA act similarly in that they inhibit G_q-stimulated PLC₃ as a result of phosphorylation of Ser¹¹⁰⁵. Moreover, PKA and PKC both inhibit G-stimulated activity by mechanisms that do not involve Ser¹¹⁰⁵.

	ACKNOWLEDGEMENTS

We thank Dr. S. McKnight and Dr. D. Haviland for providing valuable experimental materials.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant HD09618 (to B. M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Texas Medical School, P. O. Box 20708, Houston, TX 77225. Tel.: 713-500-6064; Fax: 713-500-0652; E-mail: Barbara.M.Sanborn@uth.tmc.edu.

Published, JBC Papers in Press, July 11, 2000, DOI 10.1074/jbc.M004276200

² C. Yue and B. M. Sanborn, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; PI, phosphatidylinositide; IP₃, phosphatidylinositol 1,4,5-trisphosphate; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; fMLP, formyl-Met-Leu-Phe; PMA, phorbol 12-myristate 13-acetate; Tx, thymelea- toxin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; CPT-cAMP, 8-[4-chlorophenythiol]-cAMP; MES, 4-morpholineethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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