Phosphorylation and Regulation of G-protein-activated Phospholipase C-beta 3 by cGMP-dependent Protein Kinases*

Chunzhi XiaDagger , Zhenmin BaoDagger §, Caiping Yue, Barbara M. Sanborn, and Mingyao LiuDagger ||

From the Dagger  Department of Medical Biochemistry and Genetics, Center for Cancer Biology and Nutrition, Institute of Biosciences and Technology, Texas A & M University System Health Science Center, Houston, Texas 77030, the § Department of Biology, Ocean University of Qingdao, Qingdao 266003, Peoples Republic of China, and the  Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, Houston, Texas 77030

Received for publication, July 14, 2000, and in revised form, February 7, 2001

    ABSTRACT
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INTRODUCTION
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Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, beta -adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca2+] by increasing intracellular cAMP and cGMP, activating their respective protein kinases. However, the physiological targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of intracellular Ca2+ by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-beta ) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca2+. In this study, we shown that G-protein-activated PLC enzymes are the potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-beta 2 and PLC-beta 3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-beta leads to the inhibition of G-protein-activated PLC-beta 3 activity by 50-70% in COS-7 cell transfection assays. By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites for the regulation of PLC-beta 3 by PKG (Ser26 and Ser1105). Mutation at these two sites (S26A and S1105A) of PLC-beta 3 completely blocked the phosphorylation of PLC-beta 3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-beta 3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca2+] by the NO-cGMP-dependent signaling pathway.

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G-protein-mediated intracellular Ca2+ signaling plays an important role in mediating the vascular smooth muscle tone and other cellular functions. Contraction of the smooth muscle defines the lumen in blood vessels, airways, uterus, and bladder. An abnormal increase in smooth muscle tone has been implicated in the pathogenesis of hypertension, cardiogenic shock, and other motility-related disorders (1, 2). Relaxation of the smooth muscle can result from the increase of intracellular cAMP and cGMP generated by the activation of adenylyl cyclases and guanylyl cyclases, respectively (3-8). The mechanism by which cyclic nucleotides (cAMP and cGMP) could relax the smooth muscle is believed to involve the inhibition of intracellular free Ca2+ concentration as a result of activating cyclic nucleotide-dependent protein kinases (9). However, the molecular target for the action of cyclic nucleotide-dependent protein kinases (PKA1 and PKG) in regulating intracellular Ca2+ signaling is unclear.

Phosphoinositide-specific phospholipase C (PLC) is a family of key enzymes in the generation and regulation of intracellular Ca2+ signaling, in cell growth, and differentiation (10). Molecular cloning has identified 10 mammalian PLC isozymes that can be grouped into three subfamilies (PLC-beta , PLC-gamma , and PLC-delta ) on the basis of their size, amino acid sequences, domain structure, and activation mechanisms (11, 12). The various PLC enzymes appear to be activated by different receptors and mechanisms. The PLC-gamma subfamily (gamma 1 and gamma 2) is directly phosphorylated and activated by tyrosine kinase-dependent signaling pathways (12). The four isozymes of PLC-beta are activated to various extents by the Galpha q subfamily of G-proteins that couple seven transmembrane receptors to intracellular signaling pathways (13-18). Specific activation of PLC-beta 2 and PLC-beta 3 by the G-protein beta gamma subunits of the Galpha i/o family of G-proteins has also been reported (19-21). Therefore, functionally distinct G-proteins could selectively couple different receptors to PI hydrolysis and intracellular calcium release in the same cell.

There is a wide range of interactions, both between homologous signaling pathways and between heterologous systems (22-25). These interactions provide a mechanism that allows G-protein-coupled pathways to influence each other's function and to modulate multiple receptor signaling pathways. For example, there is increasing evidence for a physiological interaction between the PI and cAMP signaling pathways. Both IP3 and diacylglycerol, products of PLC activation, affect cAMP formation. Calcium mobilized from intracellular stores by IP3 forms a complex with calmodulin. Ca2+-calmodulin can directly bind to certain forms of adenylyl cyclases to activate them (26, 27). Diacylglycerol can also activate protein kinase C that attenuates or potentiates hormone-induced cAMP accumulation in different cell types. On the other hand, there are also numerous reports that the hydrolysis of phosphatidylinositol 4,5-bisphosphates (PIP2) is regulated by cAMP and cAMP-dependent protein kinase (PKA). The effects of cAMP on agonist-stimulated phosphatidylinositide production range from stimulation to inhibition. cAMP or agents that stimulate production of cAMP (forskolin, prostaglandins E1 and E2, theophylline, prostacyclin, and beta -adrenergic agents), inhibit phosphatidylinositide breakdown and intracellular Ca2+ mobilization in a number of cells and tissues (3, 5, 28-38). However, the molecular mechanism by which the cAMP pathway regulates the PIP2 metabolism and intracellular Ca2+ mobilization is poorly understood. Our recent work (22, 39), together with others (37), has suggested that PKA could phosphorylate G-protein-activated phospholipase C isozymes, regulating the G-protein-mediated intracellular Ca2+ signaling.

Ca2+ and nitric oxide (NO) work together in the control of cell homeostasis. NO has been recognized to account for the activities of the endothelial-derived relaxing factor by increasing intracellular cGMP concentration. In a number of cells, nitric oxide and atrial natriuretic factor inhibited the release of intracellular Ca2+ from internal storage sites. The inhibitory effects of these agents appear to be mediated by increasing intracellular cGMP and by activating cGMP-dependent protein kinases (6, 9, 40-46). Potential PKG phosphorylation targets of intracellular Ca2+ signaling pathway include phospholipase C, G-proteins, Ca2+-ATPase, phospholamban, IP3 receptor ion channels, and myosin phosphatase (47). However, the molecular mechanism of PKG phosphorylation in mediating intracellular Ca2+ release has not been defined. In most cases, the physiological targets of PKG phosphorylation remain to be identified, and their function needs to be clarified. Knowledge of the molecules involved in the regulation of intracellular Ca2+ by cGMP-dependent protein kinase could be of great importance in pharmacology (48). For example, the effect of the popular drug, Viagra (sildenafil), is to increase intracellular cGMP concentration and lower intracellular Ca2+ levels via inhibiting a specific phosphodiesterase in the vascular smooth muscle. In other words, Viagra achieves its pharmacological effects of relaxing the vascular smooth muscle through the increase of intracellular cGMP and possibly cGMP-dependent protein kinase (9). However, the molecular mechanism on how an increase in cGMP affects intracellular Ca2+ and, consequently, the relaxation of the vascular smooth muscle is still under active investigation. In this report, we demonstrate that G-protein-activated PLC-beta 2 and PLC-beta 3 are directly phosphorylated by PKG in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC blocks the activation of the proteins by G-protein subunits. Furthermore, we identified two serine residues (Ser26 and Ser1105) as the PKG phosphorylation sites in PLC-beta 3. Mutation of these two sites completely abolished the phosphorylation of the protein by PKG and the inhibitory effect of PKG on the activation of PLC-beta 3 by G-protein subunits. Therefore, these data define a potential cross-talk mechanism between G-protein-mediated intracellular Ca2+ release and the cGMP/PKG signaling pathway.

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Materials-- PLC-beta antibodies, immunoblotting, and immunoprecipitation reagents were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). LipofectAMINE, DMEM, and all other cell culture reagents were obtained from Life Technologies, Inc. [3H]Inositol (22 Ci/mmol), [32P]orthophosphate, and [gamma -32P]ATP (3000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. The PLC-beta 1 clone in baculovirus was obtained from Dr. P. Sternweis (University of Texas Southwestern Medical Center). cDNAs encoding three different PKG isozymes (PKGIalpha , PKGIbeta , and PKGII) were kindly provided by Dr. Suzanne Lohmann at the University of Wurzburg, Germany) (70, 74).

Protein Purification and Site-directed Mutagenesis-- His6-tagged PLC-beta 2 and PLC-beta 3 in baculovirus (PharMingen, San Diego, CA) were constructed from the respective PLC cDNA plasmids. The expressed proteins, His6-PLC-beta 2, and His6-PLC-beta 3, were purified by chromatography over Ni2+-agarose, and Mono Q and Mono S columns as described previously for PLC-beta 1 from the membrane fraction of Sf9 cells (49). The purity of the proteins is more than 98% as judged by SDS-PAGE. Site-directed mutations at Ser26, Ser474, Ser1105 (Ser to Ala) were generated by polymerase chain reaction or using the GeneEditor kit (Promega, Madison, WI). All PLC mutant sequences were confirmed by DNA sequencing.

In Vitro Phosphorylation of PLC-beta and Phosphopeptide Mapping-- Purified His6-tagged recombinant PLC-beta (beta 1, beta 2, and beta 3) were incubated with purified PKG enzymes or PKA catalytic subunit at a molar ratio 50:1 in the presence of 1-10 µCi of [gamma -32P]ATP and 100 µM ATP in a total volume of 50 µl of kinase buffer (10 mM HEPES, pH 7.2, 10 mM MgCl2) for 30 min at 30 °C. For the time course study, 2 µg of PLC-beta proteins were incubated with PKG or PKA for the indicated time. PKG enzymes were obtained from Promega or kindly provided by Dr. S. Lohmann at the University of Würzburg, Germany. Phosphorylation reactions were terminated by addition of 4× SDS sample buffer and boiling for 5 min. Proteins were separated on 8% SDS-PAGE gels, and the phosphorylated proteins were visualized by autoradiography.

Two-dimensional phosphopeptide mapping of PLC-beta 3 phosphorylated by PKG or PKA in vitro was carried out with a Hunter thin layer electrophoresis system (CBS Scientific Company, Del Mar, CA) according to the manufacturer's instructions. Briefly, the phosphorylated PLC proteins were separated by SDS-PAGE. PLC proteins were washed and digested with trypsin for 24 h at 35 °C. The phosphopeptides were separated by two-dimensional chromatography.

Preparation of Smooth Muscle Cells and in Vivo 32P Labeling-- Smooth muscle cells were isolated from the aorta of Harlan Sprague-Dawley rats as described previously (50). Briefly, the aorta was incubated in collagenase (1.5 mg/ml) for 10 min at 37 °C before removing the adventitia. The smooth muscle cells were maintained in DMEM with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.2 mM glutamine. The cells were grown to confluence and used until the third passage to prevent the loss of endogenous PKGs. Nearly confluent smooth muscle cells (10-cm dish) were labeled with [32P]orthophosphate (0.33 mCi/ml) in phosphate-free DMEM containing 10% dialyzed fetal calf serum for 6 h. After stimulation with the membrane-permeable cGMP analogue, 8-pCPT-cGMP (100-200 µM), cells were lysed in ice-cold lysis buffer containing a mixture of protease and phosphatase inhibitors (37). Cell extracts were centrifuged at 15,000 × g for 10 min at 4 °C. Proteins immunoprecipitated with specific anti-PLC-beta 3 antibody were separated on a 7.5% SDS-PAGE and analyzed by autoradiography. The amount of PLC-beta 3 protein was normalized for sample loading by Western blot.

Cell Culture, Transfection, and PLC Activity Assays-- COS-7 cells were cultured and transfected as described previously (22). Briefly, COS-7 cells (1 × 105 cells per well) were seeded in 12-well plates in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum a day before transfection. The total amount of DNA in all transfections was 1-1.2 µg per well. The cytomegalovirus vector, pCIS, encoding beta -galactosidase was used to maintain the amount of DNA constant and the amount of a particular cDNA in each set of experiments equal. cDNAs were mixed with LipofectAMINE (Life Technologies, Inc.) in serum-free Opti-MEM (Life Technologies, Inc.), and cells were transfected for about 5 h. 24 h post-transfection, cells were washed with phosphate-buffered saline (PBS) and incubated in 0.5 ml of inositol-free DMEM medium containing 10 µCi ml-1 of myo-[2-3H] inositol (Amersham Pharmacia Biotech) and 10% dialyzed fetal bovine serum. 24 h later, cells were washed with PBS and incubated in inositol-free medium containing 10 mM LiCl for 30 min at 37 °C. For activation cGMP-dependent protein kinases, 100 µM 8-pCPT-cGMP was added to the inositol-free LiCl medium. The cells were lysed by adding ice-cold 10 mM formic acid and incubated on ice for 30 min. Then the lysate was neutralized with 15 mM NH4OH. Total inositol phosphates were separated by an anion exchange column (AG 1-X8, Bio-Rad) as described. The eluted inositol phosphates was mixed with 10 ml of scintillation mixture (BCS; Amersham Pharmacia Biotech) and counted in a liquid scintillation counter.

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INTRODUCTION
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Phosphorylation of PLC-beta by PKG in Vitro and in Vivo-- We have shown previously that PKA can directly phosphorylate PLC-beta 2 and PLC-beta 3, blocking the activation of the enzymes by G-protein subunits (22) (39). On the other hand, direct phosphorylation of PLC-beta isozymes by cGMP-dependent protein kinases has not been reported, although it has been known that an increase in intracellular cGMP can inhibit the hydrolysis of phosphatidylinositide and the release of intracellular Ca2+. In order to understand the regulation of G-protein-mediated PLC-Ca2+ signaling by pathway coupled to intracellular cGMP, we extended our studies on the phosphorylation of PLC-beta isozymes by PKG both in vitro and in the smooth muscle cells. As shown in Fig. 1A, incubation of purified PLC-beta isozymes with purified PKG demonstrated that both PLC-beta 2 and PLC-beta 3 are good substrates for PKG, whereas PLC-beta 1 was not phosphorylated by the kinase under the same conditions. To quantify PKG-stimulated PLC-beta 3 phosphorylation, purified PLC-beta 3 was phosphorylated by PKG in vitro. Fig. 1B shows the time-dependent incorporation of 32P into PLC-beta 3 protein catalyzed by PKA and PKG. Compared with the phosphorylation of PLC-beta 3 by PKA (Fig. 1B, top), PKG phosphorylation is slower and approached a plateau in about 60 min. Estimation of the Km value for PKA is about 1 µM, whereas the Km value for PKG is somewhat higher, ~12 µM when assayed with different concentrations of purified PLC-beta 3 protein. A maximum ratio of 1.3 mol of phosphate/mol of PLC-beta 3 was determined by a filter binding assay at the 60-min incubation point, suggesting two possible phosphorylation sites in PLC-beta 3 protein.


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Fig. 1.   Phosphorylation of purified PLC-beta isozymes by PKG in vitro. A, purified His6-tagged PLC-beta 1, PLC-beta 2, and PLC-beta 3 proteins were phosphorylated by PKGI (0.01 µg, 30 units, Promega) for 30 min at 30 °C. PLC-beta 2 and PLC-beta 3, but not PLC-beta 1, are good phosphorylation substrates for PKGI. A total of 2 µg of purified PLC-beta proteins were used in each reaction. 0.5 µg of protein was loaded on the gel. Similar phosphorylation was observed using PKGII (data not shown). B, time-dependent phosphorylation of PLC-beta 3 in vitro. Reactions were terminated at the indicated times (in minutes). Compared with PKA phosphorylation of PLC-beta 3 (top), phosphorylation of PLC-beta 3 by PKG is slower, reaching a plateau at ~60 min. C, phosphorylation of PLC-beta 3 by three different PKG isoforms. PLC-beta 3 was overexpressed in COS-7 cells and immunoprecipitated by specific anti-PLC-beta 3 antibody. The precipitated protein was phosphorylated by purified PKG isoforms (PKGIalpha , PKGIbeta , and PKGII) (0.05-0.1 µg/reaction) (73). Phosphorylation of immunoprecipitates by preimmune serum (without PLC-beta 3 protein) (-) and by specific anti-PLC-beta 3 antibody are shown and labeled for the presence of PLC-beta 3 protein. Putative autophosphorylated PKGs were detected along with phosphorylated PLC-beta 3 in the immunoprecipitation complexes in the assay. Samples were analyzed by SDS-PAGE and visualized by autoradiography.

There are three different isoforms of PKG that differ in their sequences, distribution, and membrane association (51). To examine whether all three isozymes of PKG can phosphorylate PLC-beta proteins, we phosphorylated overexpressed PLC-beta 3 in the presence of PKGIalpha , PLGIbeta , and PKGII, and we then immunoprecipitated PLC-beta 3 using specific anti-PLC-beta 3 antibodies. As shown in Fig. 1C, PLC-beta 3 was phosphorylated by all three isozymes of PKG in our in vitro phosphorylation assays.

To test the notion that PLC-beta 2 and PLC-beta 3 are physiological targets of cGMP-dependent protein kinases in the cell, we further analyzed whether activation of PKG can directly phosphorylate PLC-beta isoforms in cells expressing these proteins. Cell extracts from cultured smooth muscle cells were incubated in the presence of [gamma -32P]ATP. As shown in Fig. 2A, stimulation with selective PKG agonist, 8-Br-cGMP, greatly increased the phosphorylation intensities of 130-140-kDa proteins. Immunoprecipitation with specific antibodies against PLC-beta 2 and PLC-beta 3 indicated that both proteins are phosphorylated by the stimulation of 8-Br-cGMP (Fig. 2B). Together these data suggested that PLC-beta proteins could be one of the physiological targets upon stimulation of smooth muscle cell extracts by intracellular cGMP.


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Fig. 2.   Phosphorylation of PLC-beta in smooth muscle cells. A, smooth muscle cell extracts were prepared by centrifugation and incubated with [gamma -32P]ATP at 37 °C for 10 min in the presence or absence of the PKG activator, 8-Br-cGMP (1 µM). Then the phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography. EGTA (2 mM) was included in all reactions to remove calcium, an activator of proteases and protein kinase C. B, PLC-beta 2 and PLC-beta 3 were phosphorylated in smooth muscle cell extracts stimulated with 8-Br-cGMP (1 µM). The phosphorylated cell extracts were immunoprecipitated with specific anti-PLC-beta 2 and PLC-beta 3 antibodies. Precipitated PLC-beta 2 and PLC-beta 3 proteins were separated by SDS-PAGE and visualized by autoradiography. The specificity of the anti-PLC-beta 2 and anti-PLC-beta 3 antibodies was tested in vitro with purified PLC proteins and in cell lysates. C, in vivo 32P labeling of PLC-beta 3 isolated from cultured smooth muscle cells. VSMCs from rat aorta between the third and fifth passage were used so that endogenous PKG can be preserved. Western blot shows that PKG can be detected up to passage level 7 in the VSMCs. The cells were metabolically labeled with [32P]orthophosphate in phosphate-free DMEM in the presence or absence of PKG activator, 8-pCPT-cGMP (100 µM) for 3-4 h. Then the PLC-beta 3 was immunoprecipitated by specific anti-PLC-beta 3 antibody, and the proteins were separated on SDS-PAGE. Phosphorylated PLC-beta 3 was visualized by autoradiography. Similar results were obtained from three independent experiments. As a control, we did not see any specific band corresponding to the size of PLC-beta when precipitated with a preimmune serum. Bottom panel, Western blot of PLC-beta 3 using specific anti-PLC-beta 3 antibody, demonstrating an equal amount of protein was loaded in the gel.

To confirm that PLC-beta 3 is phosphorylated upon stimulation of increased cGMP in the cell, cultured smooth muscle cells before passage level 5 were metabolically labeled with [32P]orthophosphate. The expression of PKG in VSMCs before passage level 8 was confirmed by Western blot using specific anti-PKG antibodies. PLC-beta 3 protein was immunoprecipitated by a specific anti-PLC-beta 3 antibody after labeling of the cell. Stimulation of PKG by the selective activator, 8-pCPT-cGMP, substantially increased the 32P incorporation into PLC-beta 3 protein (Fig. 2C), indicating that PLC-beta 3 is a potential physiological target of cGMP-dependent protein kinases in the cell.

Inhibition of G-protein-activated PLC-beta 3 by PKG Isoforms-- To test whether cGMP and PKG are involved in the agonist-stimulated and G-protein-mediated PLC signaling pathway, we constructed mammalian expression plasmids that encode the three different PKG isoforms. Then we reconstituted the G-protein-activated PLC-Ca2+ signaling pathway in COS-7 cells by introducing cDNAs encoding G-protein subunits, PLC-beta , and PKGs into the cells. Specific activation of PLC-beta 3 by G-protein beta gamma subunits was measured by the release of inositol phosphates (IP3) (Fig. 3A). In the presence of PKG isozymes, the activation of PLC-beta 3 by G-protein beta gamma subunits was substantially reduced upon stimulation of PKGs by the membrane-permeable cGMP analogue, 8-pCPT-cGMP (Fig. 3A). Since PLC-beta 3 is also activated by Galpha q, we examined the PI turnover in COS-7 cells transfected with PLC-beta 3 and Galpha q protein in the presence or absence of PKG. As shown in Fig. 3B, transfection of empty vector, Galpha q, PLC-beta 3, or PKG alone had little effect on basal PI turnover. Cotransfection of Galpha q with PLC-beta 3 increased the production of inositol phosphates by approximately 5-fold. On the other hand, cotransfection and activation of PKG inhibited more than 60% of the activation of PLC-beta 3 by Galpha q.


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Fig. 3.   Inhibition of G-protein-activated PLC-beta by PKGs. A, activation of PLC-beta 3 by Gbeta gamma subunits was inhibited by three different PKG isoforms (PKGIalpha , PKGIbeta , and PKGII) in COS-7 cells. cDNAs transfected into the cells are listed below the column. The total amount of cDNAs added was 1.2 µg per well. The amount of cDNA for each gene was kept the same in each transfection (0.3 µg/well). pCMV-lacZ was used to make the total amount of cDNAs equal in each well (1.2 µg/well). B, PKG inhibits Galpha q-stimulated PLC-beta 3 activity in COS-7 cells. In all transfection experiments PKG was activated in the cell by 100 µM 8-pCPT-cGMP for 30-60 min. Total amount of cDNA for each gene transfected into COS-7 cells is 0.35 µg/well. Data presented are the means ± S.E. (n = 3) of three transfection repeats (wells). C, activation of PKG inhibits agonist-induced PI turnover in COS-7. COS-7 cells were transfected with M1 muscarinic receptor (0.3 µg) in the presence or absence of PKG isozymes (0.6 µg). After 48 h of transfection, [3H]inositol-labeled cells were incubated with 100 µM 8-pCPT-cGMP for 30 min. The cells were stimulated with 20 µM carbachol for 20 min (dark columns). The control cells were treated with PBS (light column). Inositol phosphates were determined as described (22, 39). All cDNAs are under the control of the cytomegalovirus promoter. The expression level of endogenous PLC-beta is not affected by the expression of PKG in the assay conditions (data not shown). Data represent the average value of triplicates. Error bars give the range of triplicates.

To examine whether the agonist-induced IP3 synthesis is indeed inhibited by the activation of PKG, we measured the production of IP3 induced by activation of the M1 muscarinic receptor in COS-7 cells in the presence or absence of overexpressed PKG. COS-7 cells were transfected with the M1 muscarinic receptors coupled to PLC-beta through Galpha q protein. As shown in Fig. 3C, when the M1 receptor was transfected in COS-7 cells and stimulated with its ligand, carbachol, the production of [3H]IP3 was increased about 5-6-fold. Cotransfection and activation of the PKG isoforms inhibited the agonist-stimulated IP3 release by 30-40% in COS-7 cells (Fig. 3C). Since COS-7 cells contain multiple PLC-beta isoforms (beta 1, beta 2, and beta 3), the observed inhibitory effects may underestimate the effects of PKG on the regulation of receptor-stimulated G-protein-coupled IP3 signaling in the cell. Together, these data suggest that activation of PKG could phosphorylate PLC and inhibit the agonist-induced IP3 production in the cell.

PKG Phosphorylates Distinct Peptides in PLC-beta 3 Compared with PKA Phosphorylation-- To examine whether PKG phosphorylates PLC-beta 3 at the same sites as PKA phosphorylation, we phosphorylated purified PLC-beta 3 protein with sequential addition of the two kinases (PKA and PKG) in the presence of non-radioactive ATP first and then of [gamma -32P]ATP. Initial phosphorylation of PLC-beta 3 by PKA in the presence of non-radioactive ATP has limited effect on the subsequent phosphorylation of the protein by PKG (Fig. 4A). However, initial phosphorylation of PLC-beta 3 by PKG in the presence of non-radioactive ATP prevents subsequent phosphorylation of the protein by PKA (Fig. 4B). These results indicate that PKG not only shares one or more phosphorylation sites in PLC proteins with PKA, but also has unique phosphorylation sites that are not blocked by initial PKA phosphorylation of the protein in the presence of non-radioactive ATP. On the other hand, the PKA phosphorylation sites in PLC-beta are shared by PKG, as shown by the inhibitory effect of initial PKG phosphorylation on the subsequent PKA phosphorylation of the PLC-beta protein (Fig. 4B).


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Fig. 4.   Sequential phosphorylation of PLC-beta by PKA and PKG. A, initial phosphorylation of PLC-beta 3 by PKA in the presence of unlabeled ATP has little effect on the subsequent phosphorylation of the protein by PKG in the presence of [gamma -32P]ATP, suggesting different sites of phosphorylation in the protein by PKA and PKG. Lane 1 (control), phosphorylation of PLC-beta 3 by PKG in the presence of [gamma -32P]ATP for 60 min. Lane 2, PLC-beta 3 was phosphorylated by PKA for 30 min in the presence of unlabeled ATP, followed by the addition of PKG and [gamma -32P]ATP for 60 min at 30 °C. B, initial phosphorylation of PLC-beta 3 by PKG dramatically reduced the subsequent phosphorylation of the proteins by PKA. Lane 1 (control), purified PLC-beta 3 protein (1 µg) was phosphorylated by PKA for 30 min at 30 °C. Lane 2, the same amount of PLC-beta 3 protein was first phosphorylated by PKG for 30 min in the presence of unlabeled ATP, followed by the addition of PKA and [gamma -32P]ATP for another 30 min.

To understand further the phosphorylation pattern of PLC-beta protein, we compared the phosphopeptide mapping of PLC-beta 3 protein phosphorylated by either PKA or PKG. As shown in Fig. 5, phosphopeptide mapping of in vitro phosphorylated PLC-beta 3 by PKA (Fig. 5A) and PKG (Fig. 5B) digested by trypsin showed largely similar patterns of peptide phosphorylation. However, compared with PKA phosphorylation, PKG has a unique phosphopeptide at position 1 in Fig. 5B. Both PKG and PKA have favored phosphorylation sites (position 3 for PKG and position 2 for PKA in Fig. 5, A and B). Together, these data suggest that PKG phosphorylates at least one unique site in PLC-beta 3 protein compared with PKA.


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Fig. 5.   Two-dimensional phosphopeptide mapping of PLC-beta 3 digested by trypsin. A, PLC-beta 3 phosphorylated by PKA. B, phosphorylation of PLC-beta 3 by PKG. PLC-beta 3 was purified from COS-7 cells by immunoprecipitation. The protein was phosphorylated in vitro by purified PKG (Promega). Phosphorylated proteins were separated by SDS-PAGE and digested with trypsin (400 µg/ml). After two-dimensional phosphopeptide mapping, phosphopeptides were visualized by autoradiography. Compared with the phosphorylation of PLC-beta 3 by PKA (A), PKG phosphorylates a unique site at point 1 in B.

Identification of the PKG Phosphorylation Sites in PLC-beta 3-- Detailed characterization of the phosphorylation sites in PLC-beta isoforms is an essential step toward understanding the mechanism of PLC-beta activation and regulation by cGMP-dependent protein kinase. In this report, we focus on the identification of phosphorylation sites in PLC-beta 3. Three putative PKG phosphorylation sites (Ser26, Ser474, and Ser1105) were found in human PLC-beta 3 (Fig. 6A). Ser26 is located in the putative PH domain of the protein. Ser474 is in the catalytic domain (X box) of the protein. Ser1105 is in the so-called G box region where Galpha q was found to interact with in PLC-beta 1 protein (52). To determine which serine residue is phosphorylated in the protein, we purified a mutant PLC-beta 3 protein (S1105A) from insect Sf9 cells. Mutation at this site abolished the phosphorylation of PLC-beta 3 by PKA (Fig. 6B), consistent with the result reported previously (39). However, the phosphorylation of purified mutant PLC-beta 3 protein (S1105A) by PKG was only reduced by ~30% compared with the phosphorylation of wild-type PLC-beta 3 protein (Fig. 6C), suggesting PKG also phosphorylates additional sites, consistent with the results of phosphopeptide mapping.


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Fig. 6.   Identification of PKG phosphorylation sites in PLC-beta 3. A, domain structure and putative phosphorylation sites of PLC-beta 3. The PH domain may play a role in interacting with Gbeta gamma and phospholipid substrates. X and Y boxes are the catalytic domains of the protein. G box is responsible for the interaction with Galpha q in PLC-beta 1. Three putative phosphorylation sites are found in PLC-beta 3 as follows: Ser26 in peptide 1 (P1), Ser474 in peptide 2 (P2), and Ser1105 in peptide 3 (P3). B, mutation at Ser1105 to Ala completely abolished the phosphorylation of the protein by PKA. Purified His6-PLC-beta 3 was phosphorylated by PKA in vitro. Phosphorylated protein was separated by SDS-PAGE and visualized by autoradiography. C, phosphorylation of the His6 PLC-beta 3 mutant (S1105A) protein by PKG. Mutation at this site reduced the PKG-catalyzed phosphorylation of PLC-beta 3 by about 30-40%. D, phosphorylation of mutant PLC-beta 3 proteins expressed and isolated from COS-7 cells. Mutation at Ser26 and Ser1105 reduced the phosphorylation of the protein by ~70% and 40%, respectively. Double mutation of Ser26 and Ser1105 to Ala completely blocked the phosphorylation of the protein by PKG. Mutation at Ser474 has little effect on the phosphorylation of the protein by PKG. Similar results were obtained from three independent experiments. Bottom panel, Western blot of PLC-beta 3 and mutants, showing equal amount of PLC proteins were loaded in the gel.

To identify the remaining phosphorylation sites, we made three single mutations at Ser26, Ser474, and Ser1105 and one double mutation (S26A,S1105A). cDNAs corresponding to the wild-type and the mutant PLC-beta 3 were cotransfected into COS-7 cells, and the proteins were isolated and purified by immunoprecipitation using specific antibodies against PLC-beta 3. Then the purified proteins were phosphorylated in vitro using purified PKG. As shown in Fig. 6D, wild-type PLC-beta 3 was a good substrate of PKG, whereas mutation at Ser26 dramatically reduced the phosphorylation of the protein. Similar to our in vitro phosphorylation assay, mutation at Ser1105 inhibited about 30-40% of the incorporation of 32P into the protein. Double mutations at serine residues 26 and 1105 (S26A,S1105A) almost completely abolished the phosphorylation of the protein by PKG, indicating that Ser26 and Ser1105 are the PKG phosphorylation sites in PLC-beta 3. On the other hand, mutation at Ser474 has little effect on the phosphorylation of the protein by PKG. Similar results were obtained when the mutant PLC-beta 3 genes were cotransfected with PKG and metabolically labeled with [32P]orthophosphate in the presence of the membrane-permeable cGMP analogue, 8-pCTP-cGMP (data not shown). These data suggest that serine 26 and serine 1105 are the two major phosphorylation sites in PLC-beta 3 catalyzed by PKG. Sequence comparison of the PLC-beta isozymes reveals that Ser26 and Ser1105 of PLC-beta 3 are not conserved among the PLC enzymes. Therefore, the mode of regulation between different PLC-beta isoforms (beta 1, beta 2, beta 3, and beta 4) could vary.

To confirm our observation that serines 26 and 1105 are functional phosphorylation sites in the protein, we cotransfected the mutant proteins with G-protein subunits into COS-7 cells and measured the activation of the mutant proteins in the presence of activated PKG. As shown in Fig. 7, A and B, mutation at the serine residues (Ser26, Ser474, and Ser1105) had no effect on the activation of PLC-beta 3 by G-protein subunits in COS-7 cell transfection assays. However, unlike the wild-type PLC-beta 3, cotransfection and activation of PKG no longer elicited inhibitory effects on the activation of the double mutant PLC-beta 3 protein (S26A,S1105A) by Galpha q (Fig. 7A) and Gbeta gamma subunits (Fig. 7B). Mutation at either Ser26 or Ser1105 alone had very limited effect on the inhibition of PKG, whereas mutation at Ser474 has no effect on the inhibition of PLC by PKG (Fig. 7, A and B). Together, these data indicate that the two serine residues (Ser26 and Ser1105) play a key role in the regulation of PLC-beta 3 enzyme by cGMP-dependent protein kinase.


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Fig. 7.   Mutation at Ser1105 and Ser26 abolished the inhibitory effect of PKG on the activation of PLC-beta 3. A, PLC-beta 3 and mutant proteins were activated by Galpha q. The inhibitory effect of PKG was affected by the mutation of PLC-beta 3 protein at Ser1105 and Ser26 or both, but not by mutation at Ser474. cDNAs corresponding to PLC-beta 3WT, PLC-beta 3 mutants, Galpha q, and PKGIbeta were transfected into COS-7 cells. The production of 3H-labeled inositol phosphates was measured as counts/min. Data presented are the means of three repeat transfections (wells) ± S.E. (n = 3). Plasmids transfected into the cells are listed below the column. The total amount of cDNAs transfected into COS-7 cells is 1.2 µg/well with 0.3 µg of cDNA for each gene. B, effect of PKG on the Gbeta gamma -stimulated activation of PLC-beta 3 mutants. PLC-beta 3 and mutant plasmids were activated by Gbeta 1gamma 2 subunits in COS-7 cell transfection assays. Mutation at Ser26 and Ser1105 to Ala completely removed the inhibitory effect of PKG on the activation of PLC-beta 3 by Gbeta 1gamma 2. cDNAs transfected into COS-7 cells are labeled below the column. Total amount of cDNAs (1 µg/well) transfected in each well are maintained the same by adding pCIS-LacZ.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the vascular smooth muscle, nitric oxide (NO) has been recognized to account for the activities of the endothelially derived relaxing factor by increasing intracellular cGMP concentration via the activation of guanylyl cyclases. NO inhibits IP3 generation and intracellular Ca2+ mobilization by increasing the intracellular cGMP concentration. The downstream target for cGMP is believed to be the PKGs, which mediate the effects of cGMP in a number of cells or tissues, including the vascular smooth muscle and platelets (9, 31, 40, 51, 53). Whereas an increase in cGMP level correlates well with a decrease in intracellular Ca2+ concentration and the relaxation of the vascular smooth muscle in a time- and concentration-dependent manner, the molecular mechanisms for cGMP-PKG to regulate intracellular Ca2+ signaling are not well defined. In this report, we demonstrate that PLC-beta 2 and PLC-beta 3 are good substrates for cGMP-dependent protein kinases, whereas PLC-beta 1 was not phosphorylated in the same assays. These results correlate well with our cell transfection assays in which activation of PLC-beta 2 and PLC-beta 3 by G-protein subunits is inhibited by coexpression and activation of PKG isozymes (regulation of PLC-beta 2 by PKG has not been shown in this paper). By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites (Ser26 and Ser1105) for the regulation of PLC-beta 3 by PKG. Mutation at these sites of PLC-beta 3 dramatically reduced the inhibitory effect of PKG on the activation of PLC by G-protein and the phosphorylation of the PLC protein by PKG. These results indicate that PKG can directly phosphorylate PLC isozymes (beta 2 and beta 3) and regulate the activation of receptor-mediated G-protein-coupled phosphatidylinositol (PI) turnover and intracellular Ca2+ release.

There are four isozymes of phospholipase C-beta that are activated to various extents by Galpha q proteins, which account for the pertussis toxin-resistant activation of PLC and intracellular Ca2+ increase. It has been shown that the C terminus of PLC-beta 1 is critical for the activation of the enzyme by Galpha q (52, 54). Deletion studies have identified a P box region (Thr903 to Gln1030) and a G box domain (Lys1031 to Leu1142) in the C terminus of PLC-beta 1. The P box of PLC-beta 1 is important for the association of PLC-beta 1 with cell membrane and the activation of the enzyme by Galpha q, whereas the G box domain is involved in the interaction with Galpha q subunits (52). We previously showed that phosphorylation of serine residues at the C-terminal domains of PLC-beta by PKA inhibited the G-protein-mediated PLC-beta activity (22, 39). Phosphorylation at Ser1105 of PLC-beta 3 accounts for the inhibitory effect of PKA on the activation of the protein by Galpha q (39). In this report, we shown that PKG also phosphorylated Ser1105 in PLC-beta 3. Phosphorylation of the protein at serine 1105 could potentially reduce or block the association of PLC-beta with G-protein alpha  subunit.

Specific activation of PLC-beta 2 and PLC-beta 3 by G-protein beta gamma subunits accounts for the pertussis toxin-sensitive PLC activation and Ca2+ release. The sites of interaction with G-protein beta gamma subunits in PLC-beta 3 are located in both the N-terminal domain and in the catalytic X domain of the PLC-beta protein (55-57). The N-terminal first 100 amino acids are predicted to form a pleckstrin homology (PH) domain (58). The PH domain isolated from PLC-delta binds to PIP2 and IP3, and deletion of the PH domain from PLC-delta inhibited anchoring of the protein to PIP2-containing membrane but did not inhibit catalysis (59-61). The role of PH domain in PLC-beta isoforms is not clear. Recent data indicate that a fragment encompassing the N-terminal PH domain bound to G-protein beta gamma subunits, and the PH domain of PLC-beta 2 determines the binding and activation by G-protein beta gamma subunits (55, 62). Therefore, phosphorylation of Ser26 in PLC-beta 3 by cGMP-dependent protein kinase could affect the interactions of the PLC protein with either G-protein beta gamma subunits or phospholipid in the membrane or with both. We are in the process of testing this hypothesis.

Nitric oxide and cyclic GMP have emerged recently as a principal focus in signal transduction, playing important regulatory roles in smooth muscle relaxation and proliferation, inhibition of platelet aggregation, neutrophil degranulation, and initiation of visual signal transduction. A number of intracellular proteins are activated by an increase in intracellular cGMP. Examples include cGMP-dependent protein kinases (PKGs), cyclic nucleotide-gated ion channels, cGMP-binding phosphodiesterases, PKA, and myosin phosphatase (47, 63, 64). In vascular smooth muscle cells, platelets, and neuronal cells, nitric oxide and atrial natriuretic factor inhibited the intracellular Ca2+ release induced by activation of G-protein-coupled receptors. The inhibitory effects of these agents appear to be mediated by increasing intracellular cGMP and by activating cGMP-dependent protein kinases. Three different isozymes of cGMP-dependent protein kinases (PKGs), PKGIalpha , PKGIbeta , and PKGII, have been cloned and characterized (51) (64). PKGIalpha and -Ibeta are alternative spliced variants, highly expressed in the vascular and gastrointestinal smooth muscle, lung, cerebellum, platelets, and heart (51, 65-68). The PKGII isoform is a membrane-associated protein, expressed in secretory epithelium and in various regions of the brain (69-71). Our in vitro studies have shown that all three PKG isoforms can directly phosphorylate PLC-beta 3 and PLC-beta 2. Cotransfection and activation of the kinases in COS-7 cells decreased the G-protein-activated IP3 release, a result of blocking PLC-beta activation. The precise physiological role of PLC-beta phosphorylation by cGMP-dependent protein kinases may depend on the localization of the two proteins in the cell or tissue.

A recent report demonstrates that cGMP kinase Ibeta can form signaling complex with IP3 receptor and IRAG (an IP3R-associated cGMP kinase substrate) in microsomal smooth muscle membrane to regulate intracellular calcium (72). The observation that PLC-beta 3 and PKGs are immunoprecipitated together suggests an interesting possibility that these proteins may form signaling complex in the cell. The notion that PLC may form signaling complex with PKG and other molecules is under investigation.

In summary, this work provides the first evidence that G-protein-activated PLC-beta isozymes are the potential physiological target for signaling pathways coupled to cGMP and cGMP-dependent protein kinases. A working model (Fig. 8) is proposed in which nitric oxide and other peptides, such as atrial natriuretic peptide and brain natriuretic peptide, stimulate guanylyl cyclases, leading to the production of cyclic GMP and the activation of cGMP-dependent protein kinases. Phosphorylation of PLC isozymes by PKG blocks the activation of the enzymes by G-protein subunits, resulting in the inhibition of agonist-stimulated intracellular Ca2+ release and the regulation of cellular functions.


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Fig. 8.   Proposed model for the regulation of G-protein-activated PLC-Ca2+ by signaling pathway coupled to cGMP-dependent protein kinase. Receptors (R) coupled to both Galpha q and Galpha i/beta gamma can activate PLC-beta isozymes, leading to the production of IP3 and diacylglycerol (DAG), key second messengers in the release of intracellular Ca2+ and the activation of protein kinase C, respectively. Reagents, such as nitric oxide (NO), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP), can stimulate guanylyl cyclases, leading to the increase of intracellular cGMP from GTP. One of the functions of cGMP is to activate cGMP-dependent protein kinases that can directly phosphorylate and inhibit G-protein-activated PLC-beta activity, blocking the hydrolysis of inositol phosphates and the release of intracellular Ca2+ and, consequently, regulating cellular functions.


    ACKNOWLEDGEMENTS

We thank Dr. Melvin I. Simon (California Institute of Technology, Pasadena, CA) for support and encouragement. We thank Dr. Suzanne Lohmann (University of Würzburg, Germany) for suggestions on the manuscript and for reagents.

    FOOTNOTES

* This work was supported in part by a Scientist Development grant from the National American Heart Association, a Basil O'Connor Starter Scholar Research award from the March of Dimes Birth Defects Foundation (to M. L.), and 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: Institute of Biosciences and Technology, Texas A & M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7505; Fax: 713-677-7512; E-mail: mliu@ibt.tamu.edu.

Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M006266200

    ABBREVIATIONS

The abbreviations used are: PKA, cAMP-dependent protein kinase; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-triphosphate; PKG, cGMP-dependent protein kinases; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; NO, nitric oxide; PH, pleckstrin homology; VSMCs, vascular smooth muscle cells; PAGE, polyacrylamide gel electrophoresis; 8-pCPT-cGMP, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate; 8-Br-cGMP, 8-bromo-cGMP.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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