Phosphorylation of Serine 1105 by Protein Kinase A Inhibits Phospholipase Cbeta 3 Stimulation by Galpha q*

Caiping YueDagger , Kimberly L. DodgeDagger , Günther Weber§, and Barbara M. SanbornDagger

From the Dagger  Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, Houston, Texas 77225 and the § Department of Molecular Medicine, Clinical Genetics Unit, CMM, L8:02, Karolinska Hospital, S-17176 Stockholm, Sweden

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

The mechanism by which protein kinase A (PKA) inhibits Galpha q-stimulated phospholipase C activity of the beta  subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta 3 by PKA results in inhibition of Galpha q-stimulated PLCbeta 3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta 3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galpha q with PLCbeta 3 or PLCbeta 3 (Ser1105 right-arrow Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galpha q, PLCbeta 3, or PLCbeta 3 (Ser1105 right-arrow Ala) mutant alone. Whereas Galpha q-stimulated PLCbeta 3 activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galpha q-stimulated PLCbeta 3 (Ser1105 right-arrow Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha q-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta 3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta 3 inhibits Galpha q-stimulated PLCbeta 3 activity. This can at least partially explain the inhibitory effect of PKA on Galpha q-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Ligand stimulation of seven transmembrane domain receptors coupled to Galpha proteins of the Galpha q or Galpha i subfamilies results in the activation of the respective heterotrimeric Galpha beta gamma protein complexes. Free Galpha q or Gbeta gamma subunits activate PLCbeta 1 isoforms to catalyze the production of IP3 and diacylglycerol from phosphatidylinositide 4,5-bisphosphate (1-3). PLCbeta 1-4 comprise the currently known mammalian phosphatidylinositide-specific PLCbeta subfamily. Although all PLCbeta s are activated by Galpha q, PLCbeta 2 and PLCbeta 3 are also stimulated by Gbeta gamma , primarily released from Galpha i (1).

Cross-talk between the G protein-PLCbeta pathway and PKA has been documented in numerous studies (4-13). Although it is generally agreed that G protein-activated PLCbeta activity can be inhibited by PKA (4-11), PKA can enhance the G protein-PLCbeta pathway in some cases (12, 13). Because PKA can inhibit phosphatidylinositide (PI) turnover activated by both Galpha q (4-8) and Galpha i (9-11) coupled receptors, it may inhibit the stimulation of both Galpha q- and Gbeta gamma -stimulated PLCbeta activity. This notion is further supported by studies with the G protein activators GTPgamma S and AlF4-. These two compounds nonselectively activate all heterotrimeric G proteins and generate free Galpha and Gbeta gamma subunits that can stimulate PLCbeta s. PKA inhibition of PI turnover initiated by GTPgamma S or AlF4- (5, 8, 14, 15) is consistent with the inhibition of Galpha q- as well as Gbeta gamma -stimulated PLCbeta activity. In addition, this phenomenon also suggests that the PKA effect is distal to receptors.

Recently, the mechanism for PKA inhibition of Gbeta gamma -stimulated PI turnover has been elucidated. Phosphorylation of PLCbeta 2 by PKA resulted in inhibition of Gbeta gamma -stimulated PI turnover (10). However, in the same study, PKA apparently did not inhibit Galpha 15- and Galpha 16-stimulated endogenous PLCbeta (beta 1 and beta 3) activity. More recently, Ali et al. (11) have reported phosphorylation of PLCbeta 3 in response to CPT-cAMP treatment in RBL-2H3 cells expressing only PLCbeta 3. CPT-cAMP inhibited Gbeta gamma -stimulated PLCbeta 3 activated by the Galpha i-coupled formylmethionylleucylphenylalanine receptor but had no effect on PAF-stimulated PLCbeta 3 activity, presumably mediated by Galpha q. These studies led to the conclusions that phosphorylation of PLCbeta 2 and PLCbeta 3 by PKA could explain the inhibition of Gbeta gamma -stimulated PI turnover by cAMP (10, 11). However, a biochemical mechanism for the inhibition by PKA of Galpha q-stimulated PLCbeta activity observed in several systems remains to be clarified. In this study, we present evidence that phosphorylation of PLCbeta 3 Ser1105 by PKA results in direct inhibition of Galpha q-stimulated PLCbeta 3 activity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- PLCbeta 3 antibody, immunoblotting, and immunoprecipitation reagents were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Lys-C was obtained from Wako Bioproducts (Richmond, VA). PKA catalytic subunit and other chemicals were purchased from Sigma. 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. Relaxin was purified from pregnant sow ovaries (16). The RBL-2H3 cell line was generously provided by Dr. H. Ali (Duke University). Galpha q was a gift from J. Hepler (Emory University), and the PLCbeta 1 clone in baculovirus was obtained from Dr. P. Sternweis (University of Texas Southwestern Medical Center). PKA catalytic subunit plasmid was kindly provided by Dr. S. McKnight (University of Washington), and Galpha q plasmid by Dr. M. Simon (California Institute of Technology).

Cloning, Site-directed Mutagenesis, and Protein Purification-- PLCbeta 3 and PLCbeta 3(His)6 in pCR3.1 vector (Invitrogen, San Diego, CA) and PLCbeta 3(His)6 in baculovirus (Pharmingen, San Diego, CA) were constructed from the PLCbeta 3 cDNA plasmid (17). Site-directed mutation of Ser1105 to Ala was achieved with the mutagenic primer (5'-AGCGCCATAACGCCATCTCGGAGG-3') using the GeneEditor kit (Promega, Madison, WI). All plasmid sequences were confirmed by DNA sequencing. PLCbeta 3(His)6 was purified essentially as described for PLCbeta 1 (18) from the membrane fraction from Sf9 cells and was 99% pure as judged by SDS-PAGE.

In Vitro Phosphorylation, Phosphoamino Acid Analysis, Peptide Mapping, and Sequencing-- 0.5, 1.5, or 2.5 µM purified recombinant PLCbeta 3(His)6 was incubated with PKA catalytic subunit at molar ratios of 20:1 or 50:1 in the presence of 1-10 µCi of [gamma -32P]ATP and 100 µM ATP in a total volume of 10 µl of PKA buffer (10 mM Tris, pH 7.0, 5 mM MgCl2) for 10 min at 30 °C. For the time course study, 1.3 µM PLCbeta 3(His)6 was incubated with PKA at a ratio of 10:1. Reactions were terminated by addition of an equal volume of 2× SDS sample buffer (15) and boiling for 5 min. Proteins were separated on SDS-PAGE gels, and the phosphorylated band was localized by autoradiography.

Two-dimensional phosphoamino acid analysis and peptide mapping of in vitro 32P-labeled PLCbeta 3(His)6 bound to PVDF membranes were carried out with a Hunter thin layer electrophoresis system (CBS Scientific Company, Del Mar, CA) according to the manufacturer's instructions. For two-dimensional peptide mapping, the membrane bound samples were digested with Lys-C (3 µg) for 24 h at 35 °C. For peptide sequencing, 150 pmol of [32P]PLCbeta 3(His)6 was digested with Lys-C (1 µg). The phosphopeptide separated by reverse-phase HPLC was sequenced at the microsequencing facility at Baylor College of Medicine (Houston, TX).

In Vivo 32P Labeling and Immunoprecipitation-- Nearly confluent PHM1-41 immortalized myometrial cells (10-cm dish) were labeled with [32P]orthophosphate (0.33 mCi/ml) in phosphate-free DMEM containing 10% dialyzed fetal calf serum for 4 h. After the treatments indicated in the figure legends, cells were lysed in 1 ml of ice-cold lysis buffer containing a mixture of protease and phosphatase inhibitors (11) and centrifuged at 15,000 × g for 5 min at 4 °C. Phosphorylated proteins immunoprecipitated with 4 µg of anti-PLCbeta 3 antibody were separated on a 7.5% SDS-PAGE gel, transferred to a PVDF membrane, and analyzed by autoradiography. PLCbeta 3 was visualized by Western blot using anti-PLCbeta 3 antibody (1:1000) to normalize for sample loading.

Cell Culture, Transfection, and PI Turnover-- COSM6 and RBL-2H3 cells were cultured and transfected as described (4, 11) with the following modifications. COSM6 cells were transfected with a total of 1.25 (see Fig. 4A) or 1.5 µg (see Fig. 4B) of plasmid DNA (using empty vector rcCMV as necessary) and 6 µl of LipofectAMINE in 0.75 ml of DMEM/well in 6-well plates, whereas 1.0 µg of total plasmid DNA and 5 µl of LipofectAMINE in 0.5 ml of DMEM were used to transfect RBL-2H3 cells. An equal volume of culture medium (4) containing 16% fetal calf serum was added 5 h later. The following day, cells were labeled with 6 µCi/well [3H]inositol in 1 ml of culture medium for 24 h at 37 °C. ZnSO4 (60 µM) was also included in the labeling medium to stimulate PKA catalytic subunit expression in COSM6 cells. After incubating with 10 mM LiCl for 10 (RBL-2H3) or 45 (COSM6) min, cells were treated as indicated in the figure legends and lysed by addition of ice-cold 10% trichloroacetic acid. The accumulation of [3H]IP3 was determined as described elsewhere (4).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

In Vitro and in Vivo Phosphorylation of PLCbeta 3 by PKA-- We have determined previously that the PKA inhibitory effect is distal to receptor and most likely affects the coupling between Galpha q and PLCbeta 1 or PLCbeta 3 isoforms in pregnant human myometrial (PHM1-41) and COSM6 cell lines (4). As shown in Fig. 1A, when incubated with PKA, C-terminal (His)6-tagged PLCbeta 3 (PLCbeta 3(His)6) purified from Sf9 cells was clearly a substrate for PKA in vitro. Similar results were also obtained with immunoprecipitation-purified recombinant PLCbeta 3 (data not shown). The phosphorylation of PLCbeta 3 was quite specific; neither highly purified recombinant Galpha q nor recombinant PLCbeta 1 was phosphorylated by PKA under similar conditions (data not shown), confirming previous observations (19, 20)


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Fig. 1.   A, in vitro phosphorylation by PKA of purified PLCbeta 3(His)6 (0.5, 1.5, and 2.5 µM for lanes 1, 3, and 2, respectively) in molar ratios of 1:20 (lane 1 and 2) or 1:50 (lane 3) for 10 min at 30 °C. Proteins separated on SDS-PAGE were analyzed by autoradiography. B, in vivo phosphorylation of endogenous PLCbeta 3 immunoprecipitated from PHM1-41 cells treated with PBS (lane C), 1.0 mM CPT-cAMP (lane CPT) for 10 min, or 1 µg/ml of relaxin (lane R) for 15 min. Proteins were separated on SDS-PAGE, transferred to a PVDF membrane, and analyzed by autoradiography (top panel). Fold stimulation after normalizing for PLCbeta 3 loading is shown in the bottom panel (data from one of two experiments). C, time course of in vitro phosphorylation of purified PLCbeta 3(His)6. The inset shows the PLCbeta 3 phosphorylation autoradiographs (Auto) and Coomassie Blue staining (Stain) at the indicated times. The densitometric analysis at each time point after normalizing for PLCbeta 3 loading is shown in the plot.

To quantify PKA-stimulated 32P incorporation from [gamma -32P]ATP, PLCbeta 3(His)6 was phosphorylated by PKA in vitro. Fig. 1C shows that the time course of 32P incorporation approached a plateau after 15 min. A maximum ratio of 0.65 mol phosphate/mol PLCbeta 3 was determined by filter binding assay at the 60-min incubation point. This is consistent with a single PKA phosphorylation site in PLCbeta 3.

To examine whether PLCbeta 3 could be phosphorylated in vivo, PHM1-41 myometrial cells were labeled with [32P]orthophosphate, and PKA was activated with the cell-permeable cAMP analogue CPT-cAMP or relaxin, a hormone that increases myometrial cell cAMP (21). Fig. 1B shows that PLCbeta 3 immunoprecipitated from cells exposed to CPT-cAMP or relaxin exhibited increased phosphorylation. After normalizing for the amount of PLCbeta 3 loaded, the treatments resulted in a 2-fold increase in PLCbeta 3 phosphorylation. A similar fold increase in PLCbeta 3 phosphorylation was recently reported in RBL-2H3 cells treated with CPT-cAMP (22). These data indicate that endogenous PLCbeta 3 can be phosphorylated in cells in response to elevated cAMP.

Identification of the PKA Phosphorylation Site-- Two-dimensional phosphoamino acid analysis with in vitro phosphorylated PLCbeta 3(His)6 revealed that only serine was phosphorylated by PKA (Fig. 2A). A similar result was also obtained with non-His-tagged recombinant PLCbeta 3 phosphorylated by PKA in COSM6 cells (data not shown). Peptide mapping of in vitro phosphorylated PLCbeta 3(His)6 digested with Lys-C revealed one major phosphorylated peptide (Fig. 2B). Importantly, an increase in phosphorylation of the same peptide (indicated by the arrow) was also detected in endogenous PLCbeta 3 in PHM1 (Fig. 2, D versus C) and COSM6 cells (Fig. 2, F versus E) treated with CPT-cAMP as well as in overexpressed PLCbeta 3 in COSM6 cells coexpressing PKA catalytic subunit (Fig. 2, H versus G). In the case of overexpressed PLC and PKA (Panels G, H), there appears to be phosphorylation of another site in the basal state that decreases when the PKA site is phosphorylated. We are in the process of determining the residue phosphorylated and the functional significance of this site. Importantly, in all three cases, activation of PKA increases phosphorylation on the site that is phosphorylated in vitro by PKA.


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Fig. 2.   A, autoradiograph showing that only 32P-labeled Ser was isolated from PLCbeta 3(His)6 phosphorylated in vitro by PKA at a molar ratio of 10:1. Dashed lines indicate the positions of phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) standards. B, autoradiograph of the two-dimensional peptide map derived from PLCbeta 3(His)6 radiolabeled by PKA in vitro as in A and digested with Lys-C. C-F, autoradiographs of two-dimensional peptide maps of immunoprecipitated endogenous PLCbeta 3 in PHM1-41 (C and D) and COSM6 (E and F) cells labeled with [32P]orthophosphate (0.75 mCi/dish, 10 cm) and treated with PBS (C and E) or 1.5 mM CPT-cAMP (D and F) for 10 min. G and H, autoradiographs of two-dimensional peptide maps of recombinant PLCbeta 3 immunoprecipitated from COSM6 cells transfected with plasmids expressing PLCbeta 3 (G) or PLCbeta 3 and PKA catalytic subunit (H). Cells were labeled with [32P]orthophosphate (0.25 mCi/dish, 35 cm) for 4 h. PLCbeta 3 was isolated and digested as described under "Experimental Procedures." The sample origins are denoted with open circles, and the peptides phosphorylated by PKA are denoted by arrows. The electrophoresis times (first dimension) were 20 (C, D, G, and H) and 25 min (E and F), respectively.

To identify the sequence of the phosphopeptide, PLCbeta 3(His)6 was phosphorylated in vitro and subjected to Lys-C digestion. A fraction containing more than 60% of the incorporated 32P was isolated by reverse-phase HPLC and sequenced. The 32P-labeled peptide was identified as RHNS1105ISEAK (Fig. 3A), in which more than 80% of Ser1105 was labeled, and no label was present in Ser1107. ~30% of the 32P found in the HPLC flow-through appeared to be free phosphate, as judged by phosphoamino acid analysis (data not shown).


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Fig. 3.   A, the sequence of the phosphopeptide isolated from PLCbeta 3(His)6 phosphorylated by PKA and the alignment of this sequence with the corresponding sequences in PLCbeta 1 and PLCbeta 2 (26). The position of [32P]phosphoserine1105 is designated by an asterisk. Ser and Thr are not found in comparable positions in either PLCbeta 1 or PLCbeta 2. B, in vitro phosphorylation by PKA of overexpressed wild type (lanes W) and Ser1105 right-arrow Ala mutant (lanes M) PLCbeta 3(His)6 purified from COSM6 cells. Protein bands (Coomassie) and their phosphorylation states (Autorad) are shown.

To confirm the phosphorylation site and avoid contamination with endogenous PLCbeta 3, His-tagged PLCbeta 3 (Ser1105 right-arrow Ala) mutant was constructed. This mutant was overexpressed in COSM6 cells, purified on a Ni-NTA column and phosphorylated by PKA in vitro. As shown in Fig. 3B, mutation of Ser1105 to Ala reduced PKA phosphorylation of PLCbeta 3 by ~90%. The small residual phosphorylation probably represents background, because it was also seen in extracts from cells transfected with empty vector and processed similarly (data not shown). We conclude from these studies that PKA phosphorylates PLCbeta 3 Ser1105 both in vivo and in vitro. Notably, this PKA phosphorylation site is not present in the corresponding sequences (20) of PLCbeta 1 or PLCbeta 2 (Fig. 3A).

Inhibition of Galpha q-stimulated PLCbeta 3 Activity by PKA-- The C terminus of PLCbeta 1 is critical for activation by Galpha q (23). Deletion studies have identified a P-box (Thr903 to Gln1030) and a G-box (Lys1031 to Leu1142) in this region. The P-box is essential for both PLCbeta 1 association with the cell membrane and its activation by Galpha q, whereas the G-box is involved in association with Galpha q subunit (24). Ser1105 of PLCbeta 3 falls in a region analogous to the G-box of PLCbeta 1. We therefore hypothesized that phosphorylation of Ser1105 by PKA might cause interference with Galpha q-PLCbeta 3 association and thereby inhibit Galpha q-stimulated PLCbeta 3 activity. To test this, PI turnover was studied in COSM6 cells transfected with Galpha q and PLCbeta 3 or PLCbeta 3 (Ser1105 right-arrow Ala) mutant in the absence and presence of PKA. As shown in Fig. 4A, transfection of empty vector (rcCMV), Galpha q, PLCbeta 3, or PLCbeta 3 (Ser1105 right-arrow Ala) mutant alone had no effect on basal PI turnover, suggesting that the proteins are primarily in their inactive forms under these conditions (25). Cotransfection of Galpha q with PLCbeta 3 produced a 5-fold increase in [3H]IP3, presumably because of the increased activation of PLCbeta 3 by Galpha q as reported previously (26). Notably, the PLCbeta 3 (Ser1105 right-arrow Ala) mutant was as effective as wild type at stimulating PI turnover. This indicates that the substitution of Ala for Ser1105 did not have a major effect on catalytic activity or G protein coupling. Importantly, when PKA catalytic subunit was also coexpressed, Galpha q-stimulated [3H]IP3 formation associated with wild type PLCbeta 3 was inhibited by ~58%, whereas no inhibition was observed with the PLCbeta 3 (Ser1105 right-arrow Ala) mutant. Increasing the amount of PKA catalytic subunit plasmid (0.5, 0.65, and 0.75 µg) resulted in a trend toward greater inhibition (58, 62, and 71%, respectively), although these values were not statistically different from each other (Fig. 4B). These data demonstrate both that PKA indeed inhibits Galpha q-stimulated PLCbeta 3 activity and that this effect requires the phosphorylation of Ser1105.


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Fig. 4.   A, PKA inhibits Galpha q-stimulated PLCbeta 3 but not mutant PLCbeta 3 (Ser1105 right-arrow Ala) activity. COSM6 cells were transiently transfected with empty vector (rcCMV) or plasmids expressing Galpha q (0.5 µg), PLCbeta 3 (P3, 0.25 µg), or PLCbeta 3 (S/A) mutant (P3 (S/A), 0.25 µg) as indicated in the absence (open bars) or presence (filled bars) of cotransfected PKA catalytic subunit plasmid (0.5 µg). Data represent duplicate determinations in one of two experiments; error bars give the range of duplicates. B, inhibition of Galpha q-stimulated PLCbeta 3 as in A, in the presence of 0.5, 0.65, or 0.75 µg of PKA catalytic subunit plasmid. Data are presented as the means ± S.E. of triplicates and were analyzed by analysis of variance and Duncan's test. Groups with different letters are different from each other at p < 0.05. C, prior treatment with CPT-cAMP (CPT) for 10 min inhibits 15 µM carbachol (Carb) or 100 nM oxytocin (OT)-stimulated (30 min) PI turnover in RBL-2H3 cells transfected with Galpha q (0.02 µg) and M1 muscarinic (M1R, 0.3 µg) or oxytocin (OTR, 0.5 µg) receptor plasmid DNA. The control cells were treated with PBS, oxytocin, or carbachol only. Data represent duplicate determinations in one of two experiments; error bars give the range of duplicates.

More evidence in support of this contention was obtained in the RBL-2H3 cell line expressing only PLCbeta 3 (11). RBL-2H3 cells were transfected with the M1 muscarinic and oxytocin receptors shown to couple in other cell types to PLCbeta through Galpha q proteins (26-29). Stimulation with the respective ligands resulted in a 4-fold increase in [3H]IP3, presumably through the coupling of the receptors to endogenous Galpha q and PLCbeta 3. Importantly, pretreating cells with CPT-cAMP, previously shown to activate endogenous PKA and result in PLCbeta 3 phosphorylation (11), completely inhibited M1 and oxytocin receptor-stimulated PI turnover (Fig. 4C). These data are consistent with our previous data in COSM6 and PHM1-41 cells (4). In myometrial membranes, oxytocin-stimulated PI turnover was determined to be essentially completely Galpha q-mediated (28).

Based on the position of Ser1105 in the enzyme, we hypothesize that its phosphorylation by PKA may perturb the association of PLCbeta 3 with Galpha q. However, we do not know at present how Ser1105 phosphorylation affects the kinetic properties of Galpha q/PLCbeta 3 coupling. It is also not yet clear what relationship this phosphorylation has to the reported inhibition of Gbeta gamma -stimulated PLCbeta 3 activity by PKA (11). These questions are under study. Interestingly, Ser954, one of the two putative PKA phosphorylation sites in PLCbeta 2, is located in the P-box. It has been suggested that PKA phosphorylation of that site may interfere with the membrane association of PLCbeta 2 (10). The close proximity of Ser1105 to the P-box may allow PKA to affect the membrane association of PLCbeta 3 as well.

Based on the ubiquitous expression of PLCbeta 3 (20), our observations could explain the inhibition of Galpha q-stimulated PI turnover by PKA observed in a variety of cells and tissues (4-11). However, the basis for the complete inhibition of Galpha q-stimulated PI turnover by PKA in cells expressing both PLCbeta 1 and PLCbeta 3 (4, 15) cannot be adequately addressed without knowing the cellular localization and relative contributions of these two isoenzymes to total PI turnover. Our data apparently contradict the reported inability of PKA to inhibit Galpha q-coupled PAF receptor-stimulated PLCbeta 3 activity in RBL-2H3 cells (11). The reason for this discrepancy is unknown at present but may reflect differences in the nature of specific receptor/G protein coupling in that cell line, differences in experimental design, or some other as yet unknown factor. Consistent with the findings reported here, we have found that coexpression of PKA catalytic subunit inhibits carbachol-stimulated PI turnover in COSM6 cells cotransfected with M1 muscarinic receptor and Galpha q (4). In contrast, Galpha 15- and Galpha 16-stimulated endogenous PLCbeta (beta 1 and beta 3) activity was not inhibited by PKA in COS7 cells (10). It is unclear how effectively the various G proteins stimulated PLCbeta 3 versus PLCbeta 1 in these cells. In reconstitution assays, Galpha 16 appears to be as effective as Galpha q in stimulating PLCbeta 1 but less effective than Galpha q in stimulating PLCbeta 3 (30). The relative contribution of PLCbeta 3 versus PLCbeta 1 to PI turnover and possible preferential coupling of Galpha q subfamily isoforms to PLCbeta 3 may account for some of the observed differences.

In summary, the data presented here establish a direct relationship between PKA-stimulated phosphorylation of Ser1105 and inhibition of PLCbeta 3 activity. This can at least partially explain the inhibitory effect of PKA on Galpha q-coupled receptor-stimulated PI turnover observed in a variety of cells and tissues.

    ACKNOWLEDGEMENTS

We thank Drs. S. McKnight, M. Simon, J. Hepler, P. Sternweis, and H. Ali for materials, F. Murad for access to the Hunter thin layer electrophoresis system, and R. Cooke for helpful advice on peptide sequence analysis.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HD09618 (to B. M. S.) and T32 HD07325 (to K. L. D.) and by funds from the Swedish Cancer Foundation (to G. W.).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 Houston Medical School, P.O. Box 20708, Houston, TX 77225. Tel.: 713-500-6064; Fax: 713-500-0652; E-mail: bsanborn{at}bmb.med.uth.tmc.edu.

1 The abbreviations used are: PLC, phospholipase C; IP3, phosphatidylinositol 1,4,5-trisphosphate; PKA, cAMP-dependent protein kinase; PI, phosphatidylinositide; GTPgamma S, guanosine 5'-[gamma -thio]trisphosphate; CPT-cAMP, 8-[4-chlorophenythio]-adenosine 3':5'-cyclic monophosphate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.

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Abstract
Introduction
Procedures
Results & Discussion
References

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