From the 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
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ABSTRACT |
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The mechanism by which protein kinase A (PKA)
inhibits Gq-stimulated phospholipase C activity of
the
subclass (PLC
) is unknown. We present evidence that
phosphorylation of PLC
3 by PKA results in inhibition of
G
q-stimulated PLC
3 activity, and we
identify the site of phosphorylation. Two-dimensional phosphoamino acid
analysis of in vitro phosphorylated PLC
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 G
q with
PLC
3 or PLC
3 (Ser1105
Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation
compared with expression of G
q, PLC
3, or
PLC
3 (Ser1105
Ala) mutant alone. Whereas
G
q-stimulated PLC
3 activity was inhibited
by 58-71% by overexpression of PKA catalytic subunit, G
q-stimulated PLC
3 (Ser1105
Ala) mutant activity was not affected. Furthermore,
phosphatidylinositide turnover stimulated by presumably
G
q-coupled M1 muscarinic and oxytocin receptors was
completely inhibited by pretreating cells with
8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLC
3. These data establish that direct phosphorylation
by PKA of Ser1105 in the putative G-box of
PLC
3 inhibits G
q-stimulated
PLC
3 activity. This can at least partially explain the
inhibitory effect of PKA on G
q-stimulated
phosphatidylinositide turnover observed in a variety of cells and
tissues.
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INTRODUCTION |
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Ligand stimulation of seven transmembrane domain receptors coupled
to G proteins of the G
q or G
i
subfamilies results in the activation of the respective heterotrimeric
G
protein complexes. Free G
q or G
subunits activate PLC
1
isoforms to catalyze the production of IP3 and
diacylglycerol from phosphatidylinositide 4,5-bisphosphate (1-3).
PLC
1-4 comprise the currently known mammalian
phosphatidylinositide-specific PLC
subfamily. Although all PLC
s
are activated by G
q, PLC
2 and
PLC
3 are also stimulated by G
, primarily released
from G
i (1).
Cross-talk between the G protein-PLC pathway and PKA has been
documented in numerous studies (4-13). Although it is generally agreed
that G protein-activated PLC
activity can be inhibited by PKA
(4-11), PKA can enhance the G protein-PLC
pathway in some cases
(12, 13). Because PKA can inhibit phosphatidylinositide (PI) turnover
activated by both G
q (4-8) and G
i
(9-11) coupled receptors, it may inhibit the stimulation of both
G
q- and G
-stimulated PLC
activity. This notion
is further supported by studies with the G protein activators GTP
S
and AlF4
. These two compounds
nonselectively activate all heterotrimeric G proteins and generate free
G
and G
subunits that can stimulate PLC
s. PKA inhibition of
PI turnover initiated by GTP
S or
AlF4
(5, 8, 14, 15) is consistent with
the inhibition of G
q- as well as G
-stimulated PLC
activity.
In addition, this phenomenon also suggests that the PKA effect is
distal to receptors.
Recently, the mechanism for PKA inhibition of G-stimulated PI
turnover has been elucidated. Phosphorylation of PLC
2 by PKA resulted in inhibition of G
-stimulated PI turnover (10). However, in the same study, PKA apparently did not inhibit
G
15- and G
16-stimulated endogenous PLC
(
1 and
3) activity. More recently, Ali
et al. (11) have reported phosphorylation of
PLC
3 in response to CPT-cAMP treatment in RBL-2H3 cells
expressing only PLC
3. CPT-cAMP inhibited
G
-stimulated PLC
3 activated by the
G
i-coupled formylmethionylleucylphenylalanine receptor but had no effect on PAF-stimulated PLC
3 activity,
presumably mediated by G
q. These studies led to the
conclusions that phosphorylation of PLC
2 and
PLC
3 by PKA could explain the inhibition of
G
-stimulated PI turnover by cAMP (10, 11). However, a biochemical
mechanism for the inhibition by PKA of G
q-stimulated
PLC
activity observed in several systems remains to be clarified. In
this study, we present evidence that phosphorylation of
PLC
3 Ser1105 by PKA results in direct
inhibition of G
q-stimulated PLC
3
activity.
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EXPERIMENTAL PROCEDURES |
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Materials--
PLC3 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 [
-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). G
q
was a gift from J. Hepler (Emory University), and the
PLC
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 G
q plasmid by Dr. M. Simon (California Institute of Technology).
Cloning, Site-directed Mutagenesis, and Protein
Purification--
PLC3 and
PLC
3(His)6 in pCR3.1 vector (Invitrogen, San
Diego, CA) and PLC
3(His)6 in baculovirus
(Pharmingen, San Diego, CA) were constructed from the
PLC
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.
PLC
3(His)6 was purified essentially as
described for PLC
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 PLC3(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 [
-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
PLC
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.
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-PLC3 antibody were separated on a 7.5% SDS-PAGE
gel, transferred to a PVDF membrane, and analyzed by autoradiography.
PLC
3 was visualized by Western blot using
anti-PLC
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).
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RESULTS AND DISCUSSION |
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In Vitro and in Vivo Phosphorylation of PLC3 by
PKA--
We have determined previously that the PKA inhibitory effect
is distal to receptor and most likely affects the coupling between G
q and PLC
1 or PLC
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
PLC
3 (PLC
3(His)6) purified from Sf9 cells was clearly a substrate for PKA in vitro. Similar results were also obtained with immunoprecipitation-purified
recombinant PLC
3 (data not shown). The phosphorylation
of PLC
3 was quite specific; neither highly purified
recombinant G
q nor recombinant PLC
1 was
phosphorylated by PKA under similar conditions (data not shown),
confirming previous observations (19, 20)
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Identification of the PKA Phosphorylation
Site--
Two-dimensional phosphoamino acid analysis with in
vitro phosphorylated PLC3(His)6
revealed that only serine was phosphorylated by PKA (Fig.
2A). A similar result was also
obtained with non-His-tagged recombinant PLC
3
phosphorylated by PKA in COSM6 cells (data not shown). Peptide mapping
of in vitro phosphorylated
PLC
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 PLC
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 PLC
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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Inhibition of Gq-stimulated PLC
3
Activity by PKA--
The C terminus of PLC
1 is critical
for activation by G
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 PLC
1 association with the
cell membrane and its activation by G
q, whereas the G-box is involved in association with G
q subunit (24).
Ser1105 of PLC
3 falls in a region analogous
to the G-box of PLC
1. We therefore hypothesized that
phosphorylation of Ser1105 by PKA might cause interference
with G
q-PLC
3 association and thereby
inhibit G
q-stimulated PLC
3 activity. To
test this, PI turnover was studied in COSM6 cells transfected with
G
q and PLC
3 or PLC
3
(Ser1105
Ala) mutant in the absence and presence of
PKA. As shown in Fig. 4A,
transfection of empty vector (rcCMV), G
q,
PLC
3, or PLC
3 (Ser1105
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 G
q with
PLC
3 produced a 5-fold increase in
[3H]IP3, presumably because of the increased
activation of PLC
3 by G
q as reported
previously (26). Notably, the PLC
3 (Ser1105
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, G
q-stimulated
[3H]IP3 formation associated with wild type
PLC
3 was inhibited by ~58%, whereas no inhibition was
observed with the PLC
3 (Ser1105
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
G
q-stimulated PLC
3 activity and that this
effect requires the phosphorylation of Ser1105.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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;
GTPS, guanosine 5'-[
-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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REFERENCES |
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