From the Afdeling Biochemie, Faculteit Geneeskunde, Campus Gasthuisberg, Katholieke Universiteit Leuven, Herestraat, B-3000 Leuven, Belgium
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ABSTRACT |
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Platelet-derived growth factor (PDGF)
stimulates protein kinase D (PKD) in a time- and
dose-dependent manner. We have used a series of PDGF
receptor mutants that display a selective impairment of the binding of
SH2-containing proteins (GTPase-activating protein, SHP-2,
phospholipase C (PLC
), or phosphatidylinositol 3'-kinase (PI3K))
to show that Tyr-1021, the PLC
-binding site, is essential for PKD
stimulation by PDGF in A431 cells. We next investigated whether any one
of these four binding sites could mediate PKD activation in the absence
of the other three sites. F5, a receptor mutant that lacks all four
binding sites for GTPase-activating protein, PLC
, PI3K, and SHP-2,
fails to activate PKD. A panel of single add-back mutants was used to
investigate if any one of these four sites could restore signaling to
PKD. Of the four sites, only the PLC
+ single
add-back receptor restored PDGF-mediated activation of PKD, and only
this add-back receptor produced diacylglycerol (DAG) in a
PDGF-dependent manner.
1,2-Dioctanoyl-sn-glycerol, a membrane-permeant DAG analog,
was found to be sufficient for activation of PKD. Taken together, these
data indicate that PLC
activation is not only necessary, but also
sufficient to mediate PDGF-induced PKD activation. Although the
presence of a pleckstrin homology domain makes PKD a potential PI3K
target, PKD was not stimulated by selective PI3K activation, and
wortmannin, an inhibitor of PI3K, did not inhibit PDGF signaling to
PKD. The activation of PKD by DAG or by the wild-type and
PLC
+ add-back PDGF receptors was inhibited by GF109203X,
suggesting a role for protein kinase C in the stimulation of PKD by
PDGF. PDGF induced a time-dependent phosphorylation of PKD
that closely correlated with activation. The PDGF-induced activation
and phosphorylation of PKD were reversed by in vitro
incubation of PKD with protein phosphatase 1 or 2A, indicating
that PDGF signaling to PKD involves the Ser/Thr phosphorylation of PKD.
Taken together, these results conclusively show that PDGF activates PKD
through a pathway that involves activation of PLC
and, subsequently,
protein kinase C.
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INTRODUCTION |
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The production of lipid second messengers is a common theme in the signal transduction of growth factors (1-5). An important task of current signal transduction research is to link these messengers to their targets or, vice versa, to find lipid messengers for proteins whose structure predicts potential lipid-binding sites.
Recently, two protein kinases were cloned (PKD1 from mouse and its human homolog, PKCµ) (6-9) that contain a kinase domain, a pleckstrin homology domain, a cysteine-rich zinc finger domain, and a putative transmembrane domain. We have demonstrated before that PKD is activated by diacylglycerol and by the tumor promotor PDB (8).
The cysteine-rich zinc fingers of the classical and novel PKC isoforms
have been shown to bind DAG and phorbol esters (10). However, zinc
fingers are also important for protein-protein interactions of PKC
and PKC
with stimulatory proteins (11, 12), and it is noteworthy
that the presence of a zinc finger in a kinase is not predictive for
the DAG/PDB stimulation of its kinase activity. The c-Raf zinc finger
mediates the interaction with phosphatidylserine-containing micelles
and 14-3-3 proteins and is required for optimal binding to Ras GTP, but
it cannot mediate PDB stimulation of Raf kinase activity (13, 14). Some
lipid-stimulated kinases can be activated by multiple lipids: PKC
and PKC
are activated not only by DAG, but also by
phosphatidylinositol 3,4,5-trisphosphate (10, 15). Akt/PKB is
stimulated by the lipid PtdIns(3,4)P2, which involves binding to the PH domain (16). Therefore, the presence of a PH domain
and zinc fingers in the N-terminal region of PKD would suggest several
possibilities for regulation of its kinase activity through interaction
with lipids or proteins. Given the large range of signaling mechanisms
that can possibly impinge upon the different domains of PKD (based on
the above-mentioned analogies), we decided to investigate which of
several growth factor signaling pathways can induce activation of
PKD.
We have chosen the -platelet-derived growth factor receptor
(
-PDGFR) as a paradigm for our studies. Binding of PDGF induces dimerization and autophosphorylation of the
-PDGFR at specific tyrosine residues. Through these specific phosphotyrosine motifs, the
phosphorylated PDGF receptor binds a large variety of SH2 proteins (for
a review, see Refs. 18 and 19). The p85 subunit of phosphatidylinositol
3'-kinase (PI3K) binds to tyrosines 740 and 751; the phosphotyrosine
phosphatase SHP-2 associates with tyrosine 1009; phospholipase C
1
binds to tyrosine 1021; and the Ras GTPase-activating protein (GAP)
associates with tyrosine 771. Furthermore, three members of the Src
kinase family (Src, Yes, and Fyn), Nck, Shc, and several as yet
unidentified proteins are known to associate with the
-PDGFR (18,
19). Using a panel of
-PDGFR mutants that are defective in the
binding of certain SH2 domain-containing proteins, it is possible to
selectively knock out or turn on specific signaling pathways so that
the functional role of a particular pathway to downstream responses can
be elucidated (20, 24, 25). Because of the large range of signaling
proteins that can bind to the
-PDGFR, this
-PDGFR mutant system
is well suited to investigate the variety of pathways that may activate newly identified components of the cellular signaling apparatus (e.g. PKD). This report shows that in A431 cells, PDGF
activates PKD through the subsequential activation of PLC
and
PKC.
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EXPERIMENTAL PROCEDURES |
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Materials-- PDGF was purchased from Upstate Biotechnology, Inc. GF109203X was obtained from Calbiochem, and G418, Lipofectin, Glutamax, and Opti-MEM from were from Life Technologies, Inc. The Biotrak DAG detection kit was from Amersham Corp. Antihemagglutinin antibodies were from Boehringer Mannheim. Protein A-TSK gel was from Affiland (Sart-Tilman, Belgium). All other materials were from Sigma.
Cell Culture and Preparation of Extracts-- Swiss 3T3 cells were grown in DMEM (1 g/liter of glucose) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM Glutamax. Cells were used 6-8 days after plating, when they were confluent and quiescent. A431 cells were grown in DMEM (4.5 g/liter of glucose) supplemented with 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM Glutamax, and 1 mg/ml G418. Before experiments, 80% confluent A431 cells were starved for 48 h in serum-free DMEM.
The A431 cell line (ATCC CRL 1555), devoid of endogenousImmunoprecipitations and Kinase Assays-- Lysates were incubated for 2 h with an antibody against the C-terminal 15 amino acids of PKD or with hemagglutinin antibodies (for hemagglutinin-tagged PKB). Immunocomplexes were captured with 15 µl of protein A-TSK gel for 1 h.
PKD immunoprecipitates were washed twice with lysis buffer A, and PKD was eluted by incubating the immunoprecipitates with lysis buffer containing a 0.5 mg/ml concentration of the immunizing peptide. 15 µl of PKD eluate was incubated for 5 min at 30 °C with 25 µl of a kinase assay mixture, resulting in a final concentration of 20 mM Tris (pH 7.4), 100 µM ATP (specific activity of 1000 cpm/pmol), 10 mM MgCl2, and 1 mg/ml syntide-2. Akt/PKB immunoprecipitates were washed once with lysis buffer, once with lysis buffer containing 0.5 M LiCl2, and twice with kinase assay buffer. The PKB immunoprecipitation pellet was incubated for 15 min with the same phosphorylation mixture as described above, except that syntide-2 was replaced by the RGRPRTTSFAE peptide corresponding to the site in glycogen synthase kinase 3-DAG Production Assay-- DAG production was measured using the Biotrak DAG detection kit, which uses [32P]phosphatidic acid yield by DAG kinase as a measure of DAG production, according to the instructions of the manufacturer. Briefly, lipids were extracted according to the method of Bligh and Dyer (27) and incubated in a DAG kinase reaction mixture containing 0.05 M imidazole (pH 6.6), 0.05 M NaCl, 12 mM MgCl2, 1 mM EGTA, and 500 µM ATP (specific activity of 50 cpm/pmol).
32Pi Labeling of Cells and Analysis of PKD Phosphorylation-- Confluent cultures of A431 cells were washed twice with DMEM (phosphate-free) and incubated in this medium containing 500 µCi/ml carrier-free 32Pi overnight (12 h). Cells were then stimulated for the indicated times with PDGF (30 ng/ml) and lysed in buffer A. Lysates were subsequently immunoprecipitated with anti-PKD antibody and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Protein Phosphatase Incubations-- PKD eluates were incubated for 30 min at 30 °C with 50 units/ml PP1C or PP2AC in the presence or absence of 1 µM microcystin. After this incubation, a PKD kinase assay was performed as indicated above in the presence of 1 µM microcystin. For visualization of the dephosphorylation of PKD by serine/threonine protein phosphatases, PKD was immunoprecipitated from lysates of 32Pi-labeled cells that were stimulated with PDGF. Immunoprecipitated PKD was then eluted from the immunocomplexes and incubated for 30 min at 30 °C with 50 units/ml PP1C or PP2AC in the presence or absence of 1 µM microcystin.
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RESULTS AND DISCUSSION |
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This work is the first report of a dissection of specific growth
factor signaling pathways that activate PKD. PDGF stimulates PKD in a
time- and dose-dependent manner, both in Swiss 3T3 cells expressing endogenous PDGF receptor and in A431 cells stably
overexpressing a retrovirally introduced PDGF receptor. PKD is
stimulated by PDGF doses as low as 5 ng/ml, with a maximum at 30 ng/ml
(Fig. 1, A and B),
which correlates well with the concentration of PDGF required for a
variety of cellular responses such as PLC, PI3K, and GAP tyrosine
phosphorylation (28). PKD activity reached a maximum after 10 min, but
remained elevated even at 90 min after addition of PDGF (Fig. 1,
C and D). Equal amounts of PKD were present in
immunoprecipitates from Swiss 3T3 cells or A431 cells stimulated for
various times with PDGF, as evidenced by Western blotting followed by
immunostaining with anti-PKD antibodies (Fig. 1, E and
F).
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Activation of PLC by the
-PDGFR Is Necessary and Sufficient
to Activate PKD--
Selective mutation of the PLC
-binding site of
the
-PDGFR into phenylalanine completely abolished the
-PDGFR-induced activation of PKD. Full activation was retained when
binding sites for SHP-2, GAP, and PI3K were selectively mutated into
phenylalanine. These results suggest that the binding of PLC
to the
-PDGFR is essential for activation of PKD (Table
I, Minus mutants).
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The PI3K Pathway Does Not Signal to PKD--
The multidomain
structure of PKD prompted us to thoroughly investigate a variety of
pathways that may impinge on this enzyme. The presence of a pleckstrin
homology domain in PKD may represent a target for modulating enzymatic
activity. It has been shown that the PH domain of Akt/PKB is crucial
for the PI3K-mediated activation of the enzyme (16, 32). Therefore, we
investigated whether PKD could be activated by selective PI3K
activation (Table II). The
PI3K+ add-back receptor, which activates PI3K without
PLC activation, failed to activate PKD, whereas it activated
Akt/PKB. Moreover, the activation of PKD by PDGF is not inhibited by
wortmannin, a known inhibitor of PI3K (33). Taken together, these data
clearly show that PKD is not a target for PI3K signaling.
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Involvement of PKC in PKD Activation--
We next investigated
whether in vivo PKD activation requires the catalytic
activity of PKC. Preincubation of A431 cells with GF109203X, a very
potent inhibitor of PKC (35, 36) but not of PKD (37), completely
abolished activation of PKD by PDGF in the WT and the
PLC+ mutant cell lines (Table
III). The activation of PKD by PDB or the
DAG analog diC8 was also completely abolished by preincubation with
GF109203X (Table III). These results indicate that PDGF causes activation of PKD through the activation of PKC.
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PDGF-induced PKD Activation Involves the Ser/Thr Phosphorylation of PKD-- PDGF induces phosphorylation of PKD, as evidenced by the incorporation of 32P in PKD immunoprecipitated from 32Pi-labeled cells that were stimulated for various times with PDGF (Fig. 3B). When comparing Fig. 1D and Fig. 3B, it is clear that the time course of incorporation of phosphate in PKD closely parallels the time course of activation of the enzyme. To show that PDGF-induced phosphorylation of PKD is required for activation of PKD, we incubated the activated PKD with the Ser/Thr-specific protein phosphatase PP1C or PP2AC. Each phosphatase was able to fully reverse the PKD activation, and this inactivation was blocked by the specific phosphatase inhibitor microcystin (Fig. 3A). To further demonstrate the effect of each of these phosphatases on the phosphorylation status of PKD, we incubated the 32Pi-labeled PKD from PDGF-stimulated cells with PP1C and PP2AC. As shown in Fig. 3A (insets), both Ser/Thr-specific phosphatases caused dephosphorylation of PKD. These data strongly suggest that a Ser/Thr phosphorylation event is involved in the activation of PKD by PDGF. Similar effects of phosphatases have been reported for other kinases such as extracellular signal-related kinase and PKB, which are also stimulated in kinase cascades (43, 44). The existence of multiple levels of control in kinase activation mechanisms is not without precedence. Akt/PKB, another PH domain-containing kinase, is activated by both protein phosphorylation (44, 45) and inositol lipid binding (16, 32). In this respect, a particularly interesting similarity between PKD and Akt/PKB regulation emerges. Both enzymes can be directly stimulated in vitro by a lipid mediator (DAG and PtdIns(3,4,5)P3, respectively), and both enzymes can be stimulated in vivo by another upstream kinase (PKC and 3-phosphoinositidedependent protein kinase 1, respectively) (16, 32, 43-45). Hence, the dual regulation of protein kinases by lipid ligands and protein phosphorylation emerges as a new regulatory theme in signal transduction.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Kazlauskas (Harvard Medical
School, Boston) for the generous gift of A431 cell lines expressing
-PDGFR mutants; Dr. J. Woodgett (Ontario Cancer Institute, Toronto)
for the PKB-pcDNA3 construct; and Drs. J Goris and M. Bollen
(Katholieke Universiteit Leuven, Belgium) for PP2A and PP1,
respectively. The expert technical assistance of V. Feytons and S. Vander Perre is also greatly appreciated.
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FOOTNOTES |
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* This work was supported by grants from the European Community (Inco-Copernicus), the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Actie Levenslijn), and the Flemish Government (Geconcerteerde Onderzoeksacties).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.
Postdoctoral Research Fellow of the Fund for Scientific Research
(Fonds voor Wetenschappelijk Onderzoek-Vlaanderen). To whom correspondence should be addressed. Tel.: 32-16-345719; Fax:
32-16-345995; E-mail: Johan.Vanlint{at}MED.KULeuven.ac.be.
§ Present address: Lithuanian Academy of Sciences, Inst. of Biochemistry, 2600 Vilnius, Lithuania.
¶ Research Director of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.
1
The abbreviations used are: PKD, protein kinase
D; PKC, protein kinase C; PDB, phorbol 12,13-dibutyrate; DAG,
diacylglycerol; PtdIns, phosphatidylinositol; PH domain, pleckstrin
homology domain; -PDGFR,
-platelet-derived growth factor
receptor; PDGF, platelet-derived growth factor; PI3K,
phosphatidylinositol 3'-kinase; GAP, Ras GTPase-activating protein;
PLC
, phospholipase C
; DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum; diC8, 1,2-dioctanoyl-sn-glycerol; PP1C/PP2AC, catalytic subunits of the type
1/type 2A protein phosphatases.
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REFERENCES |
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