Tyrosine Phosphorylation of Protein Kinase D in the Pleckstrin Homology Domain Leads to Activation*

Peter StorzDagger , Heike DöpplerDagger , Franz-Josef Johannesdagger§, and Alex TokerDagger

From the Dagger  Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 and § Fraunhofer Institute for Interfacial Engineering and Biotechnology, 70569 Stuttgart, Germany

Received for publication, December 27, 2002, and in revised form, March 7, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein kinase D (PKD) is a member of the AGC family of Ser/Thr kinases and is distantly related to protein kinase C (PKC). Formerly known as PKCµ, PKD contains protein domains not found in conventional PKC isoforms. A functional pleckstrin homology (PH) domain is critical for the regulation of PKD activity. Here we report that PKD is tyrosine-phosphorylated within the PH domain, leading to activation. This phosphorylation is mediated by a pathway that consists of the Src and Abl tyrosine kinases and occurs in response to stimulation with pervanadate and oxidative stress. Mutational analysis revealed three tyrosine phosphorylation sites (Tyr432, Tyr463, and Tyr502), which are regulated by the Src-Abl pathway, and phosphorylation of only one of these (Tyr463) leads to PKD activation. By using a phospho-specific antibody, we show that Abl directly phosphorylates PKD at Tyr463 in vitro, and in cells phosphorylation of this site is sufficient to mediate full activation of PKD. Mutation of the other two sites, Tyr432 and Tyr502, had no significant influence on PKD activity. These data reveal a tyrosine phosphorylation-dependent activation mechanism for PKD and suggest that this event contributes to the release of the autoinhibitory PKD PH domain leading to kinase activation and downstream responses.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein kinase D (PKD)1/PKCµ is a serine/threonine protein kinase with homology to conventional PKC isoforms in the regulatory domain (1, 2). However, PKD has additional protein modules not found in other PKCs, including an acidic domain, a hydrophobic domain, and a pleckstrin homology (PH) domain, whose functions are yet not fully defined (3-5). Thus, PKD together with two other homologues, PKD2 (6) and PKD3/PKCnu (7), represent a family of protein kinases whose regulation and function are distinct from conventional PKC isoenzymes (8). PKD is activated in response to numerous stimuli including platelet-derived growth factor (PDGF) (9), triggering of the B-cell receptor (10) or T-cell receptor complex (11, 12), oxidative stress (13, 14), and through G-protein-coupled receptors (15-17). Genotoxic agents have also been shown to activate PKD via a caspase 3-dependent mechanism (18, 19).

PKD plays a major role in Golgi function and organization (15, 20-22). In addition, nuclear factor-kappa B (NF-kappa B) as well as the serum-response element have been shown to be targets of PKD-dependent signaling (14, 23). PKD-mediated activation of serum-response element-driven genes was shown to occur via a Raf-1 kinase-dependent mitogen-activated protein kinase pathway (23), and the activation of PKD by mitogens suggests a role for PKD in cellular proliferation (24). Moreover, activation of NF-kappa B by PKD indicates an essential role for this kinase in promoting cellular survival in response to oxidative stress (14).

As with other members of the AGC kinase superfamily, PKD activity and function are tightly regulated by phosphorylation. Two serine residues, Ser203 and Ser916 (in murine PKD), have been mapped as autophosphorylation sites (11, 25). Furthermore, two serine residues (Ser744 and Ser748 in murine PKD), located within the activation loop, are trans-phosphorylated in cells, and this is necessary for full catalytic activity (26, 27). Recent studies (26, 28) have shown that PKCs are upstream kinases for PKD and can directly phosphorylate the activation loop serine residues. Unlike members of the PKC family, PKD lacks an autoinhibitory pseudosubstrate sequence (5). However, deletion analysis of the PKD regulatory domain revealed that the amino-terminal pleckstrin homology (PH) domain has an autoinhibitory function, such that PH domain deletion mutants are constitutively active in cells (3). Interestingly, PKCs including PKCeta , have been shown to bind to PKD, more specifically to the PH domain (29, 30). In addition, direct phosphorylation of the PKD activation loop serines by PKCepsilon has been shown to release PH domain autoinhibition (28). However, the precise mechanism by which the PH domain exerts negative regulation on PKD activity, and how this is relieved, has not been elucidated.

Here we report that tyrosine phosphorylation of PKD occurs within the PH domain, leading to activation. This phosphorylation is mediated by the Src/Abl signaling pathway in response to oxidative stress as well as pervanadate treatment of cells. We show that three tyrosine phosphorylation sites within the PKD PH domain are regulated by Src/Abl signaling, and phosphorylation of one of these (Tyr463) is responsible for PKD activation. These results provide evidence for a tyrosine phosphorylationdependent activation mechanism for PKD.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell Lines, Antibodies, and Reagents-- The HeLa cell line was from the American Type Culture Collection and maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The anti-Abl, anti-Src (polyclonal), and anti-PKD/PKCµ antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-Src (monoclonal) and anti-phosphotyrosine (4G10) were from Upstate Biotechnology, Inc. (Waltham, MA). Anti-HA was purified in-house from the 12CA5 hybridoma. The anti-pY463 has been described previously (14). The secondary POX-linked anti-mouse or anti-rabbit antibodies were from Roche Applied Science. Pervanadate was prepared by mixing 1 ml of a 20 mM sodium orthovanadate with 330 µl of 30% H2O2 (Fisher) for 10 min at room temperature, yielding a 15 mM solution of pervanadate and H2O2. Residual H2O2 was inactivated by a 15-min incubation with 10 µl of catalase (8 units/mg, Sigma). Pervanadate solutions were freshly prepared for each experiment. The PKD-specific substrate peptide used was AALVRQMSVAFFFK. Superfect (Qiagen, Valencia, CA) was used for transient transfections. Purified recombinant catalytic subunit gamma -isoform of human protein phosphatase 1 (PP-1gamma ) was from Calbiochem, and human recombinant CD45 protein-tyrosine phosphatase was from Biomol (Plymouth Meeting, PA). The active, purified Abl kinase fragment was from Calbiochem. The Src family kinase inhibitor PP1 was from Biomol, and PP2 and the control PP3 were from Calbiochem. 12-Phorbol 13-myristate acetate (PMA) was from Sigma. Bovine brain PtdSer and sn-1,2-dioleoylglycerol were from Avanti Polar Lipids (Alabaster, AL).

DNA Constructs-- Full-length PKD expression plasmids are based on an amino-terminal HA-PKD in pcDNA3, derived from human PKD/PKCµ, using PCR with the following primer pairs: 5'-GCGGGATCCATGTATCCTTATGATGTTCTTGATTATGCTAGCGCCCCTCCGGTCCTG-3' and 5'-GCGCTCGAGTCAGAGGATGCTGACACGCTC-3'. Amino-terminal HA-tagged Delta PH-PKD (HA-Delta PH-PKD) and HA-tagged PKD.K612W (HA-PKD.K612W) were created by PCR using the above pairs and using non-tagged Delta PH-PKD or PKD.K612W (both described previously (23, 31)) as templates and cloned into pcDNA3 via BamHI and XhoI. Mutagenesis was carried out by PCR using QuickChange (Stratagene, La Jolla, CA) with the following primer pairs: PKD.Y432F, 5'-GGATGGATGGTCCACTTCACCAGCAAGGACACG-3' and 5'-CGTGTCCTTGCTGGTGAAGTGGACCATCCATCC-3'; PKD.Y432E, 5'-GGATGGATGGTCCACGAAACCAGCAAGGACACG-3' and 5'-CGTGTCCTTGCTGGTTTCGTGGACCATCCATCC-3'; PKD.Y432Q, 5'-GGATGGATGGTCCACCAAACCAGCAAGGACACG-3' and 5'-CGTGTCCTTGCTGGTTTGGTGGACCATCCATCC-3'; PKD.Y443F, 5'-CTGCGGAAACGGCACTTTTGGAGATTGGATAGC-3' and 5'-GCTATCCAATCTCCAAAAGTGCCGTTTCCGCAG-3'; PKD.Y462F, 5'-GACACAGGAAGCAGGTTCTACAAGGAAATTCTT-3' and 5'-AGGAATTTCCTTGTAGAACCTGCTTCCTGTGTC-3'; PKD.Y463F, 5'-GACACAGGAAGCAGGTACTTCAAGGAAATTCCTTTATCT-3' and 5'-AGATAAAGGAATTTCCTTGAAGTACCTGCTTCCTGTGTC-3'; PKD.Y463E, 5'-GACACAGGAAGCAGGTACGAAAAGGAAATTCTTTTATCT-3' and 5'-AGATAAAGGAATTTCCTTTTCGTACCTGCTTCCTGTGTC-3'; PKD.Y463Q, 5'-GACCACGGAAGCAGGTACCAAAAGGAAATTCTTTTATCT-3' and 5'-AGATAAAGGAATTTCCTTTTGGTACCTGCTTCCTGTGTC-3'; PKD.Y501F, 5'-ATCACTACGGCAAATGTAGTGTTTTATGTGGGAGAAAATGTGGTC-3' and 5'-GACCACATTTTCTCCCACATAAAACACTACATTTGCCGTAGTGAT-3'; PKD.Y502F, 5'-ACGGCAAATGTAGTGTATTTTGTGGGAGAAAATGTGGTC-3' and 5'-GACCACATTTTCTCCCACAAAATACACTACATTTGCCGT-3'; PKD.Y502E, 5'-GCAAATGTAGTGTATGAAGTGGGAGAAAATGTG-3' and 5'-CACATTTTCTCCCACTTCATACACTACATTTGC-3'; and PKD.Y502Q, 5'-GCAAATGTAGTGTATCAAGTGGGAGAAAATGTG-3' and 5'-CACATTTTCTTCCACTTGATACACTACATTTGC-3'. All constructs were verified by DNA sequencing. v-Abl p120, Src wild-type, Src*, and Src.Y527F constructs have been described (14).

Purification of Recombinant Proteins-- Recombinant His6- and HA-tagged PKD and PKD.K612W were expressed in baculovirus-infected Sf9 insect cells. Infection, harvesting, cell lysis, and purification of proteins by Ni2+-nitrilotriacetate chromatography has been described (32).

Immunoblotting and Immunoprecipitation-- Cells were lysed in lysis buffer (50 mM Tris/HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, pH 7.4) plus protease inhibitor mixture (Sigma). Lysates were used either for immunoblot analysis or proteins of interest were immunoprecipitated by a 1-h incubation with the respective antibody (2 µg) followed by a 30-min incubation with protein G-Sepharose (Amersham Biosciences). Immune complexes were washed three times with TBS (50 mM Tris/HCl, pH 7.4, 150 mM NaCl) and resolved by SDS-PAGE or subjected to in vitro kinase assays.

Preparation of PS/DOG-- Mixed lipid vesicles were prepared by drying lipids (diacylglycerol in a PtdSer background; DOG/PtdSer, 4:140 µM) stored in chloroform/methanol (1:1, v/v) under a stream of nitrogen. Lipids were reconstituted by sonication into PKD kinase buffer in an ice bath sonicator at 4 °C for 10 min at 50% output. Fresh preparations were made for each experiment.

Phosphorylation of PKD by Abl-- Purified PKD (500 ng) and Abl kinase (25 ng) and lipids (PS/DOG) were mixed in a total volume of 40 µl of kinase buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 2 mM dithiothreitol). The kinase reaction was carried out for 20 min at 37 °C after addition of 10 µl of ATP solution (75 µM ATP in kinase buffer). The reaction was stopped with sample buffer, or PKD was immunoprecipitated, and PKD kinase assays were performed. Samples were applied to SDS-PAGE and transferred to nitrocellulose.

PKD Kinase Assays-- After immunoprecipitation (anti-PKD for endogenous and anti-HA for transfected PKD) and washing, 20 µl of kinase buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 2 mM dithiothreitol) was added to the precipitates. The kinase reaction was carried out for 20 min at room temperature after addition of 10 µl of kinase substrate mixture (150 µM PKD-specific substrate peptide, 50 µM ATP, 10 µCi of [gamma -32P]ATP in kinase buffer). To terminate, the samples were centrifuged, and the supernatants were spotted onto P81 phosphocellulose paper (Whatman). The papers were washed three times with 0.75% phosphoric acid and once with acetone and dried, and activity was determined by liquid scintillation counting. For autophosphorylation assays, the reaction was performed in the absence of substrate peptide and was stopped by the addition of sample buffer. Samples were resolved by SDS-PAGE and transferred to nitrocellulose.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pervanadate Treatment of HeLa Cells Activates PKD-- H2O2 stimulation of cells has been shown to stimulate PKD activity (13, 14). In order to investigate a potential role for tyrosine phosphorylation in the activation of PKD, HeLa cells were treated in a time- and dose-dependent manner with the phosphotyrosine phosphatase inhibitor pervanadate. Activation of PKD was monitored by phosphorylation of a PKD-specific synthetic peptide substrate (33), as well as by autophosphorylation. Substrate phosphorylation increased by 4-5-fold and autophosphorylation by 4-fold following a 10-min treatment of cells with 75 µM pervanadate (Fig. 1A). Pervanadate-induced activation of PKD peaked by 30 min of stimulation and with a quantitatively identical response to control PMA treatment of HeLa cells (Fig. 1B). The production of pervanadate requires the inactivation of H2O2 with catalase (see "Experimental Procedures"). In control experiments using catalase, we demonstrated that there is no residual H2O2 in the pervanadate preparations such that the observed PKD activation is due to pervanadate treatment alone (Fig. 1C). Overexpression of either wild-type PKD or a kinase-inactive PKD allele containing a mutation in the ATP-binding site (K612W) revealed that phosphorylation of PKD in response to pervanadate is due to PKD autophosphorylation and not due to co-immunoprecipitating kinase (Fig. 1D).


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Fig. 1.   PKD activation by pervanadate. 2 Mio HeLa cells were treated for 10 min with pervanadate in a dose-dependent manner (A) or treated with 75 µM pervanadate in a time-dependent manner (B). PKD was immunoprecipitated (IP) and autophosphorylation or substrate phosphorylation kinase assays were performed. C, 2 Mio HeLa cells were treated for 10 min with H2O2 (10 µM), catalase (5 µl of a 1 unit/ml stock), or pervanadate (75 µM) as indicated. PKD was immunoprecipitated, and an autophosphorylation kinase assay was performed. D, wild-type PKD or PKD.K612W was overexpressed, and cells were treated with pervanadate (PV) (10 min, 75 µM). PKD was immunoprecipitated, and an autophosphorylation kinase assay was performed. Immunoblots of immunoprecipitated PKD revealed equivalent expression of PKD in each experiment. Numbers represent relative PKD autophosphorylation activity compared with respective controls. All results are typical of three independent experiments.

Pervanadate-induced Tyrosine Phosphorylation of PKD Leads to Activation-- Since PKD can be activated by pervanadate and H2O2, agents that elevate intracellular phosphotyrosine levels, we determined the tyrosine phosphorylation of PKD in response to these stimuli. Pervanadate treatment increased tyrosine phosphorylation of either endogenous (Fig. 2A) or overexpressed PKD (Fig. 2B). The control phorbol ester PMA did not induce PKD tyrosine phosphorylation, despite the fact that it is a potent activator of PKD. Moreover, there was a good correlation between the kinetics of PKD activation and tyrosine phosphorylation in pervanadate-treated HeLa cells (compare Figs. 1B to 2A). Interestingly, PKD kinase activity was not necessary for pervanadate-induced tyrosine phosphorylation, suggesting that tyrosine phosphorylation occurs prior to PKD autophosphorylation (Fig. 2B). To determine whether tyrosine phosphorylation directly contributes to the activation of PKD, we stimulated HeLa cells with pervanadate and immunoprecipitated PKD. The precipitates were treated with either purified recombinant catalytic subunit gamma -isoform of human protein phosphatase 1 (PP-1gamma ) or the phosphotyrosine phosphatase (CD45). Dephosphorylation of PKD recovered from pervanadate (or H2O2, data not shown)-treated cells resulted in a significant decrease in kinase activity (Fig. 2C). PKD activity could not be completely reduced by CD45 treatment, despite its ability to reduce PKD tyrosine phosphorylation by ~80%. This indicates that either tyrosine phosphorylation of PKD is involved in the initial activation step, which is then followed by autophosphorylation, or that the upstream tyrosine kinase is co-immunoprecipitated with PKD, leading to a steady state between phosphorylation and dephosphorylation in the in vitro reaction.


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Fig. 2.   PKD tyrosine phosphorylation contributes to its activation. A, 2 Mio HeLa cells were treated with PMA (10 min, 100 nM) or with 75 µM pervanadate for the indicate time. PKD was immunoprecipitated (IP), and tyrosine phosphorylation (anti-pY; 4G10) was analyzed. B, wild-type PKD or PKD.K612W were overexpressed, and cells were treated with pervanadate (PV) (10 min, 75 µM). PKD was immunoprecipitated, and tyrosine phosphorylation (anti-pY; 4G10) was analyzed. C, 2 Mio HeLa cells were treated with 75 µM pervanadate as indicated. PKD was immunoprecipitated, and precipitates were treated for 15 min (room temperature) with phosphatases (PP-1gamma or CD45), and substrate phosphorylation kinase assays were performed or tyrosine phosphorylation (anti-pY; 4G10) was determined. The immunoblots were stripped and re-probed against PKD protein in all experiments. All results are typical of three independent experiments.

The Src/Abl Signaling Pathway Is Responsible for Tyrosine Phosphorylation and Activation of PKD-- By using overexpression of dominant negative alleles of Src and Abl, we recently showed that these tyrosine kinases contribute to PKD activation in cells (14). In order to determine whether pervanadate- and H2O2-stimulated tyrosine phosphorylation of PKD are mediated by Src and/or Abl, HeLa cells were treated with the Src kinase inhibitors PP1 and PP2 prior to stimulation. Both PP1 and PP2 blunted the activation of PKD in response to pervanadate by 50%, concomitant with a reduction of tyrosine phosphorylation (Fig. 3A). The control inactive PP3 compound had no effect on PKD activity or tyrosine phosphorylation. This underscores the requirement of Src in the activation and phosphorylation of PKD. Moreover, co-expression of constitutively active Src (Src.Y527F) or Abl (v-Abl p120) together with wild-type PKD demonstrated that both kinases mediate tyrosine phosphorylation of PKD in transfected cells (Fig. 3B).


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Fig. 3.   Src/Abl mediates pervanadate-induced tyrosine phosphorylation and activation of PKD. A, 2 Mio HeLa cells were pre-treated for 1 h with respective Src kinase family inhibitors (PP1 and PP2), control (PP3), or left untreated and then stimulated for 10 min with 75 µM pervanadate (PV). PKD was immunoprecipitated (IP) and tyrosine phosphorylation (anti-pY; 4G10) as well as activity (auto- and substrate phosphorylation) were determined. B and C, wild-type PKD and active Src (Src.Y527F) or Abl (v-Abl.p120) were overexpressed in 2 Mio HeLa cells, and PKD was immunoprecipitated and analyzed for tyrosine phosphorylation by immunoblot analysis. The nitrocellulose was the stripped and re-probed for PKD. Src and Abl expression was controlled by immunoblotting with anti-Src and anti-Abl. All results are typical of three independent experiments.

Src/Abl Mediates Tyrosine Phosphorylation of PKD in the PH Domain-- It is known that a functional PH domain is required for PKD regulation in cells (3). To determine whether the tyrosine phosphorylation of PKD occurs within this domain, we first assessed PKD phosphorylation in pervanadate-treated cells expressing either wild-type PKD or a PKD.Delta PH mutant. Deletion of the PH domain resulted in a near complete loss of PKD tyrosine phosphorylation when compared with the control wild-type protein (Fig. 4A). Similar results were obtained in cells expressing either active Abl (v-Abl p120) or active Src (Src.Y527F), whereby tyrosine phosphorylation of the PKD.Delta PH mutant was significantly blunted compared with wild-type PKD (Fig. 4B). Thus, the PH domain of PKD is the major target of pervanadate/Src/Abl-dependent tyrosine phosphorylation.


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Fig. 4.   Tyrosine phosphorylation mediated by pervanadate and Src/Abl occurs in the PKD PH domain. A, HA-tagged wild-type PKD or PKD.Delta PH was overexpressed, and cells were treated with pervanadate (10 min, 75 µM). PKD was immunoprecipitated (IP) and immunoblotted with anti-pY (4G10). The nitrocellulose membrane was stripped and re-probed with anti-PKD (PKD). B, HA-tagged wild-type (WT) PKD or PKD.Delta PH were co-expressed with active Src (Src.Y527F) or Abl (v-Abl.p120), and PKD was immunoprecipitated and analyzed for tyrosine phosphorylation. The nitrocellulose was the stripped and re-probed for PKD expression (anti-PKD). Src and Abl expression was controlled by immunoblotting with anti-Src and anti-Abl. All results are typical of three independent experiments.

Mapping of Src- and Abl-dependent Tyrosine Phosphorylation Sites in the PKD PH Domain-- We next used a mutational approach to determine which tyrosine residues within the PKD PH-domain are targets of Src and Abl. We mutated six candidate tyrosine residues (Tyr432, Tyr443, Tyr462, Tyr463, Tyr501, and Tyr502) into phenylalanine (Fig. 5A). Each of these single mutants as well as a triple mutant (PKD*; Y432F/Y463F/Y502F) were co-expressed with active Src (Src.Y527F) in HeLa cells, and the level of PKD tyrosine phosphorylation was assessed. The PKD Y432F, Y463F, and Y502F mutants showed an appreciable loss of phosphotyrosine signal compared with wild-type PKD or the other three mutants (Fig. 5B). Thus, Tyr432, Tyr463, and Tyr502 are likely targets of Src-dependent tyrosine phosphorylation. Interestingly, upon co-expression of these PKD mutants with active Abl (v-Abl p120), only the Y463F mutant showed any appreciable loss of phosphotyrosine signal (Fig. 5C). These data indicate both overlap as well as specificity in the Src- and Abl-mediated tyrosine phosphorylation of PKD.


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Fig. 5.   Mapping the Src- and Abl-mediated tyrosine phosphorylation sites in the PKD PH domain. A, schematic overview of PKD domains (HR, hydrophobic region; C1a and C1b, C1 domains; PH, pleckstrin homology; KD, kinase domain) and the pleckstrin homology domain primary and predicted secondary structure (alpha , alpha -helix; beta , beta -sheet; VL, variable region). Tyrosine residues within the PH domain are highlighted. B and C, wild-type PKD (PKD), PKD* (PKD.Y432F/Y463F/Y502F) or other indicated mutants were overexpressed in combination with active Src (Src.Y527F, B) or active Abl (v-Abl p120, C). PKD was immunoprecipitated, and tyrosine phosphorylation (anti-pY; 4G10) was determined. The immunoblots were stripped and re-probed against PKD protein (anti-PKD). Src or Abl expression was controlled by immunoblotting with anti-Src or anti-Abl. All results are typical of three independent experiments.

Src Associates with PKD in Cells-- Because both Src and Abl are able to induce the tyrosine phosphorylation of PKD in cells, we tested whether these tyrosine kinases exist in a complex with PKD. Co-immunoprecipitation experiments were carried out with both endogenous and transfected PKD. Endogenous Src was found in a complex with PKD immunoprecipitates following pervanadate stimulation of HeLa cells (Fig. 6A). Under these co-immunoprecipitation conditions, we failed to detect co-immunoprecipitation between endogenous Abl and endogenous PKD (Fig. 6A). Similar results were obtained when PKD and Src were co-transfected in HeLa cells, where overexpressed Src was co-immunoprecipitated with overexpressed PKD (Fig. 6B). A constitutive association between PKD and Src was observed when the two proteins were overexpressed, and this could not be increased further with pervanadate treatment. Interestingly, mutation of Y463F had no effect on this association. Again, overexpressed Abl could not be detected on PKD immunoprecipitates (Fig. 6B). Moreover, the kinase activity of Src was not required for this association because the kinase-inactive Src* allele was still able to co-immunoprecipitate with transfected PKD (Fig. 6C). Thus, in response to pervanadate stimulation of cells, Src translocates to a complex that contains PKD, which likely facilitates the phosphorylation event. Conversely, the phosphorylation of PKD by Abl is likely mediated by a transient association between the two kinases that cannot be recovered under these co- immunoprecipitation conditions.


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Fig. 6.   Co-immunoprecipitation of Src and PKD. A, HeLa cells were treated with pervanadate (PV) (10 min, 75 µM). Endogenous PKD was immunoprecipitated (alpha -PKD), and co-immunoprecipitation of Abl or Src was evaluated by immunoblotting with anti-Abl (alpha -Abl) or anti-Src (alpha -Src). Blots were stripped and re-probed with anti-PKD. B, wild-type (wt) PKD or PKD.Y463F were co-expressed with either wild-type Abl or wild-type Src. Cells were treated with pervanadate (10 min, 75 µM), and PKD was immunoprecipitated, and co-immunoprecipitation of Abl or Src was evaluated by immunoblotting with anti-Abl (alpha -Abl) or anti-Src (alpha -Src) or as control anti-PKD (alpha -PKD). C, wild-type Src or kinase-inactive Src (Src*) were co-expressed with wild-type PKD, PKD immunoprecipitated with anti-HA (alpha -HA), and immunoblotted with anti-Src (alpha -Src), or a control, anti-PKD (alpha -PKD). All results are typical of three independent experiments.

Abl Phosphorylates and Activates PKD through Tyr463 Phosphorylation-- Tyr463 in human PKD lies in a motif highly conserved between mammalian PKD isoforms, as well as PKD orthologues (Fig. 7A). We next tested whether Abl can directly phosphorylate PKD in vitro using a phospho-specific antibody raised against a phosphopeptide based on the Tyr463 motif. This antibody only detected PKD when phosphorylated at Tyr463 in cells but not the Y463F mutant, demonstrating that it is specific for phospho-Tyr463 (data not shown and Ref. 14). Incubation of purified PKD with purified Abl revealed that Tyr463 can be phosphorylated directly by Abl in vitro (Fig. 7B). Furthermore, PKD phosphorylated by Abl showed a 2-fold increase in protein kinase activity compared with PKD incubated alone, suggesting that Tyr463 phosphorylation partially contributes to PKD activation (Fig. 7C). This activation was on the same order of magnitude as the control activators PS/DOG. Finally, we evaluated whether cellular stimuli, which are known to activate PKD in cells, also induce PKD tyrosine phosphorylation. Stimulation of cells with pervanadate or oxidative stress (H2O2) induced tyrosine phosphorylation of PKD at Tyr463 (Fig. 7D). However, stimulation of HeLa cells with either epidermal growth factor, platelet-derived growth factor (PDGF), or insulin-like growth factor-1 did not induce any appreciable tyrosine phosphorylation of PKD, indicating that Tyr463 phosphorylation is limited to oxidative stress signaling.


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Fig. 7.   Abl directly phosphorylates PKD at residue Tyr463 resulting in increased PKD activity. A, alignment of the amino acid sequences surrounding Tyr463 in human (Homo (H.) sapiens) PKD/PKCµ and in other PKD isoforms from mouse (Mus (M) musculus) and the nematode worm (Caenorhabditis (C) elegans). The potential phosphorylation site is highlighted, and identical amino-acids are underlined. B, recombinant, purified PKD was incubated either alone or together with purified Abl in vitro with cold ATP for 20 min at 37 °C, resolved by SDS-PAGE, and immunoblotted with anti-pY463. The nitrocellulose membrane was stripped and re-probed with anti-PKD. C, in vitro activation of PKD by Abl. Recombinant, purified PKD was incubated either alone or together with purified Abl or PS/DOG micelles and cold ATP in vitro for 20 min at 37 °C. PKD was immunoprecipitated, and a substrate kinase assay was performed. Precipitates were resolved by SDS-PAGE and immunoblotted for precipitation of equal amounts of PKD. D, HeLa cells were treated with pervanadate (10 min, 75 µM, positive control), H2O2 (10 min, 10 µM), epidermal growth factor (EGF) (5 min, 50 ng/ml), PDGF (5 min, 50 ng/ml), or insulin-like growth factor-1 (IGF-1) (5 min, 50 ng/ml). PKD was immunoprecipitated (IP), and tyrosine phosphorylation (alpha -pY; 4G10) was analyzed by immunoblotting (left panel). HeLa cells were transfected with PKD and treated with H2O2 (10 min, 10 µM). PKD was immunoprecipitated, and tyrosine phosphorylation of Tyr463 (anti-pY463) was analyzed on immunoblots (right panel).

Phosphorylation of Tyr463 Mediates PKD Activation in Cells-- We next evaluated the contribution of Tyr463 phosphorylation to PKD activation in pervanadate- and Src/Abl-stimulated cells. First, pervanadate-stimulated PKD activation was partially blunted in cells expressing a Y463F mutant, when compared with wild-type PKD. Second, the triple mutant PKD* (PKD.Y432F/Y463F/Y502F) showed a quantitatively similar activity compared with Y463F (Fig. 8A). This suggests that phosphorylation of Tyr432 and Tyr502 does not contribute to PKD activation. In addition, overexpression of the Y432F or Y502F mutants did not significantly alter PKD activity in response to pervanadate treatment of HeLa cells (data not shown).


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Fig. 8.   PKD activity is dependent on Tyr463 phosphorylation. A-C, wild-type PKD (PKD) or the indicated PKD mutants were expressed, and cells were treated with pervanadate (PV) (10 min, 75 µM) as indicated. PKD was immunoprecipitated (IP) (anti-HA) and a substrate kinase assay performed. PKD expression and tyrosine phosphorylation (A, alpha -pY) was determined by immunoblotting. All results are typical of three independent experiments.

In order to test whether mutation of these three tyrosine residues to glutamate would lead to constitutive PKD activity, by effectively mimicking the negative charge induced by phosphorylation, we constructed Y432E, Y463E, and Y502E mutants. As a control, glutamine mutations were also created. Interestingly, only the PKD.Y463E mutant showed constitutive PKD activity when expressed in cells (Fig. 8B), whereas the other two glutamate mutants were essentially unchanged from wild-type PKD. There was no significant alteration of PKD kinase activity in all of the glutamine mutants (the slight increase in the Y463Q mutant was not reproducible over the course of several experiments). Moreover, the constitutive kinase activity of PKD.Y463E could not be further augmented by pervanadate stimulation of cells (Fig. 8C). Finally, to reveal a potential negative regulatory function of Tyr432 or Tyr502 phosphorylation, we also compared the kinase activity of two double mutants, PKD.Y463E/Y432F and PKD.Y463E/Y502F. In response to pervanadate-stimulation of cells, however, neither of these two mutants showed any appreciable decrease in kinase activity compared with PKD.Y463E (Fig. 8C). Again the implication is that these two tyrosine residues do not significantly contribute to PKD activation.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PKD is a member of a protein kinase family that is distinct from PKC isoenzymes and that is subject to diverse regulatory mechanisms that are required for its activation (8). In the present study, we report an activation mechanism for PKD that is mediated by tyrosine phosphorylation of residues within the PH domain. Treatment of HeLa cells with the tyrosine phosphatase inhibitor pervanadate, or with H2O2, as well as by overexpression of active alleles of Src and Abl leads to tyrosine phosphorylation and activation of PKD. By using a mutational approach, we mapped three candidate tyrosines within the PH domain that serve as targets of Src and/or Abl. These are Tyr432, Tyr463, and Tyr502 (Fig. 5). Although mutation of these single sites to non-phosphorylatable Phe residues reduced PKD tyrosine phosphorylation (Fig. 5), only mutation of Tyr463 showed an appreciable loss of protein kinase activity in transfected cells (Fig. 8). Because mutation of this residue to a Glu induced constitutive protein kinase activity, we deduced that phosphorylation of this site is both necessary and sufficient to stimulate PKD activity in cells, both in response to pervanadate and downstream of the Src/Abl pathway (Fig. 8). Thus, phosphorylation of PKD at Tyr463 represents an alternative mechanism for PKD activation and is particularly interesting in light of the fact that this residue lies within the PH domain.

Unlike PKCs, PKD lacks a classical pseudosubstrate sequence necessary for autoregulation (5). However, PKD is negatively regulated by an autoinhibitory PH domain, and deletion of this domain results in a protein with elevated constitutive protein kinase activity (24). Similar increases in PKD activity were reported by mutation of critical residues in this domain such as Trp538 (3). Recently, it was also shown that serine phosphorylation at Ser738 and Ser742 in the activation loop mediates the release of the PH domain (28). Because phosphorylation of the activation loop can be mediated directly by PKCepsilon , as demonstrated by in vitro experiments (34), this suggests a hierarchical PKC-PKD pathway that leads to PKD activation and release of the PH domain. In addition, because PKCs are also able to bind to the PKD PH domain, this may also contribute to a conformational change leading to release of the PH domain, thus exposing the activation loop (29, 30). Thus far, structural evidence for such a model has not been presented.

Our data reveal an alternative regulatory mechanism for PKD activation that may also result in the release of PH domain autoinhibition. Phosphorylation of Tyr463, either by pervanadate stimulation of cells or downstream of the Src/Abl pathway, leads to PKD activation, and this can be achieved both in vitro using purified proteins or in transfected cells. Thus, Tyr463 represents a critical feature of this activation process, because mutation to phenylalanine compromises PKD activity, and mutation to glutamate results in a constitutively active kinase. It is tempting to speculate that phosphorylation of Tyr463 leads to release of the PH domain, thus exposing the activation loop which would now be accessible to PKC for phosphorylation. This model is supported by the fact that the Y463E mutant is constitutively phosphorylated at Ser738/Ser742 when expressed in cells.2 Future studies will address the precise interplay between Tyr463 and activation loop phosphorylation.

Both in vitro and in cells, Abl appears to be a direct upstream kinase for Tyr463. Src, on the other hand, may also have a function in phosphorylating two additional sites in the PKD PH domain, Tyr432 and Tyr502. In this regard, it is interesting to note that Src translocates to a complex containing PKD in response to pervanadate stimulation of cells, whereas we have failed to detect any appreciable association of Abl with PKD under the same conditions (Fig. 6). This suggests that the phosphorylation of PKD at Tyr463 by Abl occurs transiently, and any complex between the two proteins cannot be recovered under detergent lysis conditions. Mutation of Tyr432 and Tyr502 to non-phosphorylatable residues results in a decrease in overall tyrosine phosphorylation (Fig. 5). It is worth noting that we have not directly demonstrated tyrosine phosphorylation of these sites. However, because mutation of these residues to phenylalanine does not significantly alter PKD activity, and also mutation to glutamate does not result in increased kinase activity, it appears that phosphorylation of Tyr432 or Tyr502 would not contribute to PKD activity. Similarly, mutation of Tyr432 or Tyr502 does not compromise Tyr463 phosphorylation. It is also worth noting that the loss of phosphotyrosine signal observed in the phenylalanine mutations is not due to a loss of Tyr463 phosphorylation (data not shown). A role for phosphorylation of Tyr432 and Tyr502 may be to serve as docking sites for SH2 domain-containing proteins, adding another level of complexity to the regulation of PKD in cells. Finally, it is important to note that although the Y463F PKD mutant is significantly compromised in protein kinase activity in stimulated cells, some residual activity remains (Fig. 8A). Coupled with the finding that PKD tyrosine phosphorylation is also not completely eliminated in either PKD.Delta PH (Fig. 4B) or PKD.Y463F (Fig. 8A), it is reasonable to speculate that pervanadate stimulation of cells may lead to the phosphorylation of additional tyrosine residues outside of the PH domain, and which may contribute to PKD kinase activity. Moreover, the constitutive protein kinase activity of the PKD.Delta PH mutant is only modestly increased in cells exposed to either oxidative stress, pervanadate, or upon co-transfection with active Src or Abl (data not shown). Again this modest increase may be due to additional phosphorylations outside of the PH domain or by increased activation loop phosphorylation mediated by unknown mechanism(s). Future studies will address these possibilities.

Phosphorylation of Tyr463 may also provide a docking site for SH2-containing molecules. However, we speculate that this is unlikely because the PKD.Y463E mutant is active per se in immune complex kinase assays. Instead, we postulate that the negative charge at this site induces conformational changes within the PH domain, leading to release of autoinhibition, as discussed above. This hypothesis does not exclude other models of PKD activation, such as release mediated by activation loop phosphorylation. Indeed, the precise mechanism by which the PH domain is released may depend on the upstream pathways that converge to regulate PKD. For example, in response to mitogens as well as bradykinin stimulation of cells, we have failed to detect Tyr463 phosphorylation (Fig. 7D) (14), despite the fact that these stimuli fully activate PKD by inducing activation loop phosphorylation. Conversely, both pervanadate and H2O2 stimulation of a variety of cell types leads to Tyr463 phosphorylation as well as activation loop phosphorylation (14). Thus, it is likely that in response to growth factor signaling, diacylglycerol binding and activation loop phosphorylation primarily control PKD activation, whereas in response to pervanadate and oxidative stress signaling, Tyr463 and activation loop phosphorylation are the primary determinants.

Taken together, our results point to an important function of Tyr463 in the PKD PH domain controlling its activity. By using both loss of function and gain of function approaches, we show that tyrosine phosphorylation at this residue results in increased PKD activity. We cannot fully rule out that the other two tyrosine residues Tyr432 and/or Tyr502 are also important for some aspect of PKD activity or function. The fact that both Src/Abl and PKCs may converge on PKD to tightly regulate its activation presents an attractive model that we are currently testing. The outcome of these and future studies will likely impact the diverse array of physiological responses that have been attributed to PKD.

    ACKNOWLEDGEMENTS

We thank B. Schaffhausen and A. Hausser for generously providing expression plasmids. We also thank members of the Toker laboratory for insightful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA 75134 (to A. T.) and by Deutsche Forschungsgemeinschaft Grant STO 439/1-1 (to P. 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.

We dedicate this paper to the memory of Franz-Josef Johannes, a friend, colleague, teacher, and mentor, whose contributions to the PKCµ/PKD field will be greatly missed.

dagger Deceased.

To whom correspondence should be addressed: Dept. of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-8535; Fax: 617-667-3616; E-mail: atoker@caregroup.harvard.edu.

Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M213224200

2 P. Storz and A. Toker, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PKD, protein kinase D; BCR, B-cell receptor complex; DOG, dioleoylglycerol; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PKC, protein kinase C; PMA, 12-phorbol 13-myristate acetate; PtdSer, phosphatidylserine; TCR, T-cell receptor complex; HA, hemagglutinin; PS, phosphatidylserine; SH2, Src homology 2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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