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Originally published In Press as doi:10.1074/jbc.C400575200 on February 22, 2005

J. Biol. Chem., Vol. 280, Issue 15, 15013-15019, April 15, 2005
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A Phosphorylation State-specific Antibody Recognizes Hsp27, a Novel Substrate of Protein Kinase D*

Heike Döppler{ddagger}§, Peter Storz{ddagger}§, Jing Li¶, Michael J. Comb¶, and Alex Toker{ddagger}||

From the {ddagger}Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 and Cell Signaling Technology, Beverly, Massachusetts 01915

Received for publication, December 13, 2004 , and in revised form, February 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser82 in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Approximately one-third of all proteins in eukaryotic cells are phosphorylated on either serine, threonine, or tyrosine, a reaction that is catalyzed by members of the protein kinase superfamily (1). Over 500 protein kinases comprise the human Kinome, and they fall into seven distinct families (2). The identification of protein substrates of distinct protein kinases lags significantly behind knowledge of their regulatory mechanisms. However, recent technological advances in the identification of phosphorylation sites, such chemical genetics as well as mass spectrometry, have enabled progress in this area (3). In addition, the advent of phosphorylation state-specific antibodies which recognize phosphorylated Ser/Thr residues in a sequence-specific context has provided much needed insight into the specific function of many protein kinases (4). A more recent application of this technique is the development of substrate-directed phospho-specific antibodies that recognize the optimal consensus motif of a specific protein kinase or family of kinases (5). This has been possible through the identification of consensus phosphorylation motifs by degenerate peptide library approaches, such that the knowledge that many kinases prefer to phosphorylate, for example, basophilic, acidophilic, or Ser-Pro-directed motifs has been further refined to obtain a specific consensus for amino acids surrounding the phosphorylatable Ser or Thr (6). As an example, identification of the consensus motif phosphorylated by Akt/PKB, an AGC kinase, enabled the development of an antibody which recognizes this motif and which was used to identify novel Akt/PKB substrates, such as the TSC2 gene product, tuberin (5, 7, 8).

PKD,1 originally cloned and termed PKCµ and identified as a PKC (protein kinase C) family member, comprises a family of three closely related isoforms, PKD1, PKD2, and PKD3/PKC{nu}. Based on sequence similarities, PKDs are now grouped into the CAMK (calcium and calmodulin-dependent kinases) family of kinases. PKDs regulate a plethora of cellular responses, ranging from cell growth, cell survival, Golgi organization, and trafficking and immune cell responses in B cells (reviewed in Ref. 9). Similarly, the regulation of PKD catalytic activity by cellular location, binding to adapter proteins, and by phosphorylation is also well documented. What has remained elusive, however, is the identification of specific protein substrates of PKD, which relay the signal to downstream responses. Notable exceptions are Kidins220 (kinase D-interacting substrate of 220 kDa), a neuronal PKD substrate protein (10), RIN1, a PKD substrate involved in the modulation of Ras signaling (11), and HDAC5 (histone deacetylase 5), a class II deacetylase implicated in suppression of cardiac hypertrophy (12). Clearly, numerous other PKD substrates must exist given the large number of cellular responses attributed to this protein kinase.

We took advantage of the known optimal consensus phosphorylation motif preferred by PKD to develop a substrate-directed, phospho-specific antibody that is immunoreactive against proteins phosphorylated by PKD in cells. Using this antibody, we have detected multiple phosphoproteins in stimulated cells and have used it to identify a previously unidentified PKD substrate, the heat shock protein Hsp27.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cell Culture, Antibodies, and cDNA Expression Plasmids—The HeLa and HEK293E cell lines were purchased from ATCC and maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The anti-PKD (C-20) and anti-Hsp27 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-FLAG (M2), and anti-actin from Sigma. Anti-HDAC5, anti-phospho-(Ser/Thr) Akt/PKB substrate antibody (Akt/PKB pMOTIF), and anti-phospho-Hsp27 (pSer82) antibodies were from Cell Signaling Technology (Beverly, MA). The anti-RIN1 and RIN1 expression plasmids were generous gifts of J. Colicelli and are published (11). Pervanadate was prepared as described previously (13). H2O2 (30%) was from Fisher Scientific. PMA (phorbol 12-myristate 13-acetate), bombesin, and bradykinin were from Sigma, and PDGF (platelet-derived growth factor) was from R&D Systems (Minneapolis, MN). Recombinant PKD was expressed in insect cells after infection with baculovirus harboring hemagglutinin-tagged PKD in pFAST-Bac (Invitrogen) and purified on a nickel-nitrilotriacetic acid affinity column. The vector-based PKD1 and PKD2 RNAi in the pSUPER expression vector have been described (14, 15). TransIT HeLa Monster reagent (Mirus, Madison, WI) was used for all transient transfections according to the manufacturer's instructions. Cells were stimulated or harvested 24 h after transfection. A GST-Hsp27 fusion protein was obtained by cloning Hsp27 cDNA into pGEX-4T1 using the following oligonucleotide primer pair for PCR: 5'-GCGGGATCCATGACCGAGCGCCGCGTCCCCTTC-3' and 5'-GCGCTCGAGTTACTTGGCGGCAGTCTCATCGGA-3'. The S15A and S82A mutations were introduced using the following oligonucleotide primer pair: 5'-TCGCTCCTGCGGGGCCCCGCCTGGGACCCCTTCCGCGAC-3'and 5'-GTCGCGGAAGGGGTCCCAGGCGGGGCCCCGCAGGAGCGA-3' for S15A and 5'-GCGCTCAGCCGGCAACTCGCCAGCGGGGTCTCGGAGATC-3' and 5'-GATCTCCGAGACCCCGCTGGCGAGTTGCCGGCTGAGCGC-3' for S82A. Mutagenesis was carried out using the QuikChange strategy (Stratagene), and all constructs were verified by DNA sequencing.

Antibody Production—The PKD pMOTIF antibody was raised against a synthetic phosphopeptide antigen CXXXLXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX, where X represents a position in the peptide synthesis where a mixture of all 20 amino acid (excluding C and W) were used, and where S* represent phosphoserine. The peptide was conjugated to keyhole limpet hemocyanin and used to immunize rabbits. Phosphopeptide-reactive rabbit antiserum was first purified by protein A chromatography. Further purification was carried out using immunodepletion by non-phosphopeptide resin chromatography, after which the resulting eluate was chromatographed on a phosphopeptide resin. The antibody specificity of the resulting fractions was tested for specificity toward optimal PKD substrate sequences by ELISA, immunoblotting, and peptide arrays (see below).

Peptide Arrays—Covalent membrane-bound phosphopeptide libraries were synthesized directly on nitrocellulose membrane by the Massachusetts Institute of Technology Biopolymers Laboratory. For antibody specificity determination, the parental peptide library for the array was XXLXRXXS*XXXX. The library was arrayed such that each row represents a fixed position in the indicated library, and this position was systematically fixed with a specific amino acid as indicated in single amino acid letter code above each column. The first column from the left is the parental peptide library (Fig. 1C). The purified antibody fractions eluting from the phosphopeptide chromatography were tested against this array by incubating the antibody at a dilution of 1:1000 with membranes for 4 h at room temperature in 1% bovine serum albumin in PBST (PBS + 0.2% Tween 20), followed by three washes with PBST. Secondary horseradish peroxidase-conjugated antibody was incubated at a dilution of 1:2000 in PBST for 1 h at room temperature. After three washes with PBST the signal was revealed by Lumiglo (Cell Signaling Technology).



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  		FIG. 1.
Analysis of PKD pMOTIF antibody specificity using phosphopeptide arrays. A, determination of the PKD optimal phosphorylation motif using a degenerate immobilized peptide array. In the array, each row represents a fixed position in the library, and this position was fixed with each one of the 20 naturally occurring proteogenic amino acids. The library was incubated with recombinant, purified PKD and [{gamma}-32P]ATP, washed, and exposed to a molecular imager. Each spot was quantitated and is tabulated such that intensity is expressed as a percentage of background phosphorylation. B, alignment of the optimal PKD consensus phosphorylation pMOTIF obtained in three separate studies, as indicated. The relative selectivity of individual amino acids at each position is shown in ascending order of text size. The blue R indicates that this arginine was fixed at p-3 in the degenerate library used in the Nishikawa et al. study (17). The sequence of the peptide used for immunization, derived from the PKD consensus motif, is also shown. X represents any amino acid (except cysteine and tryptophan), pS represents phosphoserine. C, phosphopeptide array for the PKD pMOTIF antibody. The immobilized phosphopeptide array was incubated with PKD pMOTIF antibody. The first column from the left represents the parental peptide library. The library was arrayed such that each row represents a fixed position in the library, and this position was fixed with each indicated amino acid.

 
Immunoblotting and Immunoprecipitation—Cells were stimulated or harvested 24 h after transfection and 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-Aldrich). The lysates were used either for immunoblot analysis or proteins were immunoprecipitated by a 1-h incubation with the respective antibody (2 µg), followed by a 30-min incubation with protein G-agarose (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 kinase assays.

In Vitro Kinase Assays—For kinase assays with GST fusion proteins or immunoprecipitated substrates, the reaction was carried out by adding 0.5 µg of purified PKD to 2 µg of purified protein in a volume of 20 µl of kinase buffer. The kinase reaction was started by adding 10 µl of kinase substrate mix (100 µM ATP (cold assay) or 100 µM ATP and 10 µCi [{gamma}-32P]ATP in kinase buffer) and was carried out for 30 min at room temperature. To terminate the kinase reaction, SDS sample buffer was added, and the samples were resolved by SDS-PAGE.

RNA Interference—RNAi plasmids for PKD1 and PKD2 silencing have been described (14, 15). To silence human Hsp27 the following oligonucleotide sequences were cloned in pSUPER: 5'-GATCCCCGGATGGCGTGGTGGAGATCTTCAAGAGAGATCTCCACCACGCCATCCTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAGGATGGCGTGGTGGAGATCTCTCTTGAAGATCTCCACCACGCCATCCGGG-3'. HEK293E and HeLa cells were transfected with pSUPER or pSUPER-RNAi using the TransIT HEK293 or HeLa-Monster reagents, respectively (Mirus). In all experiments the cells were transfected at 30% confluence. Transfection efficiencies (80–90%) were controlled using a green fluorescent protein expression vector. Reduced expression of target proteins was evaluated by immunoblotting.

ELISA—ELISA was performed according to established protocols (16). Briefly, 50 µlof1 µM synthetic phospho- and non-phosphopeptides were used to coat each well in 96-well plates. Coating was carried out overnight at 4 °C. Phospho-PKD substrate antibody was used at a 1:1000 dilution. The plates were incubated at 37 °C for 2 h after addition of primary antibody. An alkaline phosphatase-conjugated goat anti-rabbit antibody (Cell Signaling Technology) was used as a secondary antibody, and p-nitrophenyl phosphate (Sigma) was used for color development. Absorbance at 405 nm was read on an ELISA plate reader.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
To develop an antibody-based method for PKD substrate identification, we first considered the optimal substrate phosphorylation motif of PKD. An oriented degenerate peptide library approach originally devised by Cantley and colleagues (17) revealed that PKD strongly selects for aliphatic residues (leucine, valine, and isoleucine) at the –5 position relative to the phospho-acceptor. In this original study, arginine was locked at the –3 position to orient the library, but a more recent application of this technique made use of a positional scanning peptide library to reveal that, in fact, PKD does strongly select arginine at –3 (18). We have confirmed these findings using an immobilized degenerate peptide library array, which was phosphorylated by purified, recombinant PKD. The library was arrayed such that each row represents a fixed position in the library, and this position was fixed with each of the 20 proteogenic amino acids (Fig. 1A). The results confirmed that PKD strongly prefers aliphatic residues, particularly leucine, at –5, and arginine at –3. Other selectivities included basic and aliphatic amino acids at –4, and neutral non-polar amino acids at –2. Relatively minor selectivity was observed at +1, although there was a preference for hydrophobic amino acids (Fig. 1, A and B).

Thus, three independent studies have defined the optimal consensus phosphorylation motif preferred by PKD. We used this information to raise an antibody, which we term anti-PKD pMOTIF, against a synthetic phosphopeptide according to the PKD motif (Fig. 1B). Epitope mapping of the antibody was first determined by ELISA with various synthetic phospho- and non-phosphopeptides. ELISA reactivities relative to the parental phosphopeptide are shown in Table I. Non-phospho control peptides scored in the 3–5% range of control phosphopeptide, revealing that the anti-PKD pMOTIF antibody only bound to phosphopeptides. Strongest antibody binding was detected when both leucine at –5 and arginine at –3 were present, consistent with the selectivity of PKD for these residues. If leucine at –5 was absent, relatively poor binding was observed. Similarly, peptides with leucine, valine, or isoleucine at –5, but lacking arginine at –3, revealed no detectable binding above control.


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  		TABLE I
Specificity of the PKD pMOTIF antibody using various phospho- and non-phosphopeptides containing different versions of the consensus PKD substrate pMOTIF as determined by ELISA

Reactivity is expressed as a percentage of ELISA reading of each peptide relative to that of the optimal PKD consensus motif (Fig. 1C). Uppercase S or T represents non-phospho-Ser/Thr, and lowercase s or t represents phospho-Ser/Thr.

 
To further investigate the selectivity of anti-PKD pMOTIF, we used a membrane-bound peptide array that was incubated with purified antibody to evaluate relative selectivity at each individual position (Fig. 1C). In the array, each spot represents a peptide based on the parental library, with each degenerate position represented by one amino acid, except the –5 and –3 positions where leucine and arginine were locked, respectively. In addition, peptides were generated that had either phosphoserine, phosphothreonine, or phosphotyrosine at each of the fixed positions. The results reveal that anti-PKD pMOTIF has modest selectivity for amino acids at the –7 and –6 positions, although methionine and tryptophan showed some selectivity at –7, whereas histidine, asparagine, and proline were preferred at –6. As expected, only leucine was selected at –5. Although in the immunizing peptide the –4 position was left degenerate, the antibody preferentially bound basic amino acids such as arginine and lysine, as well as isoleucine, leucine, and methionine. This conforms well to aliphatic and basic amino acids preferred by PKD (Fig. 1C). As expected, peptides with arginine revealed the strongest binding at –3. At –2, glutamate, lysine, methionine, and, to a lesser extent, glutamine were preferred. Anti-PKD pMOTIF selected phosphoserine at the phospho-acceptor position, and, to a lesser extent, phosphothreonine. There was no detectable binding of phosphotyrosine or any other amino acid at this position. Carboxyl-terminal to the phospho-acceptor, there was modest selectivity for hydrophobic amino acids at +1 and +2. Taken together, these results show that anti-PKD pMOTIF binds to phosphopeptides, which accurately reflect the optimal PKD consensus motif.

We next evaluated the ability of this antibody to detect putative PKD substrates in cells. PKD is activated in response to a wide variety of agonists that stimulate activation of PKC, which in turn phosphorylates and activates PKD (19). NIH-3T3 fibroblasts were stimulated with either bombesin, bradykinin, or PDGF. We also used PMA and pervanadate, which are well known activators of PKD. In response to all of these agonists, increased phosphorylation of a number of putative PKD substrates was detected (Fig. 2A). Specifically, three proteins of ~85, 100, and 150 kDa were detected in fibroblasts stimulated with PDGF, PMA, and pervanadate. A strong immunoreactive band was also detected at ~45 kDa. Two immunoreactive bands at 25 and 27 kDa were also detected in response to all agonists. These data show that the anti-PKD pMOTIF antibody is immunoreactive against proteins in cells stimulated with agonists that are known to activate PKD.



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  		FIG. 2.
PKD pMOTIF antibody recognizes putative substrates. A, NIH-3T3 cells were stimulated with Bombesin (50 µM, 10 min), bradykinin (50 ng/ml, 10 min), PDGF (50 ng/ml, 10 min), PMA (100 nM, 10 min), or pervanadate (75 µM, 15 min). Cells were lysed, and samples were immunoblotted against the PKD pMOTIF antibody. The open arrow indicates a 27-kDa protein, and filled arrows indicate other putative PKD substrates. B, HEK293E cells were transfected with vector control (pSUPER) or PKD RNAi (pSU-PER PKD1/2) for 48 h. Cells were then stimulated with H2O2 (10 µM, 10 min), pervanadate (75 µM, 15 min), or PMA (100 nM, 10 min). Lysates were immunoblotted with the PKD pMOTIF antibody. The open arrow indicates a putative PKD substrate of 27 kDa. PKD silencing by PKD-specific RNAi and equal loading were determined in control blots using {alpha}-PKD or {alpha}-actin. C, HeLa cells were serum-starved for 24 h and then stimulated with IGF-1 (50 ng/ml) or PMA (100 nM) for 10 min. Lysates were immunoblotted with anti-Akt/PKB pMOTIF (left panel) and anti-PKD pMOTIF (right panel). Open arrows indicate putative Akt/PKB substrates, and solid arrows indicate putative PKD substrates. D, top panel, HeLa cells were transfected with RIN1 or vector control (Ctrl) and then stimulated with PMA (100 nM, 10 min) and lysates immunoblotted with anti-PKD pMOTIF or anti-RIN1. Bottom panel, HeLa cells were stimulated with PMA (100 nM, 10 min), and HDAC5 was immunoprecipitated. Immunoprecipitates were immunoblotted with anti-PKD pMOTIF, stripped, and reprobed with anti-HDAC5.

 
Because several other proteins were detected by this antibody even in unstimulated cells, probably reflecting nonspecific immunoreactivity, we next determined whether any of these putative substrates could be directly attributed to PKD activity. To achieve this, we reduced endogenous PKD expression in HEK293 cells with RNAi. Cells were stimulated with either pervanadate or H2O2, as studies have shown that exposure of cells to oxidative stress such as H2O2 potently activates PKD (14, 20). The same range of phosphoproteins revealed by the anti-PKD pMOTIF antibody in fibroblasts were also detected in control HEK293 cells (Fig. 2B). In cells transfected with PKD RNAi, there was a marked reduction in the immunoreactivity of several proteins, most notably at 27 kDa. Because the anti-PKD pMOTIF antibody is phospho-specific we conclude that these may represent novel PKD substrates.

To further validate the specificity of the PKD pMOTIF antibody, we next compared the immunoreactivity of this antibody with the Akt/PKB substrate-directed antibody, which recognizes the optimal consensus sequence RXRXXS*/T*{psi} (5). Stimulation of serum-starved HeLa cells with IGF-1, a potent agonist of the phosphoinositide 3-kinase-Akt/PKB signaling pathway, resulted in the phosphorylation of a number of proteins detected by the Akt/PKB substrate antibody (Fig. 2C, left panel). These bands were not detected in cells stimulated with PMA, which does not activate Akt/PKB. Conversely, PMA, a potent stimulus for PKD, induced the phosphorylation of a distinct subset of proteins as measured by PKD pMOTIF immunoreactivity (Fig. 2C, right panel). Stimulation of cells with IGF-1, which does not activate PKD, did not induce the phosphorylation of the same subset of proteins. Note, however, that both antibodies did detect some proteins in cells stimulated with both agonists, and these are likely to represent neither Akt/PKB nor PKD substrates. Finally, we determined whether the PKD pMOTIF antibody is immunoreactive against known PKD substrates. To this end, RIN1, a neuronal PKD substrate, was detected by PKD pMOTIF in transfected HeLa cells stimulated with PMA (Fig. 2D). Similarly, endogenous HDAC5, also a known PKD substrate, was detected by this antibody, again in response to PMA stimulation of HeLa cells. Therefore, PKD pMOTIF detects both putative and known PKD substrate proteins.

To further demonstrate the feasibility of this antibody to discover novel PKD substrates, we performed a protein BLAST search on the Swiss-Prot data base using the peptide sequences from the ELISA screen. The search consistently returned the heat shock protein Hsp27 with the highest score for a protein containing the PKD consensus phosphorylation motif. We noted that one of the most prominent immunoreactive bands in Fig. 2B migrated at 27 kDa. To validate that this indeed represents Hsp27, we first reduced expression of endogenous Hsp27 using RNAi, followed by immunoblotting with anti-PKD pMOTIF. As predicted, immunoreactivity of the 27-kDa band was significantly reduced following H2O2 stimulation of HeLa cells (Fig. 3A). Analysis of the Hsp27 amino acid sequence reveals two optimal putative PKD phosphorylation sites at Ser15 and Ser82 (Fig. 3B). Shown for comparison is the minimal PKD consensus phosphorylation sequence and the PKD phosphorylation sites in RIN1 and HDAC5. Next, we evaluated immunoreactivity of Hsp27 in cells transfected with PKD RNAi. There was a marked reduction in the phosphorylation of the 27kDa band as detected by immunoblotting total cell lysates with anti-PKD pMOTIF, whereas total Hsp27 levels were unaffected (Fig. 3C, left panel). That this band represents Hsp27 was confirmed by immunoprecipitation of Hsp27, followed by immunoblotting with PKD pMOTIF, and again there was a reduction in immunoreactivity in cells transfected with PKD RNAi (Fig. 3C, right panel). Because this antibody is phospho-specific, we conclude that PKD activation leads to the phosphorylation of Hsp27, which is detected by PKD pMOTIF.



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  		FIG. 3.
Identification of Hsp27 as a PKD substrate. A, HeLa cells were transfected with vector control (pSUPER) or Hsp27 RNAi (Hsp27-RNAi) for 48 h. Cells were then stimulated with H2O2 (10 µM, 10 min), and lysates were immunoblotted with anti-PKD pMOTIF. Lysates were also probed with anti-Hsp27 and anti-actin. B, alignment of putative PKD phosphorylation sites in Hsp27 and the PKD minimal substrate motif. C, HeLa cells were transfected with vector control (pSUPER) or PKD RNAi for 48 h. Cells were then stimulated with H2O2 (10 µM, 10 min) and lysed. Left panel, lysates were immunoblotted with anti-PKD pMOTIF antibody, stripped, and re-probed with anti-Hsp27. Lysates were also immunoblotted with anti-PKD and anti-actin for loading control. Right panel, Hsp27 immunoprecipitates were immunoblotted with anti-PKD pMOTIF, stripped, and reprobed with anti-Hsp27 (ns, nonspecific immunoreactive band). D, baculovirus-expressed, purified PKD was incubated in an in vitro kinase assay with GST, GST-Hsp27, GST-Hsp27.S15A, or GST-Hsp27.S82A followed by autoradiography (top left panel). Equal loading of GST and GST fusion proteins was controlled by Coomassie Blue staining (bottom left panel). Baculovirus-expressed, purified PKD was incubated in a cold in vitro kinase assay with GST, GST-Hsp27, or GST-Hsp27.S82A; resolved by SDS-PAGE; and immunoblotted with the anti-PKD pMOTIF and anti-pSer82 Hsp27 (right panels) E, FLAG-tagged Hsp27, Hsp27.S15A, Hsp27.S82A, or vector control was overexpressed in HeLa cells. Cells were stimulated with H2O2 (10 µM, 10 min), and Hsp27 was immunoprecipitated ({alpha}-FLAG). Hsp27 phosphorylation was determined by immunoblotting with anti-PKD pMOTIF (top panel) or with anti-pSer82 Hsp27 (bottom panel). Equal expression of Hsp27 was determined by reprobing against Hsp27 or anti-FLAG.

 
Next, we investigated on which residue Hsp27 is phosphorylated by PKD. GST-Hsp27 fusion proteins, either wild-type, Ser15 -> Ala, or Ser82 -> Ala mutations, were incubated with purified, recombinant PKD in in vitro kinase assays. Both wild-type and Ser15 -> Ala GST-Hsp27 were efficiently phosphorylated by PKD, whereas the Ser82 -> Ala mutant showed no detectable phosphorylation (Fig. 3D, left panel). This was confirmed by immunoblotting separate in vitro kinase assays either with anti-PKD pMOTIF or with a phospho-antibody specific to Ser82 in Hsp27. Both antibodies recognized wild-type Hsp27, wheras Ser82 -> Ala Hsp27 immunoreactivity was reduced (Fig. 3D, right panel). These results demonstrate that: (i) PKD directly phosphorylates Hsp27 and that (ii) Ser82, and not Ser15, is the relevant site, at least in vitro. This is also true in cells, because both wild-type and Ser15 -> Ala Hsp27 are efficiently detected by anti-PKD pMOTIF upon stimulation with H2O2, whereas no appreciable immunoreactivity was evident with the Ser82 -> Ala mutant (Fig. 3E, top panel). Again, the same result was obtained by immunoblotting with anti-pSer82 (Fig. 3E, bottom panel). We therefore conclude that Hsp27 is phosphorylated at Ser82 by PKD in stimulated cells.

Although much is known about the mechanisms of regulation of PKD and its importance in cell biology, the identification of specific protein substrates that relay the PKD signal has remained elusive. Here we have used an antibody-based method, which we speculate will aid in the identification of such substrates. We have shown that the anti-PKD pMOTIF antibody reacts with peptides that conform to the preferred phosphorylation motif of this kinase and furthermore validate its use and show that the Hsp27 protein is a previously unidentified in vivo PKD substrate. Phosphorylation of Hsp27 at Ser15 and Ser82 has previously been demonstrated in response to treatment of cells with a variety of stresses such as oxidative stress and heat shock (21, 22), and the MAPKAP kinases 2/3 have been shown to phosphorylate Hsp27 in vitro (21). However, the identity of the physiological kinase(s) for Hsp27 phosphorylation at these residues has not been determined. Using a combination of the anti-PKD pMOTIF antibody, PKD-specific RNAi, and in vitro kinase assays, we show that PKD is the relevant kinase for Hsp27 phosphorylation at Ser82. Hsp27 phosphorylation at Ser15 and Ser82 modulates oligomerization and chaperone function, leading to protection of cells from injury due stress (23). Because PKD plays a major role in protecting cells from oxidative stress (14), we further speculate that Hsp27 phosphorylation by PKD may play a key role in this response.

The use of the PKD pMOTIF antibody in combination with proteome-wide screens should yield much needed information concerning the identify of additional PKD substrates. Because other kinases in the human Kinome may reveal optimal phosphorylation motifs similar to that of PKD, it will be important to perform combinatorial screens with PKD-specific RNAi, as shown here. Additional in vitro validation by direct phosphorylation of identified putative PKD substrates will also be important to confirm the newly identified substrate. Given the present lack of any other rapid, substrate-directed methods to discover substrates of PKD in cells, this method should be well suited for analysis of PKD signaling in cells.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant CA75134 (to A. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

|| To whom correspondence should be addressed: Dept. of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Ave., RN-237, Boston, MA 02215. Tel.: 617-667-8535; Fax: 617-667-3616; E-mail: atoker{at}bidmc.harvard.edu.

1 The abbreviations used are: PKD, protein kinase D; PKC, protein kinase C; ELISA, enzyme-linked immunosorbent assay; HDAC5, histone deacetylase 5; Hsp27, heat shock protein 27; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; PMA, 12-phorbol 13-myristate acetate; RNAi, RNA interference; S*, phosphoserine; T*, phosphothreonine; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank the members of the joint Friday morning signaling group meeting at Beth Israel Deaconess Medical Center/Harvard Medical School and members of the Toker laboratory for insightful discussions and advice. We are grateful to John Colicelli for providing the RIN1 antibody and plasmids.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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