The Pleckstrin Homology Domain of Protein Kinase D Interacts Preferentially with the eta  Isoform of Protein Kinase C*

Richard T. WaldronDagger §, Teresa IglesiasDagger , and Enrique Rozengurt

From the Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095-1786 and the Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdom

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ABSTRACT
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EXPERIMENTAL PROCEDURES
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The results presented here demonstrate that protein kinase D (PKD) and PKCeta transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCeta . The presence of PKCeta in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCeta was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCepsilon , and did not bind PKCzeta . When PKCeta was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCeta immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino-terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCeta . Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCeta . These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs epsilon  and eta , thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Protein kinase C (PKC),1 a major cellular target for the potent tumor-promoting phorbol esters (1, 2), has been implicated in the mediation of diverse cellular functions, including short-term regulation of ion fluxes, receptor ligand binding, and signal transduction, and a wide range of longer term effects including proliferation, differentiation, transformation, and regulation of the mammalian cell cycle (3-6). At least 10 PKC isoforms, i.e. the classic (alpha , beta 1, beta 2, and gamma ), novel (delta , epsilon , eta , and theta ), and atypical PKCs (zeta , lambda ) have been identified by molecular cloning techniques (5, 7, 8). The different PKC isoforms exhibit distinct patterns of expression in different cell types and tissues as well as distinct subcellular distributions, leading to the view that they play distinct, rather than redundant roles in signal transduction (5, 9, 10). However, since very few of their specific substrates and binding partners have been identified, it has not yet been possible to ascribe to each isoform a unique cellular function.

The recently identified protein kinase D (PKD) is a mouse serine protein kinase with distinct structural and enzymological properties (11-13). In particular, the catalytic domain of PKD, which is distantly related to Ca2+-regulated protein kinases (11), possesses only a low degree of sequence similarity to the highly conserved regions of the kinase subdomains of the PKC family (14). Accordingly, PKD does not phosphorylate a variety of substrates utilized by PKCs indicating that PKD is a protein kinase with distinct substrate specificity (11, 12, 15). The amino-terminal region of PKD contains a tandem repeat of cysteine-rich regions that bind phorbol esters and diacylglycerol (11), similar to those found in classical and novel PKCs (16). However, unlike all PKCs, PKD does not possess a regulatory pseudosubstrate domain upstream of the first cysteine-rich motif. An additional structural feature that distinguishes PKD from the PKC family is the presence of a PH domain, interposed between the second cysteine-rich motif and the catalytic domain. PH domains are modular protein domains which mediate protein-protein and lipid-protein interactions and are found in many cytoskeletal and signal transducing proteins (17, 18). The PKD PH domain has been found to contribute to regulation of PKD catalytic activity (15).

Recently we reported that exposure of intact cells to biologically active phorbol esters, membrane-permeant diacylglycerol or bryostatin-1 induces PKD activation via a PKC-dependent pathway (19, 20). PKD activated within cells via PKC can be immunopurified from cell extracts in an active state that is independent of exogenously added cofactors (19). Growth factors and mitogenic neuropeptides that promote phospholipid turnover and diacylglycerol generation also stimulate this pathway, indicating that PKD activation is an early event in cell stimulation (21). We further substantiated the role of PKCs in PKD activation by coexpression of PKD together with constitutively active mutant PKC isoforms. These studies revealed that novel PKCepsilon and PKCeta fully activated PKD within cells, whereas classical PKCbeta 1 or atypical PKCzeta did not (19). These results raise the possibility that PKD functions downstream of novel PKCs in a previously undescribed signal transduction pathway.

Here, we demonstrate, for the first time, that coexpression of PKD with PKCeta in COS-7 cells leads to the formation of a stable PKD·PKCeta complex. Strikingly, we found very little evidence of complex formation between PKD and the PKCepsilon isoform despite its close similarity to PKCeta , and no evidence for a stable interaction between PKD and PKCzeta . Our results also demonstrate that the PH domain is critical for stable PKD·PKCeta complex formation, thus indicating that these domains can mediate highly selective protein-protein interactions.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell Culture and Transfections-- COS-7 cells were maintained by subculture in 10-cm tissue culture plates, every 3-4 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 10% CO2. For experimental dishes, confluent cells were subcultured at a density of 6 × 104 cells/ml in 6- or 10-cm dishes on the day prior to transfections. All transfections and co-transfections were carried out with equivalent amounts of DNA (6 µg/6-cm dish, 12 µg/10-cm dish), using vector pcDNA3 as the control DNA added to single transfections. Transfections were carried out in Opti-MEM (Life Technologies, Inc.) using Lipofectin (Life Technologies, Inc.) at 10 µl/6-cm dish or 20 µl/10-cm dish, added to cells in a final volume of 2.5 ml/6-cm dish or 5 ml/10-cm dish, following formation of DNA-Lipofectin complexes according to the protocol provided by the manufacturer. Cells were allowed to take up complexes in the absence of fetal bovine serum for 5-6 h or overnight, then fetal bovine serum (10% final concentration) in Opti-MEM was added to the dishes to yield a final volume of 5 ml/6-cm dish or 10 ml/10-cm dish. Cells were used for experiments after a further 48-72 h of incubation.

cDNA Constructs used in Transfections-- The constructs pcDNA3-PKD encoding PKD (12), pcDNA3-PKD/K618M encoding kinase-deficient mutant PKD (19), cysteine-rich domain mutant constructs pcDNA3-PKDDelta Cys1, pcDNA3-PKDDelta Cys2, and pcDNA3-PKDDelta CRD (22), the PKD mutant pcDNA3-PKDDelta NH2 with a deletion of the NH2-terminal first 66 residues including the putative transmembrane motif (22) and the PH domain mutants pcDNA3-PKDDelta PH lacking the entire PH domain, pcDNA3-PKDDelta 1-4beta lacking the first 4 beta  strands of the 7-stranded beta -barrel portion, and pcDNA3-PKDDelta alpha lacking the COOH-terminal alpha -helix have been described previously (15). An additional PH domain mutant PKD construct, pcDNA3-PKDDelta 1-7beta , encoding PKD lacking all seven beta -strands of the beta -barrel (deletion of amino acids 429-532) was constructed using PCR, as follows: primers carrying the AscI site (forward primer, 5'-GTCAGGCGCGCCGATGTGGCCAGGATGTGGGA-3'; reverse primer, 5'-GTCAGGCGCGCCAGCCAACAGAGGAGCCCTTG-3') were used to amplify the COOH-terminal part of the PH domain (from Asp533 to Gly557). The generated PCR product was AscI-digested and reinserted into the AscI site of pcDNA3-PKDDelta PH, resulting in the plasmid pcDNA3-PKDDelta 1-7beta . The cDNAs encoding wild-type and active mutant PKC isoforms were kind gifts from Dr. Peter Parker, Imperial Cancer Research Fund, and have been described previously (23).

Preparation of PKD PH Domain Fusion Protein-- The cDNA sequence spanning the entire PKD PH domain (aa 418-567) was amplified by PCR from wild-type PKD using specific oligonucleotide primers (forward primer, 5'-GATGGATCCGTGAAGCACACGAAGCGGAGG-3'; reverse primer, 5'-GCGGAATTCAGAAATATCTTTGTGTGAGTTGGA-3') containing restriction sites for BamHI and EcoRI, respectively (underlined). The resulting PCR product was subcloned as a BamHI-EcoRI fragment into the vector pGEX4T3 (Pharmacia Biotech Inc.) to generate the bacterial expression construct pGEX-GST-PKDPH. The 42-kDa GST-PKD PH domain fusion protein (GST-PKDPH) was expressed in Escherichia coli, purified on glutathione-agarose beads, eluted with 25 mM reduced glutathione, dialyzed against phosphate-buffered saline, and stored at -20 °C in 40% glycerol. Purity and concentration of the recombinant protein were assessed by SDS-PAGE and Coomassie Brilliant Blue staining.

Assays of PKC·GST-PKDPH Fusion Protein Binding in Vitro-- COS-7 cells transiently transfected with the different PKC isoforms were lysed 72 h after transfection by removal of growth medium from cells on ice and addition of lysis buffer (50 mM Tris-HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 10 µg/ml aprotinin, 100 µg/ml leupeptin, 1 mM AEBSF (Pefabloc), and 1% Triton X-100), and the resulting extracts were combined with either GST (control) or GST-PKDPH fusion proteins preadsorbed onto glutathione-agarose beads. After 2 h at 4 °C, the complexes were washed 8 times with lysis buffer, and bound proteins were extracted with SDS-PAGE sample buffer and subjected to SDS-PAGE and Western analysis using isoform-specific antisera to detect associated PKC, as described in figure legends.

Immunoprecipitations-- COS-7 cells transfected with wild-type or mutant PKD or co-transfected together with different PKC isoforms were lysed as described above. Small amounts (typically 1/10) of these total lysates were saved and combined with equal volumes of SDS-PAGE sample buffer (1 M Tris-HCl, pH 6.8, 6% SDS, 0.5 M EDTA, 4% 2-mercaptoethanol, 10% glycerol) for Western blot analysis. PKD was immunoprecipitated at 4 °C for 3 h with either the PA-1 antiserum (1:100 dilution) raised against the synthetic peptide EEREMKALSERVSIL that corresponds to the predicted COOH-terminal region of PKD, as described previously (12), or a 1:200 dilution of a commercial antiserum (PKD C-20, Santa Cruz Biotechnologies), which also recognizes the COOH-terminal region of PKD. PKCs were immunoprecipitated using respective PKC antisera at 1:100 dilution. Immune complexes were recovered using protein A coupled to agarose.

In Vitro Kinase Assays-- Immune complexes were washed twice with lysis buffer, then twice with kinase buffer consisting of 30 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol. Autophosphorylation reactions were initiated by combining 20 µl of immune complexes with 10 µl of a phosphorylation mixture containing [gamma -32P]ATP (3 µCi/reaction diluted with cold ATP to give a final concentration of 100 µM) in kinase buffer. Reactions were transferred to a water bath at 30 °C for 10 min, then terminated by addition of 1 ml of ice-cold kinase buffer and removed to an ice bucket. Immune complexes were recovered by centrifugation, and the proteins were extracted for SDS-PAGE analysis by addition of SDS-PAGE sample buffer. Dried SDS-PAGE gels were subjected to autoradiography to visualize radiolabeled protein bands.

For assays of exogenous substrate phosphorylation, immune complexes were processed as for autophosphorylation reactions, then substrates (either syntide-2 or epsilon -peptide at final concentrations 2.5 or 1.75 mg/ml, respectively) were added in the presence of [gamma -32P]ATP (2 µCi/reaction diluted with cold ATP to give a final concentration of 100 µM) in kinase buffer (final reaction volume, 30 µl), and transferred to a water bath at 30 °C for 10 min. Reactions were terminated by adding 100 µl of 75 mM H3PO4, and 75 µl of the mixed supernatant was spotted to Whatman P-81 phosphocellulose paper. Papers were washed thoroughly in 75 mM H3PO4, dried, and radioactivity incorporated into peptides was determined by detection of Cerenkov radiation in a scintillation counter.

Western Blot Analysis-- For Western blot analysis, immune complexes and proteins associated with glutathione-agarose/GST fusion protein complexes were washed as for in vitro kinase reactions, then extracted by boiling in SDS-PAGE sample buffer. Samples of cell lysates were directly solubilized by boiling in SDS-PAGE sample buffer. Following SDS-PAGE on 8% gels, proteins were transferred to Immobilon-P membranes (Millipore), as described previously (21) and blocked by overnight incubation with 5% non-fat dried milk in phosphate-buffered saline, pH 7.2. Membranes were incubated at room temperature for 2 h with antisera specifically recognizing either PKD or the different PKC isoforms, at a dilution of 1 µg/ml, in phosphate-buffered saline containing 3% non-fat dried milk. Immunoreactive bands were visualized using either horseradish peroxidase-conjugated anti-rabbit IgG and subsequent enhanced chemiluminescence detection or 125I-labeled protein A followed by autoradiography.

Materials-- [gamma -32P]ATP (6000 Ci/mmol) was from Amersham International (United Kingdom). Protein A-agarose and AEBSF (Pefabloc) were from Boehringer Mannheim (UK). Antisera (PKD C-20, PKCzeta C-20, PKCepsilon C-15, PKCeta C-15, and PKCalpha C-15) used in Western blot analysis were from Santa Cruz Biotechnologies, Palo Alto, CA. PKC standard proteins were from Calbiochem. Opti-MEM and Lipofectin were from Life Technologies, Inc. Glutathione-Sepharose was from Pharmacia Biotech. Syntide-2 peptide and immunizing peptide EEREMKALSERVSIL corresponding to the PKD COOH terminus were synthesized at the Imperial Cancer Research Fund. epsilon -Peptide was from Alexis Biochemicals. All other reagents were from standard suppliers or as described in the text and were the highest grade commercially available.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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An 80-kDa Phosphoprotein Associates with PKD in Cells Coexpressing PKD and PKCeta -- COS-7 cells transiently transfected with pcDNA3-PKD either alone or together with PKCepsilon , PKCeta , or PKCzeta expression constructs encoding constitutively active PKC mutants were incubated with or without 200 nM PDBu for 10 min and lysed. PKD was immunoprecipitated from the extracts and the resulting immunoprecipitates were subjected to in vitro kinase assays, followed by SDS-PAGE analysis and autoradiography to detect the prominent 110-kDa band corresponding to autophosphorylated PKD.

In agreement with previous results (12, 19-21), PKD isolated from cells transfected with PKD alone displayed low catalytic activity and PDBu treatment of these cells resulted in isolation of persistently activated PKD. Furthermore, PKD isolated from cells co-transfected with PKD together with active mutant PKCs epsilon  or eta  was in persistently activated form in the absence of PDBu treatments of these cells (Fig. 1A). Strikingly, when PKD immunoprecipitates from PKD/PKCeta co-transfected cells were assayed, an additional phosphorylated band corresponding to an apparent molecular mass of approximately 80 kDa was also apparent in the autoradiograms (Fig. 1A). This band did not appear when PKD was coexpressed with the active PKC epsilon  or zeta  mutant forms despite their level of overexpression shown by Western blot analysis (Fig. 2), and despite the fact that the PKCepsilon mutant induced PKD activation to essentially the same degree as did the PKCeta mutant (Fig. 1A). This latter result also indicates that the presence in PKD immunoprecipitates of the phosphoprotein giving rise to the 80-kDa band resulting from coexpression with PKCeta is not uniquely required for the persistent activation of PKD. Expression of PKCeta on its own followed by immunoprecipitation with the PA-1 antiserum and in vitro kinase assays did not result in the appearance of the 80-kDa band (Fig. 1A). Thus, the immunoprecipitation and subsequent labeling of the 80-kDa band required PKD.


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Fig. 1.   Detection of PKD·PKCeta complexes within PKD immunoprecipitates by in vitro kinase assays. A, exponentially growing COS-7 cells (40-60% confluent) were co-transfected with pcDNA3-PKD (PKD) together with either empty vector pcDNA3 (-) or the vectors containing the cDNAs encoding the constitutively active PKC isoforms, as indicated. At 72 h post-transfection, the cultures were either treated with 200 nM PDBu (PDB) (+) or left untreated (-) for 10 min. The cultures were then lysed and the lysates immunoprecipitated with the PA-1 antiserum and further analyzed by in vitro kinase reactions as described under "Experimental Procedures." A representative autoradiogram is shown. Similar results were obtained from each of at least three similar experiments. B, COS-7 cells were co-transfected with pCO2-PKCeta plasmid encoding the wild-type PKCeta (+) together with either pcDNA3-PKD or empty vector pcDNA3, as indicated (-). After 72 h of incubation, the cultures were lysed and the lysates immunoprecipitated with the commercially obtained anti-PKD antiserum in the absence (-) or presence (+) of the immunizing peptide (Imm Pep) used to generate the PA-1 antiserum at a concentration of 20 µg/ml lysate, and further analyzed by autophosphorylation reactions. This experiment was repeated twice with similar results. A representative autoradiogram is shown. C, left, COS-7 cells were co-transfected with pcDNA3-PKD (PKD), pcDNA3-PKDK618M (KM), or empty vector pcDNA3 (-) together with the plasmid construct encoding constitutive active PKCeta (eta *), as indicated. At 72 h post-transfection, the cultures were lysed and the lysates immunoprecipitated with the PA-1 antiserum and further analyzed by in vitro kinase reactions. This experiment was repeated three times with similar results. A representative autoradiogram is shown. C, right, COS-7 cells were co-transfected with either pcDNA3-PKD (PKD) or pcDNA3-PKDK618M (KM) together with plasmid constructs encoding wild-type PKCeta (eta ), active mutant PKCeta (eta *), or empty vector pcDNA3 (-), as indicated. At 72 h post-transfection, the cultures were either treated with 200 nM PDBu (+) or left untreated (-) for 10 min, as indicated. The cultures were then lysed and the lysates immunoprecipitated with the PA-1 antiserum and further analyzed by exogenous substrate phosphorylation assays using epsilon  peptide as described under "Experimental Procedures."


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Fig. 2.   Determination of PKD binding specificity toward different PKC isoforms by Western blotting. Exponentially growing COS-7 cells were either transfected with empty vector pcDNA3 (-) or pcDNA3-PKD (A) or co-transfected with vectors containing the cDNAs encoding either wild-type (epsilon  and eta ; C, D, and G) or constitutively active PKC isoforms (zeta *, epsilon *, and eta *; B, E, and F) together with either pcDNA3-PKD (+) or empty vector pcDNA3 (-), as indicated. At 72 h post-transfection, the cultures were lysed and the lysates were immunoprecipitated with either PA-1 antiserum (A-F) or the PKCeta antiserum (G). After washing, the immune complexes were either extracted with gel loading buffer and analyzed by SDS-PAGE followed by Western blot analysis using the isoform-specific polyclonal antisera against either the different PKCs (A-F) or PKD (G, PKD W.Blot) or analyzed by in vitro kinase assays (G, IVK), as indicated. Arrows indicate the position of PKD in anti-PKCeta immunoprecipitates (G) and PKCeta in PKD immunoprecipitates (D and F). Similar results were obtained in at least three experiments.

Initially, we considered whether this band might be the result of either nonspecific immunoprecipitation by the PA-1 antiserum or long-term overexpression of the active mutant PKCeta . To test this, we used a commercially available antiserum (rather than PA-1) to perform PKD immunoprecipitations from cells coexpressing PKD together with the wild-type PKCeta , followed by in vitro kinase assays. As shown in Fig. 1B, this assay also led to the isolation of immune complexes containing 110- and 80-kDa phosphoproteins. Thus, it was possible to substitute either the primary antibody used in the immunoprecipitation or the wild-type for the active mutant PKCeta and still retain both bands. In addition, both autophosphorylated PKD and the coprecipitated 80-kDa phosphoprotein band were eliminated when the immunoprecipitation reactions were carried out in the presence of the immunizing peptide used to generate the PA-1 antiserum (Fig. 1B).

To test whether the 80-kDa band represented a proteolytic fragment of autophosphorylated PKD, we co-transfected an expression construct, pcDNA3-PKD/K618M, which encodes a kinase-deficient mutant of PKD, together with PKCeta , and performed immunoprecipitations with the PA-1 antiserum followed by in vitro kinase assays. As shown in Fig. 1C, this assay again resulted in the appearance of the 80-kDa band. Importantly, whereas the intensity of this band was similar to that seen when the wild-type PKD was used for co-transfection, the intensity of the 110-kDa band was drastically reduced in comparison with that generated by the co-transfection with wild-type PKD2 (Fig. 1C, left). These data also indicate that the kinase activity of PKD is not required for the association with the 80-kDa phosphoprotein.

PKCeta Specifically Associates with PKD-- Previous studies indicated that epsilon -peptide, a peptide based on the pseudosubstrate domain of PKCepsilon , is a substrate for all PKCs (24), but is a poor substrate for PKD (11, 12, 15). In agreement with these studies, anti-PKD immunoprecipitates from lysates of cells transfected with PKD (or the kinase-deficient mutant, PKD/K618M) did not phosphorylate epsilon -peptide, even when PKD was activated within cells by PDBu treatments (Fig. 1C, right). Surprisingly, PKD immunoprecipitates from lysates of cells co-transfected with PKD (either wild-type or PKD/K618M) and PKCeta (either constitutive active or wild-type) contained activity which strongly phosphorylated epsilon -peptide. Since this activity was associated with both catalytically active and inactive forms of PKD, and PKD does not phosphorylate the peptide, we conclude that the activity was due to an associated protein kinase (e.g. contributed by the 80-kDa phosphoprotein) whose activity did not require the catalytic activity of PKD.

Since PKCeta has an apparent molecular mass of approximately 80 kDa in SDS-PAGE, and since the 80-kDa phosphoprotein had appeared only when PKD was coexpressed together with mutant or wild-type PKCeta , it seemed plausible that coexpression of PKD and PKCeta resulted in the formation of a PKD·PKCeta complex that persists during immunoprecipitation of PKD. To test this hypothesis directly, we used Western blot analysis to examine whether PKCeta was present in PKD immunoprecipitates. In view of the results indicating that the 80-kDa band is not detected in PKD immunoprecipitates from lysates of cells co-transfected with PKD together with either PKCzeta or PKCepsilon (Fig. 1A), we also performed Western blot analysis to assess whether these isoforms were present in PKD immunoprecipitates. Since PKCalpha is abundantly expressed in COS-7 cells, we also tested for the presence of this isoform in PKD immunoprecipitates. Lysates of cells either transfected with PKD or co-transfected with PKD and PKCs epsilon , eta , or zeta  were either examined directly by Western blot analysis using polyclonal antibodies that specifically recognize PKCs alpha , zeta , epsilon , or eta  or subjected to immunoprecipitation reactions with PA-1 antiserum followed by Western analysis. As shown in Fig. 2, D and F, anti-PKCeta immunoblotting of PKD immunoprecipitates indicated that both wild-type and mutant PKCeta associated with PKD. Similar analysis did not detect the presence of PKCs alpha , zeta , or epsilon  (either wild-type or constitutively active) in PKD immunoprecipitates from the corresponding cell lysates (Fig. 2, A-C and E). However, longer exposure of the autoradiograms did reveal a faint band of anti-PKCepsilon immunoreactivity (not shown), indicating that PKCepsilon may also associate with PKD but to a much lesser degree.

Although the experiments in Figs. 1 and 2 indicate that PKCeta was present in PKD immunoprecipitates from cells co-transfected with these two proteins, these results were obtained using antibodies directed against PKD. To substantiate further the existence of a PKD-PKCeta interaction, we also performed immunoprecipitations using an antibody directed against the opposite partner in the association, i.e. PKCeta , followed by Western analysis to detect the interacting PKD protein. As illustrated in Fig. 2G, in vitro kinase assays revealed the presence of an autophosphorylated band in the position corresponding to PKD (110 kDa) that depended on the presence of co-transfected PKD. Furthermore, Western blot analysis revealed the presence of immunoreactive PKD when both proteins were co-transfected. Thus, the results shown in Fig. 2G demonstrate the presence of PKD in PKCeta immunoprecipitates from lysates of cells co-transfected with PKD and PKCeta .

Phosphorylation of PKC and PKD Substrates by PKD Immunoprecipitates-- To further address the specificity of the PKD-PKC interaction, we measured phosphorylation of exogenous substrates by PKD immunoprecipitates from cells expressing each PKD-PKC combination. In initial experiments, we examined phosphorylation of epsilon -peptide by endogenously expressed and transfected PKC isoforms in anti-PKC immunoprecipitates. Results shown in Fig. 3A demonstrate that PKCeta or PKCepsilon immunoprecipitates from mock-transfected COS-7 cells contained low levels of epsilon -peptide phosphorylation activity that was dramatically increased by transfection of these isoforms. PKCzeta immunoprecipitates from mock-transfected cells contained similar activity to the immunoprecipitates from PKCeta - or PKCepsilon -transfected cells. These results demonstrate the ability of each PKC tested to phosphorylate epsilon -peptide.


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Fig. 3.   Determination of PKD binding specificity toward different PKC isoforms by phosphorylation of exogenous substrates. Exponentially growing COS-7 cells were transfected with either empty vector pcDNA3 (-) or plasmid constructs encoding the constitutively active PKC isoforms, or co-transfected with pcDNA3-PKD together with either empty vector or the active PKC isoforms, as indicated. At 72 h post-transfection, the cultures were lysed, the lysates were immunoprecipitated with the PA-1 antiserum, and subjected to exogenous substrate phosphorylation assays as described under "Experimental Procedures." A and B, lower panel, epsilon -peptide phosphorylation assay. B, upper panel, syntide-2 phosphorylation assay. The results are from at least three experiments, each performed in duplicate (B), or from a single experiment performed in triplicate (A).

For assays of PKD immunoprecipitates we used both epsilon -peptide, a poor substrate for PKD (Fig. 1), and syntide-2 (25, 26), a synthetic peptide previously demonstrated to be an excellent substrate for PKD (11), thereby exploiting the distinct substrate specificity of PKCs and PKD. In this way, we measured both PKD activation and the retention of PKC activity bound to PKD in the same immune complexes. In agreement with previous results, when the PA-1 antiserum was used to immunoprecipitate PKD from cells overexpressing only PKD, syntide-2 assays revealed a low basal activity that was dramatically increased upon PDBu stimulation of cells (Fig. 3B, upper graph). Again, these immunoprecipitates did not phosphorylate epsilon -peptide to any significant extent, even after the cells had been stimulated with PDBu (Fig. 3B, lower graph). In contrast with these results, when PKD was immunoprecipitated from cells co-transfected with PKD and either mutant or wild-type PKCeta , both syntide-2 and epsilon -peptide were strongly phosphorylated, indicating the presence of both PKD and PKCeta enzyme activities. These results extend those of Fig. 1 by providing evidence that PKCeta activity, and not that of PKCzeta or PKCepsilon , is preferentially associated with PKD. Thus, phosphorylation of the PKCepsilon peptide, an excellent PKC substrate, correlates with the appearance of the 80-kDa band in the in vitro kinase assays (Fig. 1) and with the detection of immunoreactive PKCeta in PKD immunoprecipitates (Fig. 2).

Interestingly, in cells co-transfected with PKD and PKCepsilon , phosphorylation of syntide-2 was also nearly maximal even in the absence of PDBu stimulation even though phosphorylation of epsilon -peptide was only slightly above control levels. This result demonstrated that, in agreement with results in Figs. 1 and 2, PKCepsilon had activated PKD during coexpression but is retained in the PKD immune complexes only slightly, implying that the involvement of PKCs in PKD activation is mediated by a transient event rather than, or in addition to, the PKD-PKC association itself. As expected, immunoprecipitates from cells co-transfected with PKD and PKCzeta phosphorylated syntide-2 in a manner indistinguishable from that from cells expressing only PKD, and did not phosphorylate epsilon -peptide. These results confirm that PKD preferentially forms complexes with PKCeta rather than with PKCepsilon , and does not form complexes with PKCzeta .

The PKD PH Domain Is Required for Formation of a PKD·PKCeta Complex-- Recent reports have shown that PH domains within the Bruton's tyrosine kinase and the serine-threonine kinase PKB/Akt can mediate association of these proteins with multiple isoforms of PKC (27, 28). In contrast, our results demonstrated that PKD preferentially associates with PKCeta . To examine whether the PH domain of PKD could mediate this specific association of PKD with PKCeta , we co-transfected COS-7 cells with wild-type PKCeta together with expression constructs encoding either intact PKD, PKD lacking the entire PH domain, or PKD with the PH domain truncated by partial deletions (15). PKD immunoprecipitates from these cells were subjected to Western blot analysis and phosphorylation of epsilon -peptide to assess the presence of PKCeta in the immune complexes (Fig. 4, A and B). In agreement with data shown in Fig. 2, wild-type PKCeta was co-immunoprecipitated with intact PKD (Fig. 4B). Similarly, these immunoprecipitates contained PKC activity, as revealed by epsilon  peptide assays (Fig. 4A). In contrast, when PKCeta was coexpressed with any of the PKD mutants containing either complete or partial deletions of the PH domain, both PKC activity (Fig. 4A) and PKCeta immunoreactivity (Fig. 4B) in PKD immunoprecipitates were sharply reduced. Control Western blots demonstrated that PKCeta was expressed in each transfection in similar amounts, as was each of the wild-type or mutant PKD proteins (Fig. 4B).


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Fig. 4.   Role of the PKD PH domain in mediating PKD·PKCeta complex formation. A, exponentially growing COS-7 cells were transfected with either pcDNA3-PKD (open bars) or co-transfected with PKDDelta PH lacking the entire PH domain (Delta PH), PKDDelta 1-4beta lacking the first amino-terminal four beta -sheets (Delta 4beta ), PKDDelta 1-7beta lacking all seven beta  sheets (Delta 7beta ), or PKDDelta alpha 1 lacking the carboxyl-terminal alpha -helix (Delta alpha 1), as indicated, together with wild type PKCeta (filled bars). At 72 h post-transfection, the cultures were lysed, the lysates were immunoprecipitated with the PA-1 antiserum, and subjected to epsilon -peptide phosphorylation assays as described under "Experimental Procedures." B, Western blot analysis of PKCeta in PKD immunoprecipitates (upper panel), PKCeta in cell lysates (center panel), and PKD mutants in cell lysates (lower panel) from the transfected cells. C, exponentially growing COS-7 cells were co-transfected with wild-type PKCeta together with PKD deletion mutants PKDDelta NH2 lacking an amino-terminal portion containing the transmembrane region (Delta NH2), PKDDelta PH (Delta PH), PKD-Delta Cys1 lacking the first cysteine-rich region (Delta Cys1), PKD-Delta Cys2 lacking the second cysteine-rich region (Delta Cys2), or PKDDelta CRD lacking the entire cysteine-rich domain (Delta CRD). (In the experiment shown, a control Western blot of cell lysates indicated that the PKDDelta CRD protein was expressed to somewhat higher levels than the other PKD truncation mutants, thereby accounting for the somewhat higher intensity of this band in the blot of PKCeta immunoprecipitates.) At 72 h post-transfection, these cells were lysed and the lysates subjected to immunoprecipitations with PKCeta or PKD antibodies, as indicated. Immunoprecipitates were washed and analyzed by SDS-PAGE prior to Western analysis. PKD immunoprecipitates were probed using the anti-PKCeta antiserum, and PKCeta immunoprecipitates were probed using the anti-PKD antiserum, as indicated. The arrow indicates the expected position for PKDDelta PH protein.

In order to determine the specificity of inhibition of PKD-PKCeta interaction by deletion of the PKD PH domain, we also examined the effect of other deletion mutants of PKD on the PKD-PKCeta interaction. COS-7 cells co-transfected with PKCeta together with PKD mutants lacking either a portion of the amino terminus containing the hydrophobic sequence of PKD, the first or second cysteine-rich regions, or the entire tandem cysteine-rich domain (22) were lysed and immunoprecipitated either with the PA-1 antiserum or with the PKCeta antiserum. As shown in Fig. 4C, Western analysis of each of these reciprocal immunoprecipitates revealed the presence of the opposite binding partner in the association. Similar to the results shown in Fig. 4, A and B, deletion of the PH domain prevented the detection of both binding partners in the respective immunoprecipitates (Fig. 4C). Taken together, these data indicate that the PH domain of PKD is necessary for efficient complex formation with PKCeta . However, it appears that the PH domain is not the only determinant of binding to PKD, as some PKCeta immunoreactivity and activity was associated with immunoprecipitates of the truncated PKD mutants (Fig. 4, A and B).

The PH Domain of PKD Selectively Binds to PKCeta -- To further examine the possibility that the PH domain of PKD interacts preferentially with PKCeta , we incubated lysates of cells transfected with activated PKC eta  or zeta  or wild-type PKC eta  or epsilon  with a fusion protein, GST-PKDPH, which contains the full-length PH domain (residues 429-557) of PKD. The recovered fusion protein was extracted and subjected to Western blot analysis to detect associated PKCs. As shown in Fig. 5A, PKCeta (activated and wild-type) proteins were retained by their ability to associate with the GST-PKDPH fusion protein. In contrast, much less PKCepsilon was recovered from parallel cell lysates with this fusion protein. Control Western blot analysis of cell lysates confirmed that PKCepsilon was indeed overexpressed in these cells, allowing us again to infer that there was a preferential binding of PKD to PKCeta over PKCepsilon . To address this issue more quantitatively, we performed Western blot analysis using different amounts of transfected cell lysates and purified protein standards (PKCepsilon and PKCeta ) to determine the absolute amounts of each PKC isoform produced in cell lysates and recovered by the fusion protein. As determined from data shown in Fig. 5B, each PKC isoform was present in the cell lysates in similar amounts (approximately 1.5 µg/ml for each PKC in the initial lysates). We then examined the binding of GST-PKDPH to PKCepsilon and PKCeta in cell lysates as a function of fusion protein concentration. These experiments demonstrated that the fusion protein bound to PKCeta at a concentration as low as 0.1 µg/ml. In contrast, very little PKCepsilon was bound to the fusion protein even at a 100-fold higher concentration (Fig. 5B).


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Fig. 5.   Binding of different PKCs to the GST-PKDPH fusion protein. COS-7 cells transiently transfected with active mutant PKCzeta (zeta *), wild type PKCepsilon (epsilon ), wild-type PKCeta (eta ), or active mutant PKCeta (eta *) were lysed after 72 h of incubation. Equivalent amounts of cell lysates were combined with immobilized GST or GST-PKDPH, as indicated and then processed by incubations, washing procedures, and Western blot analysis as described under "Experimental Procedures." A, GST or GST-PKD PH (1 µg each) was incubated with lysates from cells transfected with active mutant PKCzeta (zeta *), wild type PKCepsilon (epsilon ), wild-type PKCeta (eta ), or active mutant PKCeta (eta *) prior to further processing. B, upper panels, different amounts of standard PKC epsilon  and eta  proteins, as indicated, were subjected to Western blot analysis using the isoform-specific antisera. Middle panels, lysates were prepared from cells transfected with either PKCeta or PKCepsilon and subjected, in parallel, to Western blot analysis using the indicated amounts of cell lysates (diluted 1:1 with 2 × SDS-PAGE gel loading buffer), as indicated. Lower panels, GST (10 µg) or different amounts of GST-PKDPH (100 ng, 300 ng, 1 µg, 3 µg, 10 µg and 15 µg) were preadsorbed to glutathione-agarose beads, combined with lysates from cells transfected with either wild-type PKCeta or PKCepsilon , as indicated, and then incubated and further processed as in A. Results shown are representative of at least three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our previous studies have shown that PKD is activated in vivo by treatment with biologically active phorbol esters or multiple agonists via a PKC-dependent pathway (19-21). The results presented here demonstrate the formation of a complex between PKCeta and PKD and thus, identify a new aspect of the relationship between these two enzymes. Although further studies will be required to address the exact physiological role of this complex, the association of PKD and PKCeta could play a part in the regulation of the activity and/or subcellular localization of these enzymes. Indeed, recent findings from Cantley and co-workers (29) indicated that transfected PKD/PKCµ formed complexes with endogenous phosphatidylinositol 4-kinase and phosphatidylinositol 4-phosphate 5-kinase enzymes present in COS-7 cells. Importantly, truncation mutants of PKD/PKCµ lacking a portion of the molecule between the NH2-terminal hydrophobic region and the PH domain failed to retain the binding to the lipid kinases (29). In view of the results presented here, we conclude that different domains of the regulatory region of the PKD molecule are involved in mediating interactions with different proteins. Together, these results suggest the attractive possibility that PKD/PKCµ may act as a scaffold protein, through its different domains, promoting the assembly of signaling enzymes.

From our results, it is clear that the PH domain of PKD mediates a major portion of the binding to PKCeta , other (even larger) deletions of the PKD molecule being without effect. Recently, two important signaling proteins, PKB/Akt and BTK, have been shown to interact via their PH domains with multiple isoforms of PKC (27, 28, 30). Interestingly, there are important differences between these studies with BTK and PKB/Akt and the findings presented here with PKD. 1) The studies with BTK and PKB/Akt indicated that the PH domain of these protein kinases interacts with multiple isoforms of PKC. In contrast, our results show that PKD associates preferentially with PKCeta . In fact, the fusion protein containing only the PKD PH domain was sufficient to isolate the wild-type PKCeta from cell lysates, and in this "pull-out" experiment, the PH domain mirrored the results seen with the intact PKD, i.e. it exhibited a striking preference for binding PKCeta over the closely related PKCepsilon , and did not interact with PKCzeta . 2) The binding of BTK PH domain to PKCepsilon requires a region within the C1 regulatory domain in the vicinity of the pseudosubstrate domain of this enzyme (31). We find that PKD associates with an active PKCeta mutant with a deletion from amino acids 155 to 171 within its pseudosubstrate domain (as well as with wild-type PKCeta ). Therefore, the interaction of the PKD PH domain with PKCeta does not require the pseudosubstrate portion of the molecule.

Recently, a model for the binding of PH domains to proteins has been presented (32) in which a candidate sequence (HIKX8E), identified by sequence homology analysis, was proposed to act as a target for the PH domains. However, the putative binding sequence HIKX8E is present in both PKCeta and PKCzeta , and consequently is unlikely to play a critical role in determining the differential binding of the PH domain of PKD to different PKCs found in the present study.

In conclusion, our findings demonstrate that coexpression of PKD with PKCeta leads to the formation of a stable PKD·PKCeta complex. Strikingly, we found very little evidence of complex formation between PKD and the PKCepsilon isoform despite its close similarity to PKCeta , and no evidence for a stable interaction between PKD and PKCzeta . Our results also demonstrate that the PH domain is critical for stable PKD·PKCeta complex formation. We conclude that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs epsilon  and eta , thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.

    FOOTNOTES

* 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.

Dagger Contributed equally to the results of this study.

§ Supported by a postdoctoral research fellowship from the Imperial Cancer Research Fund. Present address: Dept. of Medicine, UCLA School of Medicine, Warren Hall, Room 11-124, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399.

To whom all correspondence should be addressed. Present address: Dept. of Medicine, UCLA School of Medicine, Warren Hall, Room 11-124, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399.

2 In results from a large number of experiments, no increases in band intensity associated with in vitro kinase analysis of the kinase-deficient mutant PKD/K618M have been seen. As we do see slight increases in the intensity of this band only when PKCeta is coexpressed, we consider likely the possibility that a small degree of transphosphorylation of the mutant by PKCeta has occurred in vitro.

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; PKD, protein kinase D; PH, pleckstrin homology; PDBu, phorbol 12,13-dibutyrate; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Weinstein, I. B. (1988) Cancer Res. 48, 4135-4143[Abstract]
  2. Nishizuka, Y. (1989) Cancer 63, 1892-1903[Medline] [Order article via Infotrieve]
  3. Rozengurt, E. (1986) Science 234, 161-166[Medline] [Order article via Infotrieve]
  4. Herschman, H. R. (1991) Annu. Rev. Biochem. 60, 281-319[CrossRef][Medline] [Order article via Infotrieve]
  5. Nishizuka, Y. (1992) Science 258, 607-614[Medline] [Order article via Infotrieve]
  6. Livneh, E., and Fishman, D. D. (1997) Eur. J. Biochem. 248, 1-9[Abstract]
  7. Dekker, L. V., Palmer, R. H., and Parker, P. J. (1995) Curr. Opin. Struct. Biol. 5, 396-402[CrossRef][Medline] [Order article via Infotrieve]
  8. Newton, A. C. (1995) J. Biol. Chem. 270, 28495-28498[Free Full Text]
  9. Hug, H., and Sarre, T. F. (1993) Biochem. J. 291, 329-343[Medline] [Order article via Infotrieve]
  10. Dekker, L. V., and Parker, P. J. (1994) Trends Biochem. Sci. 19, 73-77[CrossRef][Medline] [Order article via Infotrieve]
  11. Valverde, A. M., Sinnett-Smith, J., Van Lint, J., and Rozengurt, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8572-8576[Abstract]
  12. Van Lint, J., Sinnett-Smith, J., and Rozengurt, E. (1995) J. Biol. Chem. 270, 1455-1461[Abstract/Free Full Text]
  13. Rozengurt, E., Sinnett-Smith, J., and Zugaza, J. L. (1997) Biochem. Soc. Trans. 25, 565-571[Medline] [Order article via Infotrieve]
  14. Rozengurt, E. (1995) Cancer Surveys 24, 81-96[Medline] [Order article via Infotrieve]
  15. Iglesias, T., and Rozengurt, E. (1998) J. Biol. Chem. 273, 410-416[Abstract/Free Full Text]
  16. Hurley, J. H., Newton, A. C., Parker, P. J., Blumberg, P. M., and Nishizuka, Y. (1997) Protein Sci. 6, 477-480[Abstract/Free Full Text]
  17. Gibson, T. J., Hyvönen, M., Musacchio, A., Saraste, M., and Birney, E. (1994) Trends Biochem. Sci. 19, 349-353[CrossRef][Medline] [Order article via Infotrieve]
  18. Lemmon, M. A., Ferguson, K. M., and Schlessinger, J. (1996) Cell 85, 621-624[Medline] [Order article via Infotrieve]
  19. Zugaza, J. L., Sinnett-Smith, J., Van Lint, J., and Rozengurt, E. (1996) EMBO J. 15, 6220-6230[Abstract]
  20. Matthews, S. A., Pettit, G. R., and Rozengurt, E. (1997) J. Biol. Chem. 272, 20245-20250[Abstract/Free Full Text]
  21. Zugaza, J. L., Waldron, R. T., Sinnett-Smith, J., and Rozengurt, E. (1997) J. Biol. Chem. 272, 23952-23960[Abstract/Free Full Text]
  22. Iglesias, T., Matthews, S., and Rozengurt, E. (1998) FEBS Lett. 437, 19-23[CrossRef][Medline] [Order article via Infotrieve]
  23. Schèonwasser, D. C., Marais, R. M., Marshall, C. J., and Parker, P. J. (1998) Mol. Cell. Biol. 18, 790-798[Abstract/Free Full Text]
  24. Herget, T., Oehrlein, S. A., Pappin, D. J., Rozengurt, E., and Parker, P. J. (1995) Eur. J. Biochem. 233, 448-457[Abstract]
  25. Lorca, T., Cruzalegui, F. H., Fesquet, D., Cavadore, J. C., Méry, J., Means, A., and Dorée, M. (1993) Nature 366, 270-273[CrossRef][Medline] [Order article via Infotrieve]
  26. Mochizuki, H., Ito, T., and Hidaka, H. (1993) J. Biol. Chem. 268, 9143-9147[Abstract/Free Full Text]
  27. Yao, L., Kawakami, Y., and Kawakami, T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9175-9179[Abstract]
  28. Konishi, H., Kuroda, S., and Kikkawa, U. (1994) Biochem. Biophys. Res. Commun. 205, 1770-1775[CrossRef][Medline] [Order article via Infotrieve]
  29. Nishikawa, K., Toker, A., Wong, K., Marignani, P. A., Johannes, F. J., and Cantley, L. C. (1998) J. Biol. Chem. 273, 23126-23133[Abstract/Free Full Text]
  30. Konishi, H., Kuroda, S., Tanaka, M., Matsuzaki, H., Ono, Y., Kameyama, K., Haga, T., and Kikkawa, U. (1995) Biochem. Biophys. Res. Commun. 216, 526-534[CrossRef][Medline] [Order article via Infotrieve]
  31. Yao, L., Suzuki, H., Ozawa, K., Deng, J., Lehel, C., Fukamachi, H., Anderson, W. B., Kawakami, Y., and Kawakami, T. (1997) J. Biol. Chem. 272, 13033-13039[Abstract/Free Full Text]
  32. Alberti, S. (1998) Proteins 31, 1-9[CrossRef][Medline] [Order article via Infotrieve]


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