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

• DOI: 10.1074/jbc.274.14.9224
• Publication Date: 2002-07-26T15:00:08Z
• Authors: Richard T. Waldron; Teresa Iglesias; Enrique Rozengurt
• Keywords: Pleckstrin homology domain; Cyclin-dependent kinase 9; Protein kinase domain; c-Raf; MAP2K7

The results presented here demonstrate that protein kinase D (PKD) and PKCη transiently coexpressed in COS-7 cells form complexes can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCη. presence of immune was initially detected by vitro assays which reveal the an 80-kDa phosphorylated band addition 110-kDa corresponding autophosphorylated PKD. association between further verified Western blot analysis peptide phosphorylation exploited distinct substrate specificity PKCs By same criteria, formed only very weakly with PKCε, did not bind PKCζ. When mutants containing either complete partial deletions PH domain, both immunoreactivity PKC activity immunoprecipitates were sharply reduced. In contrast, deletion amino-terminal portion molecule, cysteine-rich region, entire domain interfere Furthermore, a glutathione S-transferase-PKDPH fusion bound preferentially These indicate discriminate closely related structures single enzyme family, e.g. novel ε η, thereby revealing previously undetected degree among protein-protein interactions mediated domains. C pleckstrin homology phorbol 12,13-dibutyrate 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride S-transferase polyacrylamide gel electrophoresis polymerase chain reaction Protein (PKC),1a major cellular target for potent tumor-promoting esters (1Weinstein I.B. Cancer Res. 1988; 48: 4135-4143PubMed Google Scholar, 2Nishizuka Y. Cancer. 1989; 63: 1892-1903Crossref PubMed Scopus (274) Scholar), has been implicated mediation diverse functions, including short-term regulation ion fluxes, receptor ligand binding, signal transduction, wide range longer term effects proliferation, differentiation, transformation, mammalian cycle (3Rozengurt E. Science. 1986; 234: 161-166Crossref (843) 4Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref (942) 5Nishizuka 1992; 258: 607-614Crossref (4207) 6Livneh Fishman D.D. Eur. J. 1997; 248: 1-9Crossref (206) Scholar). At least 10 isoforms, i.e. classic (α, β1, β2, γ), (δ, ε, θ), atypical (ζ, λ) have identified molecular cloning techniques (5Nishizuka 7Dekker L.V. Palmer R.H. Parker P.J. Curr. Opin. Struct. Biol. 1995; 5: 396-402Crossref (110) 8Newton A.C. Chem. 270: 28495-28498Abstract Full Text PDF (1461) different isoforms exhibit patterns expression types tissues as well subcellular distributions, leading view they play distinct, rather than redundant roles transduction 9Hug H. Sarre T.F. 1993; 291: 329-343Crossref (1215) Scholar,10Dekker Trends Sci. 1994; 19: 73-77Abstract (917) However, since few their substrates binding partners identified, it yet possible ascribe each isoform unique function. recently is mouse serine structural enzymological properties (11Valverde A.M. Sinnett-Smith Van Lint Rozengurt Proc. Natl. Acad. U. S. A. 91: 8572-8576Crossref (356) 12Van 1455-1461Abstract (154) 13Rozengurt Zugaza J.L. Soc. Trans. 25: 565-571Crossref (59) particular, catalytic PKD, distantly Ca2+-regulated kinases possesses low sequence similarity highly conserved regions subdomains family (14Rozengurt Surveys. 24: 81-96PubMed Accordingly, does phosphorylate variety utilized indicating 15Iglesias T. 1998; 273: 410-416Abstract (130) region contains tandem repeat diacylglycerol similar those found classical (16Hurley J.H. Newton Blumberg P.M. Nishizuka 6: 477-480Crossref (314) unlike all PKCs, possess regulatory pseudosubstrate upstream first motif. An additional feature distinguishes interposed second motif domain. domains are modular mediate lipid-protein many cytoskeletal transducing proteins (17Gibson T.J. Hyvönen M. Musacchio Saraste Birney 349-353Abstract (295) 18Lemmon M.A. Ferguson K.M. Schlessinger Cell. 1996; 85: 621-624Abstract (428) contribute (15Iglesias Recently we reported exposure intact biologically active esters, membrane-permeant bryostatin-1 induces activation via PKC-dependent pathway (19Zugaza EMBO 15: 6220-6230Crossref (220) Scholar,20Matthews S.A. Pettit G.R. 272: 20245-20250Abstract (69) activated within immunopurified extracts state independent exogenously added cofactors Growth factors mitogenic neuropeptides promote phospholipid turnover generation also stimulate this pathway, early event stimulation (21Zugaza Waldron R.T. 23952-23960Crossref (155) We substantiated role coexpression together constitutively mutant isoforms. studies revealed PKCε fully cells, whereas PKCβ1 PKCζ raise possibility functions downstream undescribed pathway. Here, demonstrate, time, leads formation stable PKD·PKCη complex. Strikingly, little evidence complex despite its close PKCη, no interaction Our critical formation, thus these selective interactions. maintained subculture 10-cm tissue culture plates, every 3–4 days Dulbecco's modified Eagle's medium supplemented 10% fetal bovine serum at 37 °C humidified atmosphere CO2. For experimental dishes, confluent subcultured density 6 × 104 cells/ml 6- dishes on day prior transfections. All transfections co-transfections carried out equivalent amounts DNA (6 μg/6-cm dish, 12 μg/10-cm dish), vector pcDNA3 control Transfections Opti-MEM (Life Technologies, Inc.) Lipofectin μl/6-cm dish 20 μl/10-cm final volume 2.5 ml/6-cm 5 ml/10-cm following DNA-Lipofectin according protocol provided manufacturer. Cells allowed take up absence 5–6 h overnight, then (10% concentration) yield dish. used experiments after 48–72 incubation. constructs pcDNA3-PKD encoding (12Van pcDNA3-PKD/K618M kinase-deficient pcDNA3-PKDΔCys1, pcDNA3-PKDΔCys2, pcDNA3-PKDΔCRD (22Iglesias Matthews FEBS Lett. 437: 19-23Crossref (60) pcDNA3-PKDΔNH2 NH2-terminal 66 residues putative transmembrane Scholar) pcDNA3-PKDΔPH lacking pcDNA3-PKDΔ1–4β 4 β strands 7-stranded β-barrel portion, pcDNA3-PKDΔα COOH-terminal α-helix described construct, pcDNA3-PKDΔ1–7β, seven β-strands (deletion amino acids 429–532) constructed PCR, follows: primers carrying AscI site (forward primer, 5′-GTCAGGCGCGCCGATGTGGCCAGGATGTGGGA-3′; reverse 5′-GTCAGGCGCGCCAGCCAACAGAGGAGCCCTTG-3′) amplify part (from Asp533 Gly557). generated PCR product wasAscI-digested reinserted into pcDNA3-PKDΔPH, resulting plasmid pcDNA3-PKDΔ1-7β. cDNAs wild-type kind gifts Dr. Peter Parker, Imperial Research Fund, (23Schèonwasser D.C. Marais R.M. Marshall C.J. Mol. 18: 790-798Crossref (670) cDNA spanning (aa 418–567) amplified oligonucleotide 5′-GATGGATCCGTGAAGCACACGAAGCGGAGG-3′; 5′-GCGGAATTCAGAAATATCTTTGTGTGAGTTGGA-3′) restriction sites BamHI EcoRI, respectively (underlined). subcloned aBamHI-EcoRI fragment pGEX4T3 (Pharmacia Biotech generate bacterial construct pGEX-GST-PKDPH. 42-kDa GST-PKD (GST-PKDPH) expressed Escherichia coli, purified glutathione-agarose beads, eluted 25 mm reduced glutathione, dialyzed against phosphate-buffered saline, stored −20 40% glycerol. Purity concentration recombinant assessed SDS-PAGE Coomassie Brilliant Blue staining. transfected lysed 72 transfection removal growth ice lysis buffer (50 Tris-HCl, pH 7.6, 2 EGTA, EDTA, 1 dithiothreitol, μg/ml aprotinin, 100 leupeptin, AEBSF (Pefabloc), 1% Triton X-100), combined GST (control) GST-PKDPH preadsorbed onto beads. After °C, washed 8 times buffer, extracted sample subjected isoform-specific detect associated PKC, figure legends. co-transfected above. Small (typically 1/10) total saved equal volumes (1 m 6.8, 6% SDS, 0.5m 4% 2-mercaptoethanol, glycerol) analysis. 3 PA-1 antiserum (1:100 dilution) raised synthetic EEREMKALSERVSIL corresponds predicted 1:200 dilution commercial (PKD C-20, Santa Cruz Biotechnologies), recognizes respective 1:100 dilution. Immune recovered A coupled agarose. twice consisting 30 7.4, MgCl2, dithiothreitol. Autophosphorylation reactions initiated combining μl mixture [γ-32P]ATP (3 μCi/reaction diluted cold ATP give μm) buffer. Reactions transferred water bath min, terminated ml ice-cold removed bucket. centrifugation, Dried gels autoradiography visualize radiolabeled bands. exogenous phosphorylation, processed autophosphorylation reactions, (either syntide-2 ε-peptide concentrations 1.75 mg/ml, respectively) (2 (final volume, μl), min. adding 75 H3PO4, mixed supernatant spotted Whatman P-81 phosphocellulose paper. Papers thoroughly mmH3PO4, dried, radioactivity incorporated peptides determined detection Cerenkov radiation scintillation counter. analysis, glutathione-agarose/GST boiling Samples directly solubilized Following 8% gels, Immobilon-P membranes (Millipore), blocked overnight incubation 5% non-fat dried milk 7.2. Membranes incubated room temperature specifically recognizing μg/ml, saline 3% milk. Immunoreactive bands visualized horseradish peroxidase-conjugated anti-rabbit IgG subsequent enhanced chemiluminescence 125I-labeled followed autoradiography. (6000 Ci/mmol) Amersham International (United Kingdom). A-agarose (Pefabloc) Boehringer Mannheim (UK). Antisera C-15, PKCα C-15) Biotechnologies, Palo Alto, CA. standard Calbiochem. Life Inc. Glutathione-Sepharose Pharmacia Biotech. Syntide-2 immunizing COOH terminus synthesized Fund. ε-Peptide Alexis Biochemicals. other reagents suppliers text highest grade commercially available. alone without 200 nm PDBu min lysed. assays, prominent agreement previous 19Zugaza 20Matthews 21Zugaza isolated displayed treatment resulted isolation persistently η treatments (Fig.1 A). when PKD/PKCη assayed, apparent mass approximately 80 kDa autoradiograms (Fig. This appear ζ forms level overexpression shown (Fig.2), fact induced essentially latter result indicates phosphoprotein giving rise uniquely required persistent Expression own immunoprecipitation appearance Thus, labeling PKD.Figure 2Determination toward blotting. Exponentially growing empty (−) (A) vectors (ε η; C, D, G) (ζ*, ε*, η*; B, E, F) (+) (−), indicated. post-transfection, cultures (A-F) (G). washing, loading analyzed polyclonal (G, W.Blot) IVK), Arrows position anti-PKCη (G) (D F). Similar obtained three experiments.View Large Image Figure ViewerDownload (PPT) Initially, considered whether might nonspecific long-term To test this, available (rather PA-1) perform immunoprecipitations coexpressing assays. As Fig. assay led 110- phosphoproteins. substitute primary antibody still retain addition, coprecipitated eliminated B). represented proteolytic pcDNA3-PKD/K618M, encodes performed again band. Importantly, intensity seen co-transfection, drastically comparison co-transfection 2In large number experiments, increases vitrokinase PKD/K618M seen. do see slight coexpressed, consider likely small transphosphorylation occurred vitro. left). data phosphoprotein. Previous indicated ε-peptide, based (24Herget Oehrlein Pappin D.J. 233: 448-457Crossref (79) but poor studies, anti-PKD (or mutant, PKD/K618M) even right). Surprisingly, constitutive wild-type) contained strongly ε-peptide. Since catalytically inactive peptide, conclude due (e.g. contributed phosphoprotein) whose require SDS-PAGE, had appeared seemed plausible persists during hypothesis directly, examine present immunoprecipitates. A), assess abundantly tested Lysates examined antibodies recognize α, ζ, 2, F, immunoblotting active) A-C E). faint anti-PKCε (not shown), may associate much lesser degree. Although Figs. two proteins, directed substantiate existence PKD-PKCη interaction, opposite partner association,i.e. interacting protein. illustrated G, (110 kDa) depended immunoreactive co-transfected. Gdemonstrate P