A Novel Tyrosine Phosphorylation Site in Protein Kinase D Contributes to Oxidative Stress-mediated Activation
2007; Elsevier BV; Volume: 282; Issue: 44 Linguagem: Inglês
10.1074/jbc.m703584200
ISSN1083-351X
Autores Tópico(s)Receptor Mechanisms and Signaling
ResumoProtein kinase D1 (PKD1) is a mediator of oxidative stress signaling where it regulates cellular detoxification and survival. Critical for the regulation of PKD1 activity in response to oxidative stress are Src- and Abl-mediated tyrosine phosphorylations that eventually lead to protein kinase Cδ (PKCδ)-mediated activation of PKD1. Here we identify Tyr95 in PKD1 as a previously undescribed phosphorylation site that is regulated by oxidative stress. Our data suggest that PKD1 phosphorylation at Tyr95 generates a binding motif for PKCδ, and that oxidative stress-mediated PKCδ/PKD interaction results in PKD1 activation loop phosphorylation and activation. We further analyzed all PKD isoforms for this mechanism and show that PKD enzymes PKD1 and PKD2 are targets for PKCδ in response to oxidative stress, and that PKD3 is not a target because it lacks the relevant tyrosine residue that generates a PKCδ interaction motif. Protein kinase D1 (PKD1) is a mediator of oxidative stress signaling where it regulates cellular detoxification and survival. Critical for the regulation of PKD1 activity in response to oxidative stress are Src- and Abl-mediated tyrosine phosphorylations that eventually lead to protein kinase Cδ (PKCδ)-mediated activation of PKD1. Here we identify Tyr95 in PKD1 as a previously undescribed phosphorylation site that is regulated by oxidative stress. Our data suggest that PKD1 phosphorylation at Tyr95 generates a binding motif for PKCδ, and that oxidative stress-mediated PKCδ/PKD interaction results in PKD1 activation loop phosphorylation and activation. We further analyzed all PKD isoforms for this mechanism and show that PKD enzymes PKD1 and PKD2 are targets for PKCδ in response to oxidative stress, and that PKD3 is not a target because it lacks the relevant tyrosine residue that generates a PKCδ interaction motif. Protein kinase D (PKD) 2The abbreviations used are: PKD, protein kinase D; PH, pleckstrin homology domain; PKC, protein kinase C; ROS, reactive oxygen species; nPKC, novel protein kinase C; GST, glutathione S-transferase; HA, hemagglutinin; pY, phosphotyrosine; CFP, cyan fluorescent protein.2The abbreviations used are: PKD, protein kinase D; PH, pleckstrin homology domain; PKC, protein kinase C; ROS, reactive oxygen species; nPKC, novel protein kinase C; GST, glutathione S-transferase; HA, hemagglutinin; pY, phosphotyrosine; CFP, cyan fluorescent protein. is a serine/threonine kinase that belongs to the family of calcium/calmodulin-dependent kinases (1Manning G. Kim D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6157) Google Scholar, 2Van Lint J. Kim A. Maeda Y. Vantus T. Sturany S. Malhotra V. Vandenheede J.R. Seufferlein T. Trends Cell Biol. 2002; 12: 193-200Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The PKD kinase family consists of three members, PKD1/PKCμ, PKD2, and PKD3/PKCν, which have some overlapping but also distinct isoform-specific functions within cells (3Chen J. Kim G. Wang Q.J. Mol. Pharmacol. 2005; 67: 152-162Crossref PubMed Scopus (40) Google Scholar, 4Hausser A. Kim P. Martens S. Link G. Toker A. Pfizenmaier K. Nat. Cell Biol. 2005; 7: 880-886Crossref PubMed Scopus (273) Google Scholar, 5Wang Q.J. Trends Pharmacol. Sci. 2006; 27: 317-323Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 6Yeaman C. Kim M.I. Wright J.R. Bard F. Bossard C. Ang A. Maeda Y. Seufferlein T. Mellman I. Nelson W.J. Malhotra V. Nat. Cell Biol. 2004; 6: 106-112Crossref PubMed Scopus (206) Google Scholar, 7Auer A. Kim J. Sturany S. von Wichert G. Van Lint J. Vandenheede J. Adler G. Seufferlein T. Mol. Biol. Cell. 2005; 16: 4375-4385Crossref PubMed Scopus (49) Google Scholar). Protein kinase D isoforms are activated in response to numerous stimuli including reactive oxygen species (ROS), growth factors (i.e. platelet-derived growth factor), activators of G protein-coupled receptors, and triggering of immune cell receptors such as the B-cell receptor or T-cell receptor complexes (8Jamora C. Kim N. Van Lint J. Laudenslager J. Vandenheede J.R. Faulkner D.J. Malhotra V. Cell. 1999; 98: 59-68Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 9Yuan J. Kim L.W. Gu J. Rozengurt E. J. Biol. Chem. 2003; 278: 4882-4891Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Storz P. Kim A. EMBO J. 2003; 22: 109-120Crossref PubMed Scopus (269) Google Scholar, 11Matthews S.A. Kim E. Cantrell D. J. Exp. Med. 2000; 191: 2075-2082Crossref PubMed Scopus (98) Google Scholar, 12Sidorenko S.P. Kim C.L. Klaus S.J. Chandran K.A. Takata M. Kurosaki T. Clark E.A. Immunity. 1996; 5: 353-363Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 13Van Lint J. Kim Y. Valius M. Merlevede W. Vandenheede J.R. J. Biol. Chem. 1998; 273: 7038-7043Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). PKD activity is regulated by autoinhibition, membrane translocation, and activating phosphorylations (reviewed by Rozengurt et al. (14Rozengurt E. Kim O. Waldron R.T. J. Biol. Chem. 2005; 280: 13205-13208Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar)). Several NH2-terminal protein domains of PKD such as the pleckstrin homology (PH) domain and the two C1 domains have negative regulatory, autoinhibitory functions and their deletion leads to constitutive activity of the enzyme (15Iglesias T. Kim E. J. Biol. Chem. 1998; 273: 410-416Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 16Hausser A. Kim G. Bamberg L. Burzlaff A. Lutz S. Pfizenmaier K. Johannes F.J. J. Cell Biol. 2002; 156: 65-74Crossref PubMed Scopus (74) Google Scholar). Although the exact molecular mechanisms are not known, it was suggested that stimulus-mediated lipid binding to the C1 domains (5Wang Q.J. Trends Pharmacol. Sci. 2006; 27: 317-323Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), protein binding to the PH domain (8Jamora C. Kim N. Van Lint J. Laudenslager J. Vandenheede J.R. Faulkner D.J. Malhotra V. Cell. 1999; 98: 59-68Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), phosphorylations of serine residues of the activation loop in the kinase domain (17Waldron R.T. Kim E. J. Biol. Chem. 2003; 278: 154-163Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), or oxidative stress-mediated tyrosine phosphorylations in the PH domain of PKD release autoinhibition (18Storz P. Kim H. Johannes F.J. Toker A. J. Biol. Chem. 2003; 278: 17969-17976Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). PKD1 is an important sensor for many inducers of oxidative stress such as Rotenone, diphenyleneiodonium, H2O2, pervanadate, and dl-buthionine-(S,R)-sulfoximine (10Storz P. Kim A. EMBO J. 2003; 22: 109-120Crossref PubMed Scopus (269) Google Scholar, 18Storz P. Kim H. Johannes F.J. Toker A. J. Biol. Chem. 2003; 278: 17969-17976Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19Storz P. Kim H. Toker A. Mol. Cell. Biol. 2005; 25: 8520-8530Crossref PubMed Scopus (187) Google Scholar, 20Storz P. Kim H. Toker A. Mol. Cell. Biol. 2004; 24: 2614-2626Crossref PubMed Scopus (201) Google Scholar, 21Waldron R.T. Kim E. J. Biol. Chem. 2000; 275: 17114-17121Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 22Waldron R.T. Kim O. Zhukova E. Rozengurt E. J. Biol. Chem. 2004; 279: 27482-27493Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Song J. Kim J. Lulla A. Evers B.M. Chung D.H. Am. J. Physiol. 2006; 290: C1469-C1476Crossref PubMed Scopus (47) Google Scholar). Importantly, PKD1 in response to activation by ROS leads to the induction of nuclear factor κB (NF-κB), a transcription factor that regulates SOD2 expression and cellular detoxification as well as cell survival (10Storz P. Kim A. EMBO J. 2003; 22: 109-120Crossref PubMed Scopus (269) Google Scholar, 19Storz P. Kim H. Toker A. Mol. Cell. Biol. 2005; 25: 8520-8530Crossref PubMed Scopus (187) Google Scholar, 24Storz P. Kim H. Ferran C. Grey S.T. Toker A. Biochem. J. 2005; 387: 47-55Crossref PubMed Scopus (49) Google Scholar). This signaling pathway could be of importance for processes regulating cell survival under oxidative stress conditions such as mechanisms that regulate aging or cancer. PKD2 also activates NF-κB in response to other stimuli such as BCR-Abl expression in myeloid leukemia cells and NF-κB, activated by this PKD isoform mediates interleukin 8 production in epithelial cells (25Chiu T.T. Kim W.Y. Moyer M.P. Strieter R.M. Rozengurt E. Am. J. Physiol. 2007; 292: C767-C777Crossref PubMed Scopus (45) Google Scholar, 26Mihailovic T. Kim M. Auer A. Van Lint J. Schmid M. Weber C. Seufferlein T. Cancer Res. 2004; 64: 8939-8944Crossref PubMed Scopus (72) Google Scholar). An initial step in ROS-mediated PKD1 activation are tyrosine phosphorylations mediated by the kinases Src and Abl (10Storz P. Kim A. EMBO J. 2003; 22: 109-120Crossref PubMed Scopus (269) Google Scholar, 18Storz P. Kim H. Johannes F.J. Toker A. J. Biol. Chem. 2003; 278: 17969-17976Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 21Waldron R.T. Kim E. J. Biol. Chem. 2000; 275: 17114-17121Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). It has been shown that Src-mediated phosphorylations in the PH domain lead to further activating phosphorylations, suggesting a conformational change as a first step in the PKD1 activation cascade (20Storz P. Kim H. Toker A. Mol. Cell. Biol. 2004; 24: 2614-2626Crossref PubMed Scopus (201) Google Scholar, 27Storz P. Kim H. Toker A. Mol. Pharmacol. 2004; 66: 870-879Crossref PubMed Scopus (99) Google Scholar). PKD1 gains full activity after phosphorylation at two serine residues in the activation loop in the kinase domain, which is mediated by novel PKC (nPKC) isoforms. Four nPKC isoforms (PKCθ, PKCε, PKCη, and PKCδ) are known within cells and all have been described to directly phosphorylate the PKD1 activation loop serines (17Waldron R.T. Kim E. J. Biol. Chem. 2003; 278: 154-163Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 20Storz P. Kim H. Toker A. Mol. Cell. Biol. 2004; 24: 2614-2626Crossref PubMed Scopus (201) Google Scholar, 28Brandlin I. Kim S. Eiseler T. Martinez-Moya M. Horschinek A. Hausser A. Link G. Rupp S. Storz P. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 2002; 277: 6490-6496Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 29Yuan J. Kim D. Cantrell D. Nel A.E. Rozengurt E. Biochem. Biophys. Res. Commun. 2002; 291: 444-452Crossref PubMed Scopus (60) Google Scholar). However, depending on the cellular context or the activating stimulus there is specificity for one isoform over the other. For example, it has been shown that PKCε and PKCη activate PKD1 in response to growth factor signaling or at the Golgi (13Van Lint J. Kim Y. Valius M. Merlevede W. Vandenheede J.R. J. Biol. Chem. 1998; 273: 7038-7043Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 17Waldron R.T. Kim E. J. Biol. Chem. 2003; 278: 154-163Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 28Brandlin I. Kim S. Eiseler T. Martinez-Moya M. Horschinek A. Hausser A. Link G. Rupp S. Storz P. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 2002; 277: 6490-6496Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 30Brandlin I. Kim T. Salowsky R. Johannes F.J. J. Biol. Chem. 2002; 277: 45451-45457Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 31Diaz Anel A.M. Kim V. J. Cell Biol. 2005; 169: 83-91Crossref PubMed Scopus (118) Google Scholar), whereas only PKCδ regulates PKD1 activity in response to oxidative stress, Rho activation, or stimulation with angiotensin II (20Storz P. Kim H. Toker A. Mol. Cell. Biol. 2004; 24: 2614-2626Crossref PubMed Scopus (201) Google Scholar, 23Song J. Kim J. Lulla A. Evers B.M. Chung D.H. Am. J. Physiol. 2006; 290: C1469-C1476Crossref PubMed Scopus (47) Google Scholar, 32Tan M. Kim X. Ohba M. Cui M.Z. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2271-2276Crossref PubMed Scopus (22) Google Scholar). It was suggested that a nPKC/PKD activation complex is necessary to facilitate activation loop phosphorylation and full activation of PKD. The nPKC isoform PKCη, for example, associates with the PH domain of PKD1 in response to growth factor signaling (33Waldron R.T. Kim T. Rozengurt E. J. Biol. Chem. 1999; 274: 9224-9230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). However, the sites of interaction of PKD with other novel PKC isoforms have not been mapped so far and it is particularly unclear how PKCδ interacts with PKD1 in response to ROS. Recently, the C2 domain of protein kinase Cδ was described as a novel phosphotyrosine binding domain (34Benes C.H. Kim N. Elia A.E. Dharia T. Cantley L.C. Soltoff S.P. Cell. 2005; 121: 271-280Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In response to overexpression of Src this domain facilitates the interaction of PKCδ with the Src-binding glycoprotein CDCP1. The C2 domain interacts with a minimal phosphotyrosine consensus motif that was described as (V/I)-pY-(Q/R)-X-(Y/F)-X, whereby the tyrosine residue is phosphorylated by Src (34Benes C.H. Kim N. Elia A.E. Dharia T. Cantley L.C. Soltoff S.P. Cell. 2005; 121: 271-280Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Although additional proteins containing this motif such as MUC-1, Abl, and PLD2 all have been shown to associate with PKCδ, the experimental proof for interaction of these proteins with the C2 domain of PKCδ through this phosphotyrosine motif is still lacking (34Benes C.H. Kim N. Elia A.E. Dharia T. Cantley L.C. Soltoff S.P. Cell. 2005; 121: 271-280Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). We here demonstrate that PKCδ interacts with PKD1, whereas a PKCδ lacking the C2 domain does not bind PKD1. This data suggests that the mechanism of how PKCδ binds to and activates PKD1 in response to oxidative stress is via its C2 domain. We describe a novel phosphotyrosine residue within a C2-binding motif in PKD1 that is phosphorylated by Src and regulates the interaction of PKD1 with PKCδ. We further show that this interaction is prerequisite for oxidative stress-mediated regulation of the PKD isoforms PKD1 and PKD2, but not for PKD3. Cell Lines, Antibodies, and Reagents—HeLa cells were from the American Type Culture Collection and were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The anti-Abl, anti-PKCδ, anti-PKCε, anti-PKCθ, anti-PKCη, anti-GST, and anti-PKD/PKCμ (C-20) antibodies were from Santa Cruz (Santa Cruz, CA), anti-Src from Upstate Biotechnology (Waltham, MA). Anti-HA and anti-FLAG were from Sigma. Anti-pS744/748 (recognizes the phosphorylated activation loop in PKD1/2/3) antibody was from BIOSOURCE/Invitrogen. The rabbit polyclonal pY95 antibody was raised against a KFPECGFpYGMYD-amide (amino acids 88–99 in human PKD1) peptide (21 Century Biochemicals, Marlboro, MA). The rabbit polyclonal pY463 antibody was a kind gift from A. Toker (Harvard Medical School, Boston, MA). The secondary horseradish peroxidase-linked anti-mouse or anti-rabbit antibodies were from Roche (Indianapolis, IN). H2O2 was purchased from Fisher Scientific and PP2 was from Calbiochem (La Jolla, CA). 12-Phorbol 13-myristate acetate was from Sigma and pervanadate was prepared as described previously (18Storz P. Kim H. Johannes F.J. Toker A. J. Biol. Chem. 2003; 278: 17969-17976Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Mirus HeLa-Monster Reagent (Madison, WI) was used for transient transfections. DNA Constructs—Full-length human PKD1 expression plasmids are based on an amino-terminal HA-tagged PKD1 cDNA cloned in pcDNA3 via BamHI and XhoI sites (18Storz P. Kim H. Johannes F.J. Toker A. J. Biol. Chem. 2003; 278: 17969-17976Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Amino-terminal HA-tagged PKD1 deleted in amino acids 1–321 (HA-PKD1-Δ1–321) was generated using 5′-gcgggatccgatatgtatccttatgatgttcctgattatgctggcgaagtgaccattaatgga-3′ and 5′-ggcctcgagtcagaggatgctgacacgctcacc-3′ oligonucleotides as primer pairs; amino-terminal HA-tagged-PKD1 deleted in the acidic region (HA-PKD1-ΔAR) and amino-terminal HA-tagged PKD1 deleted in the PH domain (HA-PKD1-ΔPH) were generated using 5′-gcgggatccatgtatccttatgatgttcttgattatgctagcgcccctccggtcctg-3′ and 5′-ggcctcgagtcagaggatgctgacacgctcacc-3′ oligonucleotides as primer pairs and a PKD1-ΔAR or PKD1-ΔPH as a template; amino-terminal HA-tagged PKD1 deleted in the kinase domain (HA-PKD1-ΔKIN) was generated using 5′-gcgggatccatgtatccttatgatgttcctgattatgctatgagcgcccctccggtcctg-3′ and 5′-gcgctcgagtcatcctgtacccacggaggagcc-3′ oligonucleotides as primer pairs. PCR products were cloned into pcDNA3 via BamHI and XhoI. The PKD1-Y463F and PKD1-Y463E expression constructs have been described before (10Storz P. Kim A. EMBO J. 2003; 22: 109-120Crossref PubMed Scopus (269) Google Scholar, 18Storz P. Kim H. Johannes F.J. Toker A. J. Biol. Chem. 2003; 278: 17969-17976Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Mutagenesis was carried out by PCR using the above described pcDNA3-HA-PKD1 construct or a pcDNA3-HA-PKD1-Y463E construct as template and the QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA) with 5′-tcccctgaatgtggtttcttcggaatgtatgataagatc-3′ and 5′-gatcttatcatacattccgaagaaaccacattcagggga-3′ oligonucleotides as primer pairs for Y95F mutation; 5′-tcccctgaatgtggtttccagggaatgtatgataagatc-3′ and 5′-gatcttatcatacattccctggaaaccacattcagggga-3′ oligonucleotides as primer pairs for Y95Q mutation; 5′-tcccctgaatgtggtttcgagggaatgtatgataagatc-3′ and 5′-gatcttatcatacattccctcgaaaccacattcagggga-3′ oligonucleotides as primer pairs for Y95E mutation; 5′-ggtttctacggaatgtttgataagatcctgctt-3′ and 5′-aagcaggatcttatcaaacattccgaaacc-3′ oligonucleotides as primer pairs for Y98F mutation; 5′-tgtggtttctacggaatgcaagataagatcctgcttttt-3′ and 5′-aaaaagcaggatcttatcttgcattccgtagaaaccaca-3′ oligonucleotides as primer pairs for Y98Q mutation. Furthermore, 5′-tttccagagtgtggattctatggcatgtatgacaaaatt-3′ and 5′-aattttgtcatacatgccatagaatccacactctggaaa-3′ oligonucleotides were used as primer pairs with GST-PKD3 as a template to generate a GST-PKD3-F103Y expression construct. Wild-type GST-PKD3 and FLAG-PKD2 expression constructs have been obtained from Dr. V. Malhotra and Dr. T. Seufferlein and have been described elsewhere (6Yeaman C. Kim M.I. Wright J.R. Bard F. Bossard C. Ang A. Maeda Y. Seufferlein T. Mellman I. Nelson W.J. Malhotra V. Nat. Cell Biol. 2004; 6: 106-112Crossref PubMed Scopus (206) Google Scholar, 35Sturany S. Kim J. Muller F. Wilda M. Hameister H. Hocker M. Brey A. Gern U. Vandenheede J. Gress T. Adler G. Seufferlein T. J. Biol. Chem. 2001; 276: 3310-3318Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). FLAG-tagged wild-type PKCδ and PKCδ-ΔC2 expression constructs were obtained by PCR using cyan fluorescent protein (CFP)-tagged wild-type PKCδ as template and 5′-gcgggatccatggactataaggacgatgatgacaaagcgccgttcctgcgcatcgcctcc-3′ or 5′-gcgggatccatggactataaggacgatgatgacaaacgcagtgaggacgaggccaag-3′ and 5′-gcgctcgagtcaatcttccaggcggtgctcgaa-3′ as primers and cloned via BamHI and XhoI into the expression vector pcDNA4/TO. Both CFP-tagged constructs were obtained from Dr. A. Newton (36Giorgione J.R. Kim J.H. McCammon J.A. Newton A.C. J. Biol. Chem. 2006; 281: 1660-1669Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Constitutively active PKCδ was obtained from Dr. S. Ohno, constitutively active Abl from Dr. N. Rosenberg, and wild-type, dominant-negative (Src-V295R-Y527F) or constitutively active Src (Src-Y527F) from Dr. J. Brugge. All constructs were verified by DNA sequencing. Immunoblotting, Immunoprecipitation, and GST Pull-down Assays—Cells were lysed in lysis buffer (50 mm Tris/HCl, pH 7.4, 1% Triton X-100, 150 mm NaCl, 5 mm EDTA, pH 7.4) plus Protease Inhibitor Mixture (Sigma). Lysates were used either for immunoblot analysis or proteins of interest were immunoprecipitated by a 1-h incubation with the respective antibody (2 μg) followed by a 30-min incubation with protein G-Sepharose (Amersham Biosciences). Immune complexes were washed three times with ice-cold TBS (50 mm Tris/HCl, pH 7.4, 150 mm NaCl), and resolved by SDS-PAGE. For GST pull-down assays lysates were incubated for 2 h with GST-Sepharose beads (Amersham Biosciences) and complexes were washed three times with ice-cold TBS (50 mm Tris/HCl, pH 7.4, 150 mm NaCl), and resolved by SDS-PAGE. Immunofluorescence—Cells were transfected (5 μg of DNA) and 24 h after transfection plated on glass coverslips at a density of 50,000 cells/well in a 24-well plate. The next day cells were washed twice with phosphate-buffered saline and fixed in 3.5% paraformaldehyde (15 min, 37 °C). Following permeabilization (0.1% Triton X-100, 10 min) cells were blocked with 3% bovine serum albumin and 0.05% Tween 200 in phosphate-buffered saline (blocking solution) for 30 min at room temperature. The coverslips were then incubated with primary antibody diluted in blocking solution anti-HA (rat), 1:2,000, overnight at 4 °C. Cells were then washed five times with phosphate-buffered saline and incubated with secondary antibody diluted 1:500 in blocking solution (donkey anti-rat IgG Alexa Fluor 488) for 2 h at room temperature. After extensive washes in phosphate-buffered saline coverslips were mounted in Fluormount-G (Southern Biotech, Birmingham, AL) and examined. The NH2 Terminus of PKD1 Contains a PKCδ Binding Motif—PKD1 has important roles in the protective response to oxidative stress induced by hydrogen peroxide, where it promotes cellular survival and detoxification (37Storz P. Trends Cell Biol. 2007; 17: 13-18Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). H2O2 has been shown to lead to Src and PKCδ activation, which both in turn can phosphorylate PKD1 (19Storz P. Kim H. Toker A. Mol. Cell. Biol. 2005; 25: 8520-8530Crossref PubMed Scopus (187) Google Scholar). In this activation mechanism Src causes priming phosphorylations that facilitate PKCδ-mediated phosphorylations, which lead to a fully active PKD enzyme (20Storz P. Kim H. Toker A. Mol. Cell. Biol. 2004; 24: 2614-2626Crossref PubMed Scopus (201) Google Scholar, 27Storz P. Kim H. Toker A. Mol. Pharmacol. 2004; 66: 870-879Crossref PubMed Scopus (99) Google Scholar). However, the mechanisms of how PKCδ interacts with and activates PKD1 in response to oxidative stress are not known. First, we determined which nPKC isoforms besides PKCδ are capable to bind PKD1 in response to oxidative stress. We found that only the δ isoform binds to PKD1 after treatment of cells with H2O2 (Fig. 1A). Then, to map the region of interaction of both enzymes we analyzed the interaction of endogenous PKCδ with PKD1 deletion mutants after stimulation of cells with hydrogen peroxide. Therefore we expressed wild-type PKD1 or mutants deleted in the acidic region (ΔAR), the pleckstrin homology domain (ΔPH), the kinase domain (ΔKIN), and the NH2 terminus (Δ1–321) (Fig. 1, B and C). We found that in response to oxidative stress, PKCδ interacts within a region containing the first 321 amino acids of PKD1 (Fig. 1C). This NH2-terminal region of PKD1 contains the lipid-binding C1a and C1b domains as well as a putative transmembrane region and a 14-3-3 binding motif (38Hausser A. Kim P. Link G. Stoll H. Liu Y.C. Altman A. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 1999; 274: 9258-9264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 39Johannes F.J. Kim J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar). Within the NH2 terminus we also found a sequence containing two tyrosine residues (Phe94-Tyr95-Gly96-Met97-Tyr98-Asp99), similar to the recently described minimal consensus motif (V/I)-pY-(Q/R)-X-(Y/F)-X that facilitates interaction of proteins with the PKCδ C2 domain (Fig. 2A) (34Benes C.H. Kim N. Elia A.E. Dharia T. Cantley L.C. Soltoff S.P. Cell. 2005; 121: 271-280Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). We mutated both tyrosines in this motif to phenylalanine (PKD1-Y95F; PKD1-Y98F) or to glutamine (PKD1-Y95Q; PKD1-Y98Q) and analyzed the interaction of these PKD1 mutants with PKCδ in response to oxidative stress (Fig. 2B). Interestingly, PKD1 mutants with tyrosine to phenylalanine or glutamine mutations at residue 95 lost their ability to interact with PKCδ suggesting that a tyrosine at this position is required. On the other hand a PKD1 mutant with tyrosine at residue Tyr98 mutated to phenylalanine was still capable of interacting with PKCδ in response to oxidative stress. However, a tyrosine 98 to glutamine PKD1 mutant (PKD1-Y98Q) lost its ability to interact with PKCδ, suggesting, that position 98 allows tyrosine or phenylalanine, which concurs with the requirements of the consensus motif for PKCδ binding (Fig. 2A).FIGURE 2Importance of PKD1 tyrosine residue 95 for the interaction with PKCδ. A, comparison of the consensus motif that allows binding of the C2 domain of PKCδ to phosphotyrosine residues and a similar motif of amino acids 94 to 99 (FYGLYD) identified in PKD1, with Tyr95 as a potential phosphorylation site. B, HA-tagged wild-type PKD1 or PKD1 mutants (PKD1-Y95F, PKD1-Y95Q, PKD1-Y98F, and PKD1-Y98Q) were overexpressed in HeLa cells and cells were treated with hydrogen peroxide (10 min, 10 mm). PKD1 was immunoprecipitated (anti-HA) and samples were analyzed for co-immunoprecipitation of PKCδ by immunoblotting with anti-PKCδ. The nitrocellulose was then stripped and re-probed for PKD expression (anti-PKD). All results are typical of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oxidative Stress-mediated Phosphorylation of PKD1 at Tyrosine Residue 95—Tyr95 mediates binding of PKD1 to PKCδ in response to oxidative stress. Furthermore, the Tyr95-Gly-Met/Leu-Tyr/Phe sequence in PKD1 shows similarity to the minimal consensus sequence ((V/I)-pY-(Q/R)-IX-(Y/F)-X) for PKCδ-binding proteins. We next analyzed if PKD1 is phosphorylated at tyrosine residue 95 in this potential PKCδ C2 domain binding motif. To analyze phosphorylation of this site in response to oxidative stress, we generated a phosphospecific antibody that specifically recognizes PKD phosphorylated at tyrosine residue 95 (anti-pY95). To demonstrate phosphorylation of tyrosine 95 in response to oxidative stress we transfected wild-type PKD1, PKD1-Y95F, and PKD1-Y98F mutants and analyzed the phosphorylation of Tyr95 after induction of oxidative stress. As expected, a PKD1-Y95F mutant was not phosphorylated in response to oxidative stress, whereas wild-type PKD1 and a PKD1-Y98F mutant were tyrosine phosphorylated and were recognized by the antibody (Fig. 3A). This experiment also indicates specificity of the pY95 antibody for the phosphorylated Tyr95 residue. Because a mutational analysis always bears the possibility that point mutants are mislocalized within cells, we analyzed cellular localization of the wild-type PKD1 and the Y95F mutant and found no significant differences in cellular localization either before or after hydrogen peroxide treatment (Fig. 3B). We also used the anti-pY95 antibody to analyze oxidative stress-treated cells for tyrosine phosphorylation of endogenous PKD1 and PKD2 at this residue and found that both are phosphorylated at this tyrosine residue in response to oxidative stress (Fig. 3C). Tyrosine phosphorylation of Tyr95 did not occur in response to treatment of cells with phorbol ester, which mimic diacylglycerol formation and thus activate PKC isoforms and PKD in some signaling pathways (supplemental Fig. 1). Because we have shown in previous studies that tyrosine kinases Src and Abl are upstream of PKD1 in response to oxidative stress signaling and that both can directly phosphorylate certain tyrosine residues in PKD1, we next analyzed if the tyrosine phosphorylation of PKD1 at Tyr95 is mediated by one of these kinases. We expressed constitutive-active Abl or Src and analyzed if this leads to PKD1 phosphorylation at residue Tyr95 and found that Src, but not Abl mediates Tyr95 phosphorylation of overexpressed and endogenous PKD1 (Fig. 4, A and B). This is in accordance with Benes et al. (34Benes C.H. Kim N. Elia A.E. Dharia T. Cantley L.C. Soltoff S.P. Cell. 2005; 121: 271-280Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), who identified Src as the kinase that leads to the phosphorylation of the tyrosine residue of the PKCδ binding motif of other targets that interacts with the C2 domain of PKCδ. Moreover, the inhibition of Src with PP2 led to an inhibition of oxidative stress-mediated phosphorylation of PKD1 at Tyr95, further indicating the importance of Src in this activation mechanism (Fig. 4C). Finally, to determine whether Src is the major regulator of PKD1 pY95 phosphorylation, we compared cells transfected with wildtype (WT-Src) and dominant-negative Src (DN-Src) and determined PKD1 Tyr95 phosphorylation in response to oxidative stress. Dominant-negative Src completely blocked PKD1 phosphorylation, indicating that Src (but not other Src-like kinases) is the mediator of PKD1 phosphorylation at pY95 (Fig. 4D). A more detailed analysis of how Src contributes to the phosphorylation of this site will be the subject of future studies. PKCδ Interaction with PKD Isoforms Occurs via a pY-Gly-Met/Leu-Tyr Motif—We next analyzed the amino acid sequences of all three PKD isoforms, PKD1/PKCμ, PKD2, and PKD3/PKCν for the potential PKCδ interactio
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