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Global Detection of Protein Kinase D-dependent Phosphorylation Events in Nocodazole-treated Human Cells

2012; Elsevier BV; Volume: 11; Issue: 5 Linguagem: Inglês

10.1074/mcp.m111.016014

ISSN

1535-9484

Autores

Mirita Franz‐Wachtel, Stephan A. Eisler, Karsten Krug, Silke Wahl, Alejandro Carpy, Alfred Nordheim, Klaus Pfizenmaier, Angelika Haußer, Boris Maček,

Tópico(s)

Biochemical and Molecular Research

Resumo

Protein kinase D (PKD) is a cytosolic serine/threonine kinase implicated in regulation of several cellular processes such as response to oxidative stress, directed cell migration, invasion, differentiation, and fission of the vesicles at the trans-Golgi network. Its variety of functions must be mediated by numerous substrates; however, only a couple of PKD substrates have been identified so far. Here we perform stable isotope labeling of amino acids in cell culture-based quantitative phosphoproteomic analysis to detect phosphorylation events dependent on PKD1 activity in human cells. We compare relative phosphorylation levels between constitutively active and kinase dead PKD1 strains of HEK293 cells, both treated with nocodazole, a microtubule-depolymerizing reagent that disrupts the Golgi complex and activates PKD1. We identify 124 phosphorylation sites that are significantly down-regulated upon decrease of PKD1 activity and show that the PKD target motif is significantly enriched among down-regulated phosphorylation events, pointing to the presence of direct PKD1 substrates. We further perform PKD1 target motif analysis, showing that a proline residue at position +1 relative to the phosphorylation site serves as an inhibitory cue for PKD1 activity. Among PKD1-dependent phosphorylation events, we detect predominantly proteins with localization at Golgi membranes and function in protein sorting, among them several sorting nexins and members of the insulin-like growth factor 2 receptor pathway. This study presents the first global detection of PKD1-dependent phosphorylation events and provides a wealth of information for functional follow-up of PKD1 activity upon disruption of the Golgi network in human cells. Protein kinase D (PKD) is a cytosolic serine/threonine kinase implicated in regulation of several cellular processes such as response to oxidative stress, directed cell migration, invasion, differentiation, and fission of the vesicles at the trans-Golgi network. Its variety of functions must be mediated by numerous substrates; however, only a couple of PKD substrates have been identified so far. Here we perform stable isotope labeling of amino acids in cell culture-based quantitative phosphoproteomic analysis to detect phosphorylation events dependent on PKD1 activity in human cells. We compare relative phosphorylation levels between constitutively active and kinase dead PKD1 strains of HEK293 cells, both treated with nocodazole, a microtubule-depolymerizing reagent that disrupts the Golgi complex and activates PKD1. We identify 124 phosphorylation sites that are significantly down-regulated upon decrease of PKD1 activity and show that the PKD target motif is significantly enriched among down-regulated phosphorylation events, pointing to the presence of direct PKD1 substrates. We further perform PKD1 target motif analysis, showing that a proline residue at position +1 relative to the phosphorylation site serves as an inhibitory cue for PKD1 activity. Among PKD1-dependent phosphorylation events, we detect predominantly proteins with localization at Golgi membranes and function in protein sorting, among them several sorting nexins and members of the insulin-like growth factor 2 receptor pathway. This study presents the first global detection of PKD1-dependent phosphorylation events and provides a wealth of information for functional follow-up of PKD1 activity upon disruption of the Golgi network in human cells. The protein kinase D (PKD) 1The abbreviations used are:PKDprotein kinase DPKD1kdPKD1 kinase deadPKD1caPKD1 constitutively activeTGNtrans-Golgi networkSILACstable isotope labeling by amino acids in cell cultureGFPgreen fluorescent proteinEGFPenhanced GFPCFPcyan fluorescent proteinYFPyellow fluorescent proteinSCXstrong cation exchangep-sitephosphorylation site; International Protein Index (IPI). 1The abbreviations used are:PKDprotein kinase DPKD1kdPKD1 kinase deadPKD1caPKD1 constitutively activeTGNtrans-Golgi networkSILACstable isotope labeling by amino acids in cell cultureGFPgreen fluorescent proteinEGFPenhanced GFPCFPcyan fluorescent proteinYFPyellow fluorescent proteinSCXstrong cation exchangep-sitephosphorylation site; International Protein Index (IPI). family comprises three closely related members: PKD1, PKD2, and PKD3, which belong to the calcium- and calmodulin-dependent kinase family of serine/threonine kinases (1Fu Y. Rubin C.S. Protein kinase D: Coupling extracellular stimuli to the regulation of cell physiology.EMBO Rep. 2011; 12: 785-796Crossref PubMed Scopus (101) Google Scholar). Dependent on stimulus and cell type, active PKD localizes to organelles such as the Golgi complex, mitochondria, nucleus, plasma membrane, and the F-actin cytoskeleton to control various cellular processes including survival responses to oxidative stress (2Storz P. Döppler H. Toker A. Protein kinase D mediates mitochondrion-to-nucleus signaling and detoxification from mitochondrial reactive oxygen species.Mol. Cell. Biol. 2005; 25: 8520-8530Crossref PubMed Scopus (190) Google Scholar), directed cell migration (3Eiseler T. Schmid M.A. Topbas F. Pfizenmaier K. Hausser A. PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration.FEBS Lett. 2007; 581: 4279-4287Crossref PubMed Scopus (47) Google Scholar, 4Eiseler T. Döppler H. Yan I.K. Kitatani K. Mizuno K. Storz P. Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot.Nat. Cell Biol. 2009; 11: 545-556Crossref PubMed Scopus (183) Google Scholar, 5Peterburs P. Heering J. Link G. Pfizenmaier K. Olayioye M.A. Hausser A. Protein kinase D regulates cell migration by direct phosphorylation of the cofilin phosphatase slingshot 1 like.Cancer Res. 2009; 69: 5634-5638Crossref PubMed Scopus (97) Google Scholar), invasion (6Eiseler T. Döppler H. Yan I.K. Goodison S. Storz P. Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion.Breast Cancer Res. 2009; 11: R13Crossref PubMed Scopus (120) Google Scholar, 7Zhang K. Ye C. Zhou Q. Zheng R. Lv X. Chen Y. Hu Z. Guo H. Zhang Z. Wang Y. Tan R. Liu Y. PKD1 inhibits cancer cells migration and invasion via Wnt signaling pathway in vitro.Cell Biochem. Funct. 2007; 25: 767-774Crossref PubMed Scopus (26) Google Scholar), differentiation (8Sucharov C.C. Langer S. Bristow M. Leinwand L. Shuttling of HDAC5 in H9C2 cells regulates YY1 function through CaMKIV/PKD and PP2A.Am. J. Physiol. Cell Physiol. 2006; 291: C1029-C1037Crossref PubMed Scopus (39) Google Scholar, 9Jensen E.D. Gopalakrishnan R. Westendorf J.J. Bone morphogenic protein 2 activates protein kinase D to regulate histone deacetylase 7 localization and repression of Runx2.J. Biol. Chem. 2009; 284: 2225-2234Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Kleger A. Loebnitz C. Pusapati G.V. Armacki M. Müller M. Tümpel S. Illing A. Hartmann D. Brunner C. Liebau S. Rudolph K.L. Adler G. Seufferlein T. Protein kinase D2 is an essential regulator of murine myoblast differentiation.PLoS One. 2011; 6: e14599Crossref PubMed Scopus (17) Google Scholar), and fission of the cell surface-directed transport carriers at the trans-Golgi network (TGN) (11Liljedahl M. Maeda Y. Colanzi A. Ayala I. Van Lint J. Malhotra V. Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network.Cell. 2001; 104: 409-420Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 12Yeaman C. Ayala M.I. Wright J.R. Bard F. Bossard C. Ang A. Maeda Y. Seufferlein T. Mellman I. Nelson W.J. Malhotra V. Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network.Nat. Cell Biol. 2004; 6: 106-112Crossref PubMed Scopus (208) Google Scholar). In most cases, involvement of PKD in these processes has been demonstrated by expression of a kinase dead PKD protein (PKDkd), which acts in a dominant-negative fashion toward the endogenous PKD proteins and thereby presents a functional knock-out. For example, expression of PKD1kd induces the formation of tubule-like structures, thus blocking secretion of basolateral cargo at the Golgi complex (11Liljedahl M. Maeda Y. Colanzi A. Ayala I. Van Lint J. Malhotra V. Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network.Cell. 2001; 104: 409-420Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Conversely, expression of a constitutively active PKD (PKD1ca) induces extensive fragmentation of Golgi membranes (13Bossard C. Bresson D. Polishchuk R.S. Malhotra V. Dimeric PKD regulates membrane fission to form transport carriers at the TGN.J. Cell Biol. 2007; 179: 1123-1131Crossref PubMed Scopus (108) Google Scholar). Likewise, PKD1kd enhances directed migration of breast cancer cells, whereas PKD1ca suppresses migration of these cells (3Eiseler T. Schmid M.A. Topbas F. Pfizenmaier K. Hausser A. PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration.FEBS Lett. 2007; 581: 4279-4287Crossref PubMed Scopus (47) Google Scholar, 4Eiseler T. Döppler H. Yan I.K. Kitatani K. Mizuno K. Storz P. Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot.Nat. Cell Biol. 2009; 11: 545-556Crossref PubMed Scopus (183) Google Scholar). protein kinase D PKD1 kinase dead PKD1 constitutively active trans-Golgi network stable isotope labeling by amino acids in cell culture green fluorescent protein enhanced GFP cyan fluorescent protein yellow fluorescent protein strong cation exchange phosphorylation site; International Protein Index (IPI). protein kinase D PKD1 kinase dead PKD1 constitutively active trans-Golgi network stable isotope labeling by amino acids in cell culture green fluorescent protein enhanced GFP cyan fluorescent protein yellow fluorescent protein strong cation exchange phosphorylation site; International Protein Index (IPI). These multiple functions of PKD are obviously mediated through numerous substrates. During the past years the knowledge on these substrates has increased dramatically. For instance, it was shown that PKD controls directed cell migration by direct phosphorylation of the cofilin phosphatase slingshot 1 (4Eiseler T. Döppler H. Yan I.K. Kitatani K. Mizuno K. Storz P. Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot.Nat. Cell Biol. 2009; 11: 545-556Crossref PubMed Scopus (183) Google Scholar, 5Peterburs P. Heering J. Link G. Pfizenmaier K. Olayioye M.A. Hausser A. Protein kinase D regulates cell migration by direct phosphorylation of the cofilin phosphatase slingshot 1 like.Cancer Res. 2009; 69: 5634-5638Crossref PubMed Scopus (97) Google Scholar, 14Barišić S. Nagel A.C. Franz-Wachtel M. Macek B. Preiss A. Link G. Maier D. Hausser A. Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1.EMBO Rep. 2011; 12: 527-533Crossref PubMed Scopus (19) Google Scholar), the kinase PAK4 (15Spratley S.J. Bastea L.I. Döppler H. Mizuno K. Storz P. Protein kinase D regulates cofilin activity through p21-activated kinase 4.J. Biol. Chem. 2011; 286: 34254-34261Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), cortactin (16Eiseler T. Hausser A. De Kimpe L. Van Lint J. Pfizenmaier K. Protein kinase D controls actin polymerization and cell motility through phosphorylation of cortactin.J. Biol. Chem. 2010; 285: 18672-18683Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and the tumor suppressor RIN1 (17Ziegler S. Eiseler T. Scholz R.P. Beck A. Link G. Hausser A. A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration.Mol. Biol. Cell. 2011; 22: 570-580Crossref PubMed Google Scholar), thereby affecting dynamic F-actin remodeling at the leading edge. At the TGN, PKD directly phosphorylates the lipid kinase PI4KIIIβ (18Hausser A. Storz P. Märtens S. Link G. Toker A. Pfizenmaier K. Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIbeta at the Golgi complex.Nat. Cell Biol. 2005; 7: 880-886Crossref PubMed Scopus (273) Google Scholar) and the lipid transfer proteins CERT (19Fugmann T. Hausser A. Schöffler P. Schmid S. Pfizenmaier K. Olayioye M.A. Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein.J. Cell Biol. 2007; 178: 15-22Crossref PubMed Scopus (162) Google Scholar) and OSBP (20Nhek S. Ngo M. Yang X. Ng M.M. Field S.J. Asara J.M. Ridgway N.D. Toker A. Regulation of oxysterol-binding protein Golgi localization through protein kinase D-mediated phosphorylation.Mol. Biol. Cell. 2010; 21: 2327-2337Crossref PubMed Scopus (90) Google Scholar), thus mediating the fission of vesicles destined for the cell surface. However, knockdown of CERT did not suppress soluble cargo secretion as effectively as a kinase dead, dominant-negative PKD1 variant (19Fugmann T. Hausser A. Schöffler P. Schmid S. Pfizenmaier K. Olayioye M.A. Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein.J. Cell Biol. 2007; 178: 15-22Crossref PubMed Scopus (162) Google Scholar), demonstrating that yet unidentified PKD substrates contribute to proper Golgi function. The microtubule-depolymerizing reagent nocodazole, which disrupts the Golgi complex to generate Golgi mini-stacks, activates PKD, and this nocodazole-dependent fragmentation of the Golgi can be blocked by expression of a kinase dead PKD1 protein (21Fuchs Y.F. Eisler S.A. Link G. Schlicker O. Bunt G. Pfizenmaier K. Hausser A. A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal.Traffic. 2009; 10: 858-867Crossref PubMed Scopus (21) Google Scholar). The PKD signaling pathways involved in nocodazole-dependent Golgi dispersal, however, remain to be investigated. Mass spectrometry-based proteomics is increasingly used in global detection of kinase substrates in eukaryotic cells. Modern, gel-free biochemical approaches for phosphopeptide enrichment (22Macek B. Mann M. Olsen J.V. Global and site-specific quantitative phosphoproteomics: Principles and applications.Annu. Rev. Pharmacol. Toxicol. 2009; 49: 199-221Crossref PubMed Scopus (345) Google Scholar) are used in combination with specific inactivation of kinases to perform quantitative phosphoproteomic readouts of kinase activity. Specific inhibition of analog-sensitive kinases (23Bishop A.C. Buzko O. Shokat K.M. Magic bullets for protein kinases.Trends Cell Biol. 2001; 11: 167-172Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 24Koch A. Hauf S. Strategies for the identification of kinase substrates using analog-sensitive kinases.Eur. J. Cell Biol. 2010; 89: 184-193Crossref PubMed Scopus (22) Google Scholar) and subsequent SILAC-based quantitative phosphoproteomics has recently been used to identify CDK1- and Aurora-dependent phosphorylation events in budding and fission yeast, respectively (25Holt L.J. Tuch B.B. Villén J. Johnson A.D. Gygi S.P. Morgan D.O. Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution.Science. 2009; 325: 1682-1686Crossref PubMed Scopus (664) Google Scholar, 26Koch A. Krug K. Pengelley S. Macek B. Hauf S. Mitotic substrates of the kinase aurora with roles in chromatin regulation identified through quantitative phosphoproteomics of fission yeast.Sci Signal. 2011; 4: rs6Crossref PubMed Scopus (92) Google Scholar). Likewise, chemical inhibition of endogenous kinases has been used to identify phosphorylation events downstream of the mTORC1, Polo-like, and Aurora kinases in human cells (27Hsu P.P. Kang S.A. Rameseder J. Zhang Y. Ottina K.A. Lim D. Peterson T.R. Choi Y. Gray N.S. Yaffe M.B. Marto J.A. Sabatini D.M. The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling.Science. 2011; 332: 1317-1322Crossref PubMed Scopus (812) Google Scholar, 28Yu Y. Yoon S.O. Poulogiannis G. Yang Q. Ma X.M. Villén J. Kubica N. Hoffman G.R. Cantley L.C. Gygi S.P. Blenis J. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling.Science. 2011; 332: 1322-1326Crossref PubMed Scopus (656) Google Scholar, 29Kettenbach A.N. Schweppe D.K. Faherty B.K. Pechenick D. Pletnev A.A. Gerber S.A. Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells.Sci Signal. 2011; 4: rs5Crossref PubMed Scopus (363) Google Scholar). In a groundbreaking study, a combination of the above approaches was used to systematically inactivate all protein kinases and phosphatases in budding yeast and derive the first comprehensive kinase/phosphatase substrate network of an eukaryotic microorganism (30Bodenmiller B. Wanka S. Kraft C. Urban J. Campbell D. Pedrioli P.G. Gerrits B. Picotti P. Lam H. Vitek O. Brusniak M.Y. Roschitzki B. Zhang C. Shokat K.M. Schlapbach R. Colman-Lerner A. Nolan G.P. Nesvizhskii A.I. Peter M. Loewith R. von Mering C. Aebersold R. Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.Sci Signal. 2010; 3: rs4Crossref PubMed Scopus (250) Google Scholar). Here we perform a global, SILAC-based quantitative phosphoproteomic study to identify phosphorylation events dependent on PKD activity. We compare two nocodazole-treated HEK293 strains, expressing constitutively active or dominant-negative (kinase dead) forms of PKD1. We demonstrate that 124 phosphorylation events are down-regulated upon the loss of PKD activity under conditions of nocodazole-dependent Golgi complex fragmentation. We perform functional enrichment analysis and show that proteins with localization and function at the membrane are significantly enriched among detected PKD targets. We further classify the PKD-dependent phosphorylation events based on their level of confidence, perform analysis of PKD1 target sequence motifs, and show that proline residues at position +1 relative to a PKD1-dependent phosphorylation site act as an inhibitory cue for PKD activity. Flp-In T-Rex-293 cells (Invitrogen) were grown in Dulbecco's modified Eagle's medium containing 10% FCS, zeocin at 100 μg/ml, and blasticidin at 15 μg/ml. These cells stably express the Tet repressor and contain a single FRT site and were used to generate the Flp-In-PKD1 lines. The cells were cotransfected with pcDNA5/FRT/TO-EGFP-PKD1ΔPH (PKD1 constitutively active or PKD1ca) (31Hausser A. Link G. Bamberg L. Burzlaff A. Lutz S. Pfizenmaier K. Johannes F.J. Structural requirements for localization and activation of protein kinase C mu (PKC mu) at the Golgi compartment.J. Cell Biol. 2002; 156: 65-74Crossref PubMed Scopus (74) Google Scholar) and PKD1K612W (PKD1 kinase dead or PKD1kd) (31Hausser A. Link G. Bamberg L. Burzlaff A. Lutz S. Pfizenmaier K. Johannes F.J. Structural requirements for localization and activation of protein kinase C mu (PKC mu) at the Golgi compartment.J. Cell Biol. 2002; 156: 65-74Crossref PubMed Scopus (74) Google Scholar), respectively, and the Flp recombinase expression plasmid pOG44 at a ratio of 1:10 and then selected with hygromycin at 100 μg/ml. Induction of protein expression with doxycycline was at 10 ng/ml. All of the cell lines were maintained in custom-made SILAC Dulbecco's modified Eagle's medium for 14 days. PKD1 constitutively active cells (FlpIN T-Rex293 PKD1ΔPH-GFP or PKD1ca) were labeled with SILAC heavy label (Lys8–Arg10); Parental FlpIN T-Rex293 cells were labeled with SILAC medium-heavy label (Lys4–Arg6); and PKD1 kinase dead cells (PKD1kd-GFP or PKD1kd) were labeled with the SILAC light label (Lys0–Arg0). Incorporation levels of the SILAC labels were in all cases higher than 95%. For phosphoproteome analysis, cells were plated on 15-cm dishes (7.5*106 cells/dish) followed by induction of protein expression 24 h later for additional 24 h. Prior to harvesting, the cells were left untreated or were stimulated with nocodazole (5 ng/μl) for 30 min as described previously (21Fuchs Y.F. Eisler S.A. Link G. Schlicker O. Bunt G. Pfizenmaier K. Hausser A. A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal.Traffic. 2009; 10: 858-867Crossref PubMed Scopus (21) Google Scholar). Three different SILAC experiments were performed. First, nocodazole-stimulated PKD1kd and nocodazole-stimulated PKD1ca cells were compared with each other, [PKDca/PKDkd]Noco+. Second, unstimulated PKD1kd-expressing cells were compared with unstimulated parental cells, [parental/PKDkd]Noco−. Finally, nocodazole-stimulated PKD1kd-expressing cells were compared with nocodazole-stimulated parental cells, [parental/PKDkd]Noco+. The cells were lysed in lysis buffer (6 m urea, 2 m thiourea, 10 mm Tris, pH 8.0, 1% octylglucoside, 1- Complete protease inhibitor mixture; missing paren Roche Applied Science) and 1× PhosSTOP phosphatase inhibitor mixture (Roche Applied Science)) for 30 min at room temperature and then sonified for 1 min on ice (Bandelin SONOPULS HD 200, Program MS73D). The proteins were precipitated using ice-cold acetone/methanol for 30 min on ice. The proteins were pelleted by centrifugation (2000 × g, 20 min, 4 °C) and washed three times with 80% ice-cold acetone. Dried proteins were resolved in digestion buffer (6 m urea, 2 m thiourea, 10 mm Tris, pH 8.0). HEK293T cells were maintained in Dulbecco's modified Eagle's medium SILAC heavy or SILAC light medium for 14 days (incorporation > 95%). The cells were plated on 15-cm dishes (15 × 106 cells/dish), and 24 h later the cells were harvested in phosphorylation buffer (50 mm Tris, pH 7.5, 10 mm MgCl2, 1× complete protease inhibitor) and lysed by sonification for 7 min in ice-cold water (Bio Rupter, Diagenode, 30-s cycles mean intensity). The lysates were clarified by centrifugation (16,000 × g, 10 min, room temperature) and incubated 20 min at room temperature to allow protein dephosphorylation by endogenous phosphatases to occur. Subsequently, 1 mg of lysate (2.5-ml volume) was incubated with 700 μl of purified active PKD1 (32Dieterich S. Herget T. Link G. Böttinger H. Pfizenmaier K. Johannes F.J. In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu.FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus (76) Google Scholar) (SILAC heavy) or PBS (SILAC light). Furthermore, DTT (2 mm), ATP (5 mm), and PhosSTOP were added to the reaction mix and incubated 30 min at 37 °C. Acetone/methanol protein precipitation was performed as described above. The dried protein pellets were resolved in water. SDS-PAGE and Western blot analysis have been described elsewhere (17Ziegler S. Eiseler T. Scholz R.P. Beck A. Link G. Hausser A. A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration.Mol. Biol. Cell. 2011; 22: 570-580Crossref PubMed Google Scholar). The proteins were visualized using the ECL detection system (Pierce) according to the manufacturer's specifications. Infrared dye-labeled secondary antibodies were visualized using the Odyssey infrared imaging system (LICOR Biosciences). The antibody specific for PKD autophosphorylation at serine 910 has been described elsewhere (21Fuchs Y.F. Eisler S.A. Link G. Schlicker O. Bunt G. Pfizenmaier K. Hausser A. A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal.Traffic. 2009; 10: 858-867Crossref PubMed Scopus (21) Google Scholar). The PKD pMOTIF antibody was from Cell Signaling. The GFP-specific mouse monoclonal antibody was obtained from Roche Applied Science. The tubulin α-specific monoclonal antibody was from Neomarkers. Secondary antibodies used were goat anti-rabbit IgG HRP-coupled (Dianova, Germany) and goat anti-mouse infrared dye-labeled secondary antibodies (LICOR Biosciences). The antibodies specific for phosphorylated serine 294 in PI4KIIIβ and phosphorylated serine 910 in human PKD1 were described elsewhere (18Hausser A. Storz P. Märtens S. Link G. Toker A. Pfizenmaier K. Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIbeta at the Golgi complex.Nat. Cell Biol. 2005; 7: 880-886Crossref PubMed Scopus (273) Google Scholar). FRET measurements were performed with an Infinite 200 reader (TECAN, Mainz, Germany). Parental and PKD1kd-GFP-expressing HEK293FlpIN cells were transiently transfected with G-PKDrep-live (33Eisler S.A. Fuchs Y.F. Pfizenmaier K. Hausser A. G-PKDrep-live, a genetically encoded FRET reporter to measure PKD activity at the trans-Golgi network.Biotechnol. J. 2012; 7: 148-154Crossref PubMed Scopus (9) Google Scholar) using TransIT293 (Mirus, Houston, TX) according to the manufacturer's instructions. Expression of PKD1kd-EGFP was induced 24 later by doxocyline (10 ng/ml). 48 h later, the cells were stimulated with nocodazole (5 μg/ml) for 30 min. The cells were solubilized in lysis buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm EDTA, 0.2% Triton X-100, Complete protease inhibitor and PhosSTOP (Roche Applied Science)), and the lysates were clarified by centrifugation at 16,000 × g for 15 min. 100 μl of lysate were measured in a black flat-bottomed 96-well plate. mCFP was excited at 430 nm, and the emission of mCFP and cpVENUS was detected at 460 nm (CFP) and 550 nm (YFP-C), respectively. The YFP-C/CFP ratio was calculated for each sample. The background of nontransfected cell lysates was subtracted for each cell type. Parental and HEK293FlpIn cells expressing PKD1kd-GFP were transiently transfected with a plasmid encoding G-PKDrep (21Fuchs Y.F. Eisler S.A. Link G. Schlicker O. Bunt G. Pfizenmaier K. Hausser A. A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal.Traffic. 2009; 10: 858-867Crossref PubMed Scopus (21) Google Scholar) using TransIT293 (Mirus) according to the manufacturer's instructions. Expression of PKD1kd-EGFP was induced 24 h later by doxocycline (10 ng/ml). 48 h later, the cells were stimulated with nocodazole (5 μg/ml) for 30 min. The cells were solubilized in lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1% Triton X-100, or Nonidet P-40, 1 mm EGTA, 1 mm EDTA, Complete protease inhibitor and PhosSTOP), and SDS-PAGE and Western blotting were performed as described above. Quantitative analysis was performed with the Odyssey software (Licor-Biosciences, Germany). Phosphorylation of G-PKDrep was normalized with the level of expression. The densitometry of the control sample was arbitrarily set to 1.0. HEK293FlpIN cells expressing PKD1kd-EGFP were grown on coverslips, washed with PBS, fixed in 4% para-formaldehyde at room temperature for 15 min, washed, permeabilized with 0.1% Triton X-100 (5 min at room temperature) and blocked with blocking buffer (5% fetal calf serum in PBS) for 30 min. The cells were incubated with the primary antibodies (giantin-specific polyclonal rabbit, Abcam; and p230-specific monoclonal mouse antibody, BD Biosciences) in blocking buffer for 2 h, washed, incubated with secondary antibodies (Alexa 546-coupled anti-mouse and anti-rabbit IgG; Invitrogen) in blocking buffer for 1 h, washed, and mounted in ProLong Gold (Invitrogen) supplemented with 1 μg/ml 4′,6′-diamino-2-phenylindole. The cells were analyzed by confocal laser scanning microscopy (LSM 710; Zeiss). EGFP was excited with the 488-nm line of the argon laser, and fluorescence was detected at 490–550 nm. Alexa 546 was excited at 561 nm using a diode-pumped solid state laser, and emission was detected at 566–680 nm. 4′,6′-diamino-2-phenylindole was excited with the 405-nm line of the argon laser, and fluorescence was detected at 415–485 nm. Protein extracts derived from the "light" and "heavy" labeled cell cultures were mixed in a 1:1 ratio according to measured protein amounts. Ten milligrams of the mixture was digested in solution with trypsin as described previously (34Borchert N. Dieterich C. Krug K. Schütz W. Jung S. Nordheim A. Sommer R.J. Macek B. Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models.Genome Res. 2010; 20: 837-846Crossref PubMed Scopus (129) Google Scholar). For proteome analysis, 2% of the total tryptic peptides were fractionated by isoelectric focusing on an OffGel 3100 Fractionator (Agilent) according to the manufacturer's instructions. Focusing was done with 24 cm (24 well) Immobiline DryStrips pH 3–10 (Bio-Rad) at a maximum current of 50 μA for 50 kVh. In SILAC experiments [parental/PKD1kd]Noco− and [parental/PKD1kd]Noco+ 100 μg of protein extracts were separated on a one-dimensional gel and in-gel digested by trypsin as described previously (34Borchert N. Dieterich C. Krug K. Schütz W. Jung S. Nordheim A. Sommer R.J. Macek B. Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models.Genome Res. 2010; 20: 837-846Crossref PubMed Scopus (129) Google Scholar). Peptide fractions were collected and desalted separately using C18 StageTips (35Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2569) Google Scholar). Phosphopeptide enrichment and phosphoproteome analysis was done as described previously (36Olsen J.V. Macek B. High accuracy mass spectrometry in large-scale analysis of protein phosphorylation.Methods Mol. Biol. 2009; 492: 131-142Crossref PubMed Scopus (45) Google Scholar) with minor modifications: ∼95% of the peptides were separated by strong cation exchange (SCX) chromatography with a gradient of 0 to 35% SCX sol

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