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

[1]  K. Pfizenmaier,et al.  G‐PKDrep‐live, a genetically encoded FRET reporter to measure PKD activity at the trans‐Golgi‐network , 2012, Biotechnology journal.

[2]  P. Storz,et al.  Protein Kinase D Regulates Cofilin Activity through p21-activated Kinase 4* , 2011, The Journal of Biological Chemistry.

[3]  C. Rubin,et al.  Protein kinase D: coupling extracellular stimuli to the regulation of cell physiology , 2011, EMBO reports.

[4]  Devin K Schweppe,et al.  Quantitative Phosphoproteomics Identifies Substrates and Functional Modules of Aurora and Polo-Like Kinase Activities in Mitotic Cells , 2011, Science Signaling.

[5]  B. Maček,et al.  Mitotic Substrates of the Kinase Aurora with Roles in Chromatin Regulation Identified Through Quantitative Phosphoproteomics of Fission Yeast , 2011, Science Signaling.

[6]  S. Gygi,et al.  Phosphoproteomic Analysis Identifies Grb10 as an mTORC1 Substrate That Negatively Regulates Insulin Signaling , 2011, Science.

[7]  B. Maček,et al.  Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1 , 2011, EMBO reports.

[8]  D. Sabatini,et al.  The mTOR-Regulated Phosphoproteome Reveals a Mechanism of mTORC1-Mediated Inhibition of Growth Factor Signaling , 2011, Science.

[9]  Rolf-Peter Scholz,et al.  A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration , 2011, Molecular biology of the cell.

[10]  Stefan Tümpel,et al.  Protein Kinase D2 Is an Essential Regulator of Murine Myoblast Differentiation , 2011, PloS one.

[11]  Patrick G. A. Pedrioli,et al.  Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast , 2010, Science Signaling.

[12]  John M. Asara,et al.  Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation , 2010, Molecular biology of the cell.

[13]  R. Sommer,et al.  Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models. , 2010, Genome research.

[14]  K. Pfizenmaier,et al.  Protein Kinase D Controls Actin Polymerization and Cell Motility through Phosphorylation of Cortactin* , 2010, The Journal of Biological Chemistry.

[15]  André Koch,et al.  Strategies for the identification of kinase substrates using analog-sensitive kinases. , 2010, European journal of cell biology.

[16]  S. Gygi,et al.  Global Analysis of Cdk1 Substrate Phosphorylation Sites Provides Insights into Evolution , 2009, Science.

[17]  K. Pfizenmaier,et al.  Protein kinase D regulates cell migration by direct phosphorylation of the cofilin phosphatase slingshot 1 like. , 2009, Cancer research.

[18]  K. Pfizenmaier,et al.  A Golgi PKD Activity Reporter Reveals a Crucial Role of PKD in Nocodazole‐Induced Golgi Dispersal , 2009, Traffic.

[19]  J. Bamburg.  Faculty Opinions recommendation of Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot. , 2009 .

[20]  M. Mann,et al.  Global and site-specific quantitative phosphoproteomics: principles and applications. , 2009, Annual review of pharmacology and toxicology.

[21]  T. Seufferlein,et al.  Characterization of cortactin as an in vivo protein kinase D substrate: interdependence of sites and potentiation by Src. , 2009, Cellular signalling.

[22]  J. Westendorf,et al.  Bone Morphogenic Protein 2 Activates Protein Kinase D to Regulate Histone Deacetylase 7 Localization and Repression of Runx2* , 2009, Journal of Biological Chemistry.

[23]  B. Maček,et al.  High accuracy mass spectrometry in large-scale analysis of protein phosphorylation. , 2009, Methods in molecular biology.

[24]  S. Goodison,et al.  Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion , 2009, Breast Cancer Research.

[25]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[26]  C. Machamer,et al.  Acute Perturbations in Golgi Organization Impact De Novo Sphingomyelin Synthesis , 2008, Traffic.

[27]  V. Malhotra,et al.  Dimeric PKD regulates membrane fission to form transport carriers at the TGN , 2007, The Journal of cell biology.

[28]  Qin Zhou,et al.  PKD1 inhibits cancer cells migration and invasion via Wnt signaling pathway in vitro , 2007, Cell biochemistry and function.

[29]  K. Pfizenmaier,et al.  PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration , 2007, FEBS letters.

[30]  K. Pfizenmaier,et al.  Regulation of secretory transport by protein kinase D–mediated phosphorylation of the ceramide transfer protein , 2007, The Journal of cell biology.

[31]  J. Whitelegge,et al.  Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D. , 2007, Biochemical and biophysical research communications.

[32]  Steven P Gygi,et al.  Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry , 2007, Nature Methods.

[33]  M. Mann,et al.  Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips , 2007, Nature Protocols.

[34]  L. Leinwand,et al.  Shuttling of HDAC5 in H9C2 cells regulates YY1 function through CaMKIV/PKD and PP2A. , 2006, American journal of physiology. Cell physiology.

[35]  M. Mann,et al.  Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap*S , 2005, Molecular & Cellular Proteomics.

[36]  Peter Storz,et al.  Protein Kinase D Mediates Mitochondrion-to-Nucleus Signaling and Detoxification from Mitochondrial Reactive Oxygen Species , 2005, Molecular and Cellular Biology.

[37]  Peter Storz,et al.  Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIβ at the Golgi complex , 2005, Nature Cell Biology.

[38]  Jing Chen,et al.  Actin and Arf1-dependent recruitment of a cortactin–dynamin complex to the Golgi regulates post-Golgi transport , 2005, Nature Cell Biology.

[39]  A. Toker,et al.  A Phosphorylation State-specific Antibody Recognizes Hsp27, a Novel Substrate of Protein Kinase D* , 2005, Journal of Biological Chemistry.

[40]  Peter Storz,et al.  Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIbeta at the Golgi complex. , 2005, Nature cell biology.

[41]  J. Shabanowitz,et al.  A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. , 2004, Analytical chemistry.

[42]  V. Malhotra,et al.  Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network , 2004, Nature Cell Biology.

[43]  E. Rozengurt,et al.  Protein kinase D complexes with C-Jun N-terminal kinase via activation loop phosphorylation and phosphorylates the C-Jun N-terminus , 2002, Oncogene.

[44]  Klaus Pfizenmaier,et al.  Structural requirements for localization and activation of protein kinase C μ (PKCμ) at the Golgi compartment , 2002, The Journal of cell biology.

[45]  V. Malhotra,et al.  Recruitment of protein kinase D to the trans‐Golgi network via the first cysteine‐rich domain , 2001, The EMBO journal.

[46]  Anthony C. Bishop,et al.  Magic bullets for protein kinases. , 2001, Trends in cell biology.

[47]  V. Malhotra,et al.  Protein Kinase D Regulates the Fission of Cell Surface Destined Transport Carriers from the Trans-Golgi Network , 2001, Cell.

[48]  S. Pfeffer,et al.  Mapmodulin, cytoplasmic dynein, and microtubules enhance the transport of mannose 6-phosphate receptors from endosomes to the trans-golgi network. , 1999, Molecular biology of the cell.

[49]  Zhou Songyang,et al.  Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes* , 1997, The Journal of Biological Chemistry.

[50]  K. Pfizenmaier,et al.  In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase Cμ , 1996, FEBS letters.

[51]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[52]  A. Linstedt,et al.  Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa. , 1993, Molecular biology of the cell.

[53]  P. Gleeson,et al.  Human autoantibodies as reagents to conserved Golgi components. Characterization of a peripheral, 230-kDa compartment-specific Golgi protein. , 1992, The Journal of biological chemistry.