title: Global detection of protein kinase D-dependent phosphorylation events in nocodazole-treated human cells.
abstract: 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.
authors.affiliation: Proteome Center Tuebingen, University of Tuebingen, Tuebingen, Germany.
authors.forename: Mirita
authors.initials: M
authors.lastname: Franz-Wachtel
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Eisler
authors.forename: Karsten
authors.initials: K
authors.lastname: Krug
authors.forename: Silke
authors.initials: S
authors.lastname: Wahl
authors.forename: Alejandro
authors.initials: A
authors.lastname: Carpy
authors.forename: Alfred
authors.initials: A
authors.lastname: Nordheim
authors.forename: Klaus
authors.initials: K
authors.lastname: Pfizenmaier
authors.forename: Angelika
authors.initials: A
authors.lastname: Hausser
authors.forename: Boris
authors.initials: B
authors.lastname: Macek
mesh_terms: D020816:Amino Acid Motifs; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology; D057809:HEK293 Cells; D006801:Humans; D015739:Nocodazole / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000378:metabolism*; D050396:TRPP Cation Channels / Q000235:genetics / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology*
chemical_list: D015739:Nocodazole; D010750:Phosphoproteins; D020543:Proteome; D050396:TRPP Cation Channels; D050257:Tubulin Modulators