title: Protein Interaction Network of Human Protein Kinase D2 Revealed by Chemical Cross-Linking/Mass Spectrometry.
abstract: We investigated the interaction network of human PKD2 in the cytosol and in Golgi-enriched subcellular protein fractions by an affinity enrichment strategy combined with chemical cross-linking/mass spectrometry (MS). Analysis of the subproteomes revealed the presence of distinct proteins in the cytosolic and Golgi fractions. The covalent fixation of transient or weak interactors by chemical cross-linking allowed capturing interaction partners that might otherwise disappear during conventional pull-down experiments. In total, 31 interaction partners were identified for PKD2, including glycogen synthase kinase-3 beta (GSK3B), 14-3-3 protein gamma (YWHAG), and the alpha isoform of 55 kDa regulatory subunit B of protein phosphatase 2A (PPP2R2A). Remarkably, the entire seven-subunit Arp2/3 complex (ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, ACTR3, ACTR2) as well as ARPC1A and ARPC5L, which are putative substitutes of ARPC1B and ARPC5, were identified. We provide evidence of a direct protein-protein interaction between PKD2 and Arp2/3. Our findings will pave the way for further structural and functional studies of PKD2 complexes, especially the PKD2/Arp2/3 interaction, to elucidate the role of PKD2 for transport processes at the trans-Golgi network. Data are available via ProteomeXchange with identifiers PXD003909 (enrichment from cytosolic fractions), PXD003913 (enrichment from Golgi fractions), and PXD003917 (subcellular fractionation).
authors: Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg , Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany.
authors: Björn
authors: B
authors: Häupl
authors: Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg , Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany.
authors: Christian H
authors: CH
authors: Ihling
authors: Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg , Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany.
authors: Andrea
authors: A
authors: Sinz
mesh_terms: D051377:Actin-Related Protein 2 / Q000378:metabolism*; D051378:Actin-Related Protein 3 / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000737:chemistry; D006056:Golgi Apparatus / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D060066:Protein Interaction Maps; D000091283:Protein Kinase D2; D011494:Protein Kinases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D000091283:Protein Kinase D2; D011494:Protein Kinases
keywords: Arp 2/3 complex; affinity enrichment; chemical cross-linking; interaction network; mass spectrometry; protein kinase D