title: A high throughput proteomics screen identifies novel substrates of death-associated protein kinase.
abstract: Death-associated protein kinase (DAPk) is a Ser/Thr kinase whose activity is necessary for different cell death phenotypes. Although its contribution to cell death is well established, only a handful of direct substrates have been identified; these do not fully account for the multiple cellular effects of DAPk. To identify such substrates on a large scale, we developed an in vitro, unbiased, proteomics-based assay to search for novel DAPk substrates. Biochemical fractionation and mass spectrometric analysis were used to purify and identify several potential substrates from HeLa cell lysate. Here we report the identification of two such candidate substrates, the ribosomal protein L5 and MCM3, a replication licensing factor. Although L5 proved to be a weak substrate, MCM3 was efficiently and specifically phosphorylated by DAPk on a unique site, Ser160. Significantly DAPk phosphorylated this site in vivo upon overexpression in 293T cells. Activation of endogenous DAPk by increasing intracellular Ca2+ also led to increased phosphorylation of MCM3. Importantly short hairpin RNA-mediated knockdown of endogenous DAPk blocked both basal phosphorylation and Ca2+-induced phosphorylation, indicating that DAPk is both necessary and sufficient for MCM3 Ser160 phosphorylation in vivo. Identification of MCM3 as an in vivo DAPk substrate indicates the usefulness of this approach for identification of physiologically relevant substrates that may shed light on novel functions of the kinase.
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, 76100 Rehovot, Israel.
authors.forename: Shani
authors.initials: S
authors.lastname: Bialik
authors.forename: Hanna
authors.initials: H
authors.lastname: Berissi
authors.forename: Adi
authors.initials: A
authors.lastname: Kimchi
mesh_terms: D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D064550:Death-Associated Protein Kinases; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D051017:Apoptosis Regulatory Proteins; D012694:Serine; D012313:RNA; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases