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.
Highlights
Death-associated protein kinase (DAPk) is a Ser/Thr kinase whose activity is necessary for different cell death phenotypes
Death-associated protein kinase (DAPk),1 a Ca2ϩ/calmodulin (CaM)-activated Ser/Thr kinase that localizes to the cytoskeleton, has been linked to cell death and is a potent tumor suppressor
The strongest phosphorylation was observed at a band of 20 kDa (S2), which corresponds to the molecular mass of myosin II regulatory light chain (MLC), a known in vitro and in vivo substrate of DAPk [13, 15, 16]
Summary
Plasmids and Reagents—Plasmids encoding N-terminal FLAGand hemagglutinin-tagged human full-length DAPk (pcDNA3-DAPk), the activated kinase deleted of its calmodulin regulatory domain (pcDNA3-DAPk⌬CaM), or the catalytically inactive mutant (pcDNA3-DAPkK42A) have been described previously [10]. 1% of the dialyzed protein was subjected to an in vitro kinase assay with recombinant catalytic domain of DAPk (DK1) to monitor the presence of particular substrates. Full-length FLAG-DAPk or FLAG-tagged substrates were expressed in 293T cells, which were lysed in B buffer (20 mM HEPES, pH 7.6, 100 mM KCl, 0.5 mM EDTA, 0.4% Nonidet P-40, 20% glycerol) supplemented with protease inhibitors. Western Blot Analysis—Total cell lysates, protein immunoprecipitates, or kinase assays were resolved on 7.5 or 10% polyacrylamide gels, transferred to nitrocellulose membranes blots, and incubated with monoclonal antibodies to MCM3 (Stressgen), DAPk (clone 55, Sigma), actin (Sigma), or ZIPk (BD Transduction Laboratories) or with affinity-purified rabbit polyclonal antibodies to the MCM3 phosphopeptide KKTIERRYpS160DLT (where pS is phosphoserine) (generated by PhosphoSolutions, Aurora, Co). Secondary antibodies consisted of horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson ImmunoResearch Laboratories), which were detected by SuperSignal enhanced chemiluminescence (Pierce)
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