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• W2000338731 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. 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. Death-associated protein kinase (DAPk), 1The abbreviations used are: DAPk, death-associated protein kinase; CaM, calmodulin; CaMKK, Ca2+/CaM-dependent protein kinase kinase; MLC, myosin II regulatory light chain; ZIPk, zipper-interacting protein kinase; shRNA, short hairpin RNA; WT, wild type; KESTREL, kinase substrate tracking and elucidation. a Ca2+/calmodulin (CaM)-activated Ser/Thr kinase that localizes to the cytoskeleton, has been linked to cell death and is a potent tumor suppressor (for a review, see Ref. 1Bialik S. Kimchi A. The death-associated protein kinases: structure, function and beyond.Annu. Rev. Biochem. 2006; 75: 189-210Crossref PubMed Scopus (356) Google Scholar). Originally identified in a screen for genes whose functions were necessary for interferon-γ-induced death (2Deiss L.P. Feinstein E. Berissi H. Cohen O. Kimchi A. Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the γ interferon-induced cell death.Genes Dev. 1995; 9: 15-30Crossref PubMed Scopus (538) Google Scholar), it has since been shown to be necessary for the regulation or execution of cell death in response to numerous stimuli, including death receptor activation (3Cohen O. Inbal B. Kissil J.L. Raveh T. Berissi H. Spivak-Kroizaman T. Feinstein E. Kimchi A. DAP-kinase participates in TNF-α- and Fas-induced apoptosis and its function requires the death domain.J. Cell Biol. 1999; 146: 141-148PubMed Scopus (0) Google Scholar), transforming growth factor-β (4Jang C.W. Chen C.H. Chen C.C. Chen J.Y. Su Y.H. Chen R.H. TGF-β induces apoptosis through Smad-mediated expression of DAP-kinase.Nat. Cell Biol. 2002; 4: 51-58Crossref PubMed Scopus (349) Google Scholar), oncogene expression (5Raveh T. Droguett G. Horwitz M.S. DePinho R.A. Kimchi A. DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation.Nat. Cell Biol. 2001; 3: 1-7Crossref PubMed Scopus (316) Google Scholar), UNC5H2 signaling (6Llambi F. Lourenco F.C. Gozuacik D. Guix C. Pays L. Del Rio G. Kimchi A. Mehlen P. The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase.EMBO J. 2005; 24: 1192-1201Crossref PubMed Scopus (133) Google Scholar), ceramide (7Yamamoto M. Hioki T. Ishii T. Nakajima-Iijima S. Uchino S. DAP kinase activity is critical for C2-ceramide-induced apoptosis in PC12 cells.Eur. J. Biochem. 2002; 269: 139-147Crossref PubMed Scopus (58) Google Scholar, 8Pelled D. Raveh T. Riebeling C. Fridkin M. Berissi H. Futerman A.H. Kimchi A. Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.J. Biol. Chem. 2002; 277: 1957-1961Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and matrix detachment (9Inbal B. Cohen O. Polak-Charcon S. Kopolovic J. Vadai E. Eisenbach L. Kimchi A. DAP kinase links the control of apoptosis to metastasis.Nature. 1997; 390: 180-184Crossref PubMed Scopus (370) Google Scholar). The specific cellular phenotype induced by DAPk activity can vary from one cell setting to another. It has been linked to both type I apoptotic and type II autophagic cell death in both caspase-dependent and caspase-independent manners (1Bialik S. Kimchi A. The death-associated protein kinases: structure, function and beyond.Annu. Rev. Biochem. 2006; 75: 189-210Crossref PubMed Scopus (356) Google Scholar). Specifically DAPk expression can lead to various actin-dependent death-associated morphologic changes, which include membrane blebbing and cell rounding (e.g. Refs. 10Cohen O. Feinstein E. Kimchi A. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity.EMBO J. 1997; 16: 998-1008Crossref PubMed Scopus (380) Google Scholar and 11Inbal B. Bialik S. Sabanay I. Shani G. Kimchi A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death.J. Cell Biol. 2002; 157: 455-468Crossref PubMed Scopus (422) Google Scholar). Detachment from the extracellular matrix often accompanies these phenotypes due to inhibition of integrin function (12Wang W.J. Kuo J.C. Yao C.C. Chen R.H. DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals.J. Cell Biol. 2002; 159: 169-179Crossref PubMed Scopus (141) Google Scholar). Even in the absence of cell death, the effects of DAPk on the cytoskeleton can lead to the induction of stress fiber formation (13Kuo J.C. Lin J.R. Staddon J.M. Hosoya H. Chen R.H. Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase.J. Cell Sci. 2003; 116: 4777-4790Crossref PubMed Scopus (70) Google Scholar) and interference with cell polarity and directed cell motility (14Kuo J. Wang W. Yao C. Wu P. Chen R. The tumor suppressor DAPK inhibits cell motility by blocking the integrin-mediated polarity pathway.J. Cell Biol. 2006; 172: 619-631Crossref PubMed Scopus (96) Google Scholar). DAPk has also been linked to p53-dependent apoptosis through the induction of p53 in a p19ARF-dependent manner (5Raveh T. Droguett G. Horwitz M.S. DePinho R.A. Kimchi A. DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation.Nat. Cell Biol. 2001; 3: 1-7Crossref PubMed Scopus (316) Google Scholar) and can lead to the up-regulation of autophagy (11Inbal B. Bialik S. Sabanay I. Shani G. Kimchi A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death.J. Cell Biol. 2002; 157: 455-468Crossref PubMed Scopus (422) Google Scholar). In addition to the chromatin fragmentation that accompanies caspase-dependent apoptosis (e.g. Refs. 4Jang C.W. Chen C.H. Chen C.C. Chen J.Y. Su Y.H. Chen R.H. TGF-β induces apoptosis through Smad-mediated expression of DAP-kinase.Nat. Cell Biol. 2002; 4: 51-58Crossref PubMed Scopus (349) Google Scholar, 5Raveh T. Droguett G. Horwitz M.S. DePinho R.A. Kimchi A. DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation.Nat. Cell Biol. 2001; 3: 1-7Crossref PubMed Scopus (316) Google Scholar, 6Llambi F. Lourenco F.C. Gozuacik D. Guix C. Pays L. Del Rio G. Kimchi A. Mehlen P. The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase.EMBO J. 2005; 24: 1192-1201Crossref PubMed Scopus (133) Google Scholar), DAPk expression can also lead to caspase-independent nuclear changes that include chromatin condensation (11Inbal B. Bialik S. Sabanay I. Shani G. Kimchi A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death.J. Cell Biol. 2002; 157: 455-468Crossref PubMed Scopus (422) Google Scholar). Each of these phenotypes can be considered an independent functional arm of DAPk. Catalytic activity is required for all DAPk-associated phenotypes, implying that phosphorylation of specific substrates mediates the various functional arms. To date, only a limited number of substrates have been identified. These include myosin II regulatory light chain (MLC) whose phosphorylation and subsequent activation of myosin-based contractility leads to membrane blebbing (10Cohen O. Feinstein E. Kimchi A. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity.EMBO J. 1997; 16: 998-1008Crossref PubMed Scopus (380) Google Scholar, 13Kuo J.C. Lin J.R. Staddon J.M. Hosoya H. Chen R.H. Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase.J. Cell Sci. 2003; 116: 4777-4790Crossref PubMed Scopus (70) Google Scholar, 15Bialik S. Bresnick A.R. Kimchi A. DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation.Cell Death Differ. 2004; 11: 631-644Crossref PubMed Scopus (79) Google Scholar, 16Jin Y. Blue E.K. Dixon S. Hou L. Wysolmerski R.B. Gallagher P.J. Identification of a new form of death-associated protein kinase that promotes cell survival.J. Biol. Chem. 2001; 276: 39667-39678Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). A second substrate identified is syntaxin-1A, a component of the v-SNARE (vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, which mediates docking and fusion of synaptic vesicles with the membrane (17Tian J.H. Das S. Sheng Z.H. Ca2+-dependent phosphorylation of syntaxin-1A by the death-associated protein (DAP) kinase regulates its interaction with Munc18.J. Biol. Chem. 2003; 278: 26265-26274Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Although kinetically syntaxin-1A is an efficient in vitro DAPk substrate, its physiologic relevance is not yet known. DAPk was also shown to phosphorylate ribosomal protein S6, thereby reducing translation rates in reticulocyte lysates (18Schumacher A. Velentza A. Watterson D. Dresios J. Death-associated protein kinase phosphorylates mammalian ribosomal protein S6 and reduces protein synthesis.Biochemistry. 2006; 45: 13614-13621Crossref PubMed Scopus (25) Google Scholar). DAPk may also participate in kinase signaling cascades as it has been shown to phosphorylate and regulate other kinases. For example, phosphorylation of the highly related zipper-interacting protein kinase (ZIPk) influences its intracellular localization and enhances its death-promoting activity (19Shani G. Marash L. Gozuacik D. Bialik S. Teitelbaum L. Shohat G. Kimchi A. Death-associated protein kinase phosphorylates ZIP kinase, forming a unique kinase hierarchy to activate its cell death functions.Mol. Cell. Biol. 2004; 24: 8611-8626Crossref PubMed Scopus (97) Google Scholar). DAPk can also efficiently phosphorylate Ca2+/CaM-dependent protein kinase kinase (CaMKK) in vitro and in vivo; this inhibits the ability of CaMKK to undergo CaM-activated autophosphorylation in vitro (20Schumacher A.M. Schavocky J.P. Velentza A.V. Mirzoeva S. Watterson D.M. A calmodulin-regulated protein kinase linked to neuron survival is a substrate for the calmodulin-regulated death-associated protein kinase.Biochemistry. 2004; 43: 8116-8124Crossref PubMed Scopus (34) Google Scholar). During oxidative stress, DAPk phosphorylates protein kinase D, leading to activation of c-Jun N-terminal kinase (JNK) and subsequently caspase-independent cell death (21Eisenberg-Lerner A. Kimchi A. DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress.Cell Death Differ. 2007; 14: 1908-1915Crossref PubMed Scopus (87) Google Scholar). Although identification of these substrates has shed light on some of the mechanisms of action of DAPk, there are still many gaps that remain in our understanding of how DAPk activity leads to the multiple functional outcomes discussed above. To fully fill in these gaps, a more thorough understanding of the complete substrate profile of DAPk needs to be attained. Here we undertook a large scale, unbiased proteomics-based screen whose aim was to identify DAPk substrates in vitro to be followed by in vivo confirmation. This was based on a recently described method for searching for kinase substrates, called KESTREL, which has been successfully used to identify substrates for several closely related kinases (22Knebel A. Morrice N. Cohen P. A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38δ.EMBO J. 2001; 20: 4360-4369Crossref PubMed Scopus (211) Google Scholar, 23Cohen P. Knebel A. KESTREL: a powerful method for identifying the physiological substrates of protein kinases.Biochem. J. 2006; 393: 1-6Crossref PubMed Scopus (66) Google Scholar). In this manner, we identified two novel DAPk substrates, ribosomal protein L5 and the MCM3 replication initiation factor. Plasmids encoding N-terminal FLAG- and hemagglutinin-tagged human full-length DAPk (pcDNA3-DAPk), the activated kinase deleted of its calmodulin regulatory domain (pcDNA3-DAPkc:workingBhatia202008-augasmbuploadj-elbm0001-0142CaM), or the catalytically inactive mutant (pcDNA3-DAPkK42A) have been described previously (10Cohen O. Feinstein E. Kimchi A. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity.EMBO J. 1997; 16: 998-1008Crossref PubMed Scopus (380) Google Scholar). A pASK-IBA3 vector encoding the catalytic domain of DAPk (amino acids 1–285) tagged at its C terminus with streptavidin was obtained from M. Watterson (Northwestern University, Chicago, IL) and used to produce recombinant DK1. pcDNA3 encoding the non-relevant protein luciferase was used as a control. FLAG-tagged L5 was generated by PCR cloning of the rat L5 cDNA (a kind gift from O. Meyuhas, Hebrew University, Jerusalem, Israel) into pcDNA3. FLAG-tagged human MCM3 in pcDNA3 was a kind gift from Dr. M. Gossen (Max Delbruck Center for Molecular Medicine, Berlin, Germany) (25Schories B. Engel K. Dorken B. Gossen M. Bommert K. Characterization of apoptosis-induced Mcm3 and Cdc6 cleavage reveals a proapoptotic effect for one Mcm3 fragment.Cell Death Differ. 2004; 11: 940-942Crossref PubMed Scopus (17) Google Scholar). Ser160 was mutated to either Ala or Asp by PCR-based site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene) according to the recommended protocol. pSuper-based vectors containing shRNA targeting DAPk (nucleotides 5776–5794, GenBank™ accession number NM_004938.2) or Hc-Red (nucleotides 99–117, GenBank accession number AF363776) were used for knockdown experiments. Chemicals and inhibitors were purchase from Sigma-Aldrich unless otherwise indicated. 293T human embryonic kidney cells and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin (Invitrogen). Cells were transiently transfected by the calcium phosphate precipitation method. When indicated, cells were treated with 5 μm ionomycin for 30 min. For experiments using shRNA, cells were transfected with pSuper-based vectors encoding DAPk shRNA or control Hc-Red shRNA for 5 days before treatment with ionomycin. For large scale purification, HeLa cells were grown until confluency, collected, lysed in lysis buffer (50 mm Tris, pH 7.6, 150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40 in the presence of 1% protease inhibitor mixture and 1 mm PMSF), and sonicated. Following dialysis against 25 mm Tris, pH 7.4, NH4SO4 was added to the lysate to achieve a final concentration of 25% salt. Proteins were allowed to precipitate on ice, and the pellet was collected by centrifugation. The resulting supernatant was then subjected to further salt precipitation repeatedly to obtain 40, 50, 60, and 80% NH4SO4 precipitants. All pellets were resuspended in 25 mm Tris, pH 7.4, and further dialyzed against 25 mm Tris, pH 7.4. 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. The remaining portion of each salt precipitant was applied to a HiTrap phenyl-HP hydrophobic column (GE Healthcare) after addition of NH4SO4 to a final concentration of 1 m. The column was washed with buffer, and then proteins were eluted over a NH4SO4 concentration gradient ranging from 1 to 0 m. Fractions collected were dialyzed against 25 mm Tris, pH 7.4, and concentrated on a YM-10 CentriPlus Spin column (Millipore) to ∼1/10 volume. A small portion (1–5%) of the total protein from selected fractions was assayed with DK1 for the presence of substrate. Fractions selected for the highest substrate content were resolved by SDS-PAGE in parallel with the products of the in vitro kinase assay. The half of the gel containing the kinase assay was silver-stained to detect the lower levels of protein present, whereas the remaining half of the gel was stained with GelCode (Pierce). The location of each substrate on the gel was denoted by comparison of the autoradiogram and silver-stained gels, and the equivalent portion of the gel was then excised from the GelCode-stained gel. The protein sampled was digested with trypsin, fractionated into individual peptides by liquid chromatography, and then analyzed by mass spectrometry (MS/MS) at the Biological Mass Spectrometry unit of the Weizmann Institute of Science using an API Q-Star Pulsari electrospray-quadruple TOF tandem mass spectrometer with a collision cell (Applied Biosystems/MDS Sciex) equipped with a nanoelectrospray source (MDS Proteomics, Odense, Denmark). Peak lists were generated from the raw data using Analyst QS1.1 and BioAnalyst 1.1.5 (MDS Sciex) and Mascot 2.1 (Matrix Science) software. Centroid and deisotoping parameters were used, and peaks less than 10% of the maximum were removed from analysis. A Mascot MS/MS ion search of the Swiss-Prot 51.0 human database (27,0778 entries, 99,412,397 residues) was performed with a peptide tolerance of ±0.8 Da and a MS/MS tolerance of ±0.8 Da. Up to two missed cleavages, four positive charges, and modifications by oxidation, deamidation, and carbamidomethylation were allowed. DK1 was expressed in TOP10 bacteria (Invitrogen) upon induction with tetracycline and affinity-purified from bacterial lysates using the StrepTactin column (Genosys Biotechnologies) according to the manufacturer’s instructions. 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. For experiments using DAPk K42A, which expresses much lower levels than the WT kinase, cells expressing either K42A or WT DAPk constructs were first treated with the actin-depolymerizing agent latrunculin B (20 μm) for 30 min to release all of the kinase from the actin cytoskeleton and then immediately lysed in B buffer. Extracts were immunoprecipitated with an anti-FLAG M2 monoclonal antibody conjugated to protein G beads, and proteins were eluted with excess FLAG peptide. MCM3-bound beads were first washed stringently in 0.5 m KCl and 0.5 m LiCl2 to remove co-precipitating kinases that resulted in high basal levels of phosphorylation. For immunoprecipitation of phosphorylated MCM3, cells were lysed in PLB (10 mm NaPO4, pH 7.5, 5 mm EDTA, 100 mm NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors (100 nm okadaic acid, 20 mm β-glycerophosphate, 1 mm NaF) and immunoprecipitated with monoclonal anti-MCM3 antibodies (Stressgen) prebound to protein G-agarose. Kinase assays of total cell lysate or fractionated lysates followed a modified protocol based on the KESTREL method described previously (22Knebel A. Morrice N. Cohen P. A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38δ.EMBO J. 2001; 20: 4360-4369Crossref PubMed Scopus (211) Google Scholar). In brief, samples were incubated with DK1 (0.5 mg) in DK kinase buffer (50 mm β-glycerophosphate, 20 mm MnCl2) in the presence of 57 nm (5 μCi) [γ-33P]ATP (Amersham Biosciences) and the following inhibitors for 2 min at 30 °C: 1 mm PMSF, 1% protease inhibitor mixture, 1 mm NaF, 5 mm EGTA, and 10 μm protease inhibitor cocktail (PI) (Sigma-Aldrich). Reactions were terminated by the addition of sample buffer and boiled prior to electrophoresis on SDS-polyacrylamide gels. Depending on the amount of protein present, gels were either silver-stained or stained with GelCode, dried, and exposed to MR x-ray film (Eastman Kodak Co.). For kinase assays using purified FLAG-tagged substrate, reactions were incubated for 10 min with DK1 as described above. For assays using full-length DAPk as the kinase, immunopurified FLAG-DAPk and FLAG-tagged substrate at kinase:substrate molar ratios of ∼1:5 or 1:10 were incubated for 10 min at 30 °C in kinase buffer (50 mm Hepes, pH 7.5, 20 mm MgCl2) supplemented with 0.16 μCi/μl [γ-33P]ATP, 50 μm ATP, 1 μm bovine calmodulin, and 0.5 mm CaCl2. For stoichiometric analysis, kinase assays were performed over a time course ranging from 1 min to 2 h. Reactions were terminated by boiling in SDS loading buffer and resolved on 7.5% acrylamide gels, which were stained with GelCode and dried. Levels of ATP incorporated were measured by phosphorimaging and comparison with a standard curve of known ATP concentrations. For determination of Km and Vmax values, a peptide derived from the MCM3 phosphorylation site was synthesized and used in P-81 Whatman filter assays. 20 ng of DK1 was incubated for 15 min at 30 °C with increasing concentrations of peptide (0.25–1000 μm) in reaction buffer (50 mm Hepes, pH 7.5, 20 mm MgCl2, 100 μm ATP, 4 μCi [γ-33P]ATP). Reactions were applied to P-81 filters and washed extensively in 1% phosphoric acid. Total counts were measured by scintillation counting. Total levels of ATP incorporated were calculated and plotted against substrate concentration. Double reciprocal plots were generated and analyzed by linear regression to obtain kinetic parameters. 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). To identify novel substrates of DAPk whose phosphorylation may mediate the various functional cellular effects of DAPk, an unbiased, high throughput screen was undertaken. An in vitro kinase assay was performed on HeLa cell lysate in the presence of a recombinant protein that consisted of the catalytic domain of DAPk (DK1). DK1 lacks the regulatory domains of DAPk and is constitutively active even in the absence of Ca2+/CaM. Furthermore it lacks the major autophosphorylation sites of the full-length kinase (24Shohat G. Spivak-Kroizman T. Cohen O. Bialik S. Shani G. Berrisi H. Eisenstein M. Kimchi A. The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism.J. Biol. Chem. 2001; 276: 47460-47467Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). To suppress the activity of endogenous kinases present in the lysates, specific kinase inhibitors were added to the reaction buffer (i.e. PKI to inhibit cAMP-dependent protein kinase and EGTA to inhibit Ca2+-dependent kinases). Furthermore the reactions were run under stringent conditions including short incubations times and the use of limiting quantities of Mn2+-ATP (γ-33P-labeled). Preliminary experiments confirmed that the replacement of the usual Mg2+ with Mn2+ in the reaction buffer had no effect on DK1 activity (data not shown). The addition of DK1 to the reaction mixture led to the phosphorylation of up to nine prominent substrates, which are referred to as S1–S9 (Fig. 1). The strongest phosphorylation was observed at a band of 20 kDa (S2), which corresponds to the molecular mass of MLC, a known in vitro and in vivo substrate of DAPk (13Kuo J.C. Lin J.R. Staddon J.M. Hosoya H. Chen R.H. Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase.J. Cell Sci. 2003; 116: 4777-4790Crossref PubMed Scopus (70) Google Scholar, 15Bialik S. Bresnick A.R. Kimchi A. DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation.Cell Death Differ. 2004; 11: 631-644Crossref PubMed Scopus (79) Google Scholar, 16Jin Y. Blue E.K. Dixon S. Hou L. Wysolmerski R.B. Gallagher P.J. Identification of a new form of death-associated protein kinase that promotes cell survival.J. Biol. Chem. 2001; 276: 39667-39678Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In fact, a protein at the same position as S2 was recognized by antibodies to MLC, suggesting that S2 is MLC (data not shown). The identification in this manner of a known DAPk substrate validates this strategy as an effective tool for identifying relevant substrates. To semipurify substrates to enable identification, HeLa cell lysate was subjected to consecutive fractionation steps. The first round consisted of stepwise precipitation with NH4SO4 (i.e. 25, 40, 50, 60, and 80% final concentration). The majority of proteins precipitated at the 50 and 60% NH4SO4 concentrations. Each salt fraction was then applied to a phenyl-HP hydrophobic column, and proteins were eluted with decreasing concentrations of NH4SO4. DK1 kinase assays were performed on 1–5% of the total protein in selected fractions. Resolution of the kinase reactions by electrophoresis followed by silver staining of the gel and autoradiography revealed the elution profiles of individual substrates. For example, the strongest S5 signal was observed to elute in fractions 19–22 upon fractionation of the 60% salt cut (Fig. 2A). Interestingly S5 was a weak and barely detectable substrate in the original total cell lysate yet gave rise to a strongly phosphorylated band after the final fractionation. This enhanced reactivity may be due to enrichment of the total amount of protein present or its isolation from additional cellular factors that inhibit its phosphorylation. The S9 substrate, which was most prominent in the 50% NH4SO4 fraction, eluted in fractions 32–46 upon application to the column with particular enrichment in fractions 36–42, corresponding to 0 mm salt (Fig. 2B). To identify the proteins corresponding to individual substrates, specific fractions were subjected to more precise resolution by SDS-PAGE. For example, for S5 identification, fraction 20 from the corresponding column was resolved on a 12% gel. For S9 identification, fractions 36 and 41, from either end of the range of elution, were each resolved on 6% gels. In each case, the band that ran at the position of the substrate was excised from each lane and analyzed by mass spectrometry. The complete results for S5 and S9 are presented in Tables I and II. In each case, several candidate proteins were present in the band of interest, migrating at the position of the DAPk substrate. The abundance of a particular protein in the sample (i.e. a high score in the MS results) does not necessarily correlate with its likelihood to be the true substrate; a strong phosphorylation signal may indicate a highly efficient substrate rather than high levels of protein present in the fraction. T" @default.
• W2000338731 created "2016-06-24" @default.
• W2000338731 creator A5050374760 @default.
• W2000338731 creator A5063517445 @default.
• W2000338731 creator A5073557575 @default.
• W2000338731 date "2008-06-01" @default.
• W2000338731 modified "2023-10-15" @default.
• W2000338731 title "A High Throughput Proteomics Screen Identifies Novel Substrates of Death-associated Protein Kinase" @default.
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