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A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1.

ABSTRACT: The cell-cycle checkpoint kinase Chk1 is essential in mammalian cells due to its roles in controlling processes such as DNA replication, mitosis and DNA-damage responses. Despite its paramount importance, how Chk1 controls these functions remains unclear, mainly because very few Chk1 substrates have hitherto been identified.Here, we combine a chemical genetics approach with high-resolution mass spectrometry to identify novel Chk1 substrates and their phosphorylation sites. The list of targets produced reveals the potential impact of Chk1 function not only on processes where Chk1 was already known to be involved, but also on other key cellular events such as transcription, RNA splicing and cell fate determination. In addition, we validate and explore the phosphorylation of transcriptional co-repressor KAP1 Ser473 as a novel DNA-damage-induced Chk1 site.By providing a substantial set of potential Chk1 substrates, we present opportunities for studying unanticipated functions for Chk1 in controlling a wide range of cellular processes. We also refine the Chk1 consensus sequence, facilitating the future prediction of Chk1 target sites. In addition, our identification of KAP1 Ser473 phosphorylation as a robust readout for Chk1 activity could be used to explore the in vivo effects of Chk1 inhibitors that are being developed for clinical evaluation.

SUBMITTER: Blasius M

PROVIDER: S-EPMC3245618 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1.

Blasius Melanie M Forment Josep V JV Thakkar Neha N Wagner Sebastian A SA Choudhary Chunaram C Jackson Stephen P SP

Genome biology 20110818 8

<h4>Background</h4>The cell-cycle checkpoint kinase Chk1 is essential in mammalian cells due to its roles in controlling processes such as DNA replication, mitosis and DNA-damage responses. Despite its paramount importance, how Chk1 controls these functions remains unclear, mainly because very few Chk1 substrates have hitherto been identified.<h4>Results</h4>Here, we combine a chemical genetics approach with high-resolution mass spectrometry to identify novel Chk1 substrates and their phosphoryl ...[more]

PMID: 21851590

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Project description:UnlabelledThe Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death.ImportanceKSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

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A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1.

Publications

A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1.

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