Journal: Cell Death & Disease

Article Title: DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

doi: 10.1038/cddis.2014.231

Figure Lengend Snippet: CDK1 phosphorylation is maintained in CHK1-depleted HCT116 cells during DNA replication stress. Western blot analysis of indicated S-phase regulatory proteins from extracts of HCT116 or HCT116 p53−/− cells pretreated with CHK1 or control siRNAs and exposed or not exposed to thymidine (2 mM, 24 h). β -Actin levels are presented as loading controls

Article Snippet: Proteins were detected using the enhanced chemiluminiscence detection system (ECL, Amersham) according to the manufacturer's recommendations using anti-CHK1 (#2345), pAurora: Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (#2914), p21 (#2947), pSer345 CHK1 (#2348), CHK1 (#2360), pThr68 CHK2 (#2661), CHK2 (#2662), pTyr15 CDK1 (#9111) from Cell Signaling, Hitchin, UK; anti-CDC45 (sc-20685), CDK1 (sc-54), Cyclin B1 (sc-245), pSer10 H3 (sc-8656), p53 (sc-126) from Santa Cruz Biotechnology (Wembley, UK) and anti-RPA34 (NA19L) from Calbiochem (Feltham, UK); or anti- β -actin (A-5060) from Sigma-Aldrich (Poole, UK).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Cell Death & Disease

Article Title: DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

doi: 10.1038/cddis.2014.231

Figure Lengend Snippet: Model for control of premature mitosis during DNA replication stress. Mitosis is triggered when activated CDK1 binds to its regulatory partner cyclinB. During DNA replication stress, activation of ATR elicits phosphorylation of CHK1 that, in turn, phosphorylates CDC25A, targeting it for ubiquitin-mediated degradation. In the absence of CDC25A, pTyr15 CDK1 is not dephosphorylated to active CDK1 thus preventing the onset of mitosis. In CHK1-depleted HCT116 cells, CDK1 phosphorylation at Tyr15 is unexpectedly sustained, which should suppress the initiation of mitosis. However, our work indicates that activated CHK1 also suppresses phosphorylation of Aurora B to prevent premature entry into mitosis in cells arrested in S-phase (left). When CHK1 is depleted, Aurora B autophosphorylation is no longer suppressed leading to its activation, histone H3 phosphorylation and premature chromosome condensation (right)

Article Snippet: Proteins were detected using the enhanced chemiluminiscence detection system (ECL, Amersham) according to the manufacturer's recommendations using anti-CHK1 (#2345), pAurora: Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (#2914), p21 (#2947), pSer345 CHK1 (#2348), CHK1 (#2360), pThr68 CHK2 (#2661), CHK2 (#2662), pTyr15 CDK1 (#9111) from Cell Signaling, Hitchin, UK; anti-CDC45 (sc-20685), CDK1 (sc-54), Cyclin B1 (sc-245), pSer10 H3 (sc-8656), p53 (sc-126) from Santa Cruz Biotechnology (Wembley, UK) and anti-RPA34 (NA19L) from Calbiochem (Feltham, UK); or anti- β -actin (A-5060) from Sigma-Aldrich (Poole, UK).

Techniques: Control, Activation Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal:

Article Title: Phosphorylation-Independent Inhibition of Cdc28p by the Tyrosine Kinase Swe1p in the Morphogenesis Checkpoint

doi:

Figure Lengend Snippet: A CDC28E12K mutant has a normal growth profile but is resistant to SWE1 overexpression. (A) Sequence homology of the amino terminus of S. cerevisiae (Sc) Cdc28p with the other Cdks in S. cerevisiae and with S. pombe and human (Hs) Cdks. The conserved tyrosine (Y) residue phosphorylated by Wee1 family kinases and the glutamic acid (E) residue mutated to lysine (K) in the CDC28 mutants isolated from the zds1Δ zds2Δ suppression screen are shaded. S. cerevisiae Kin28p, Ctk1p, and Ssn3p and human Cdk4 do not have a glutamic acid at this position; however, the human Cdk4 does have an adjacent glutamic acid residue that is boxed but not shaded. Ctk1p and Ssn3p have large amino-terminal regions, and the sequence shown begins at residue 179 for Ctk1p and 71 for Ssn3p. (B) Growth rates of exponentially proliferating wild-type and CDC28E12K strains. (C) Flow cytometric analysis of the DNA content of exponentially proliferating wild-type (WT) and CDC28E12K cells. (D) Percentage of budded and anaphase cells, calculated from microscopic examination of the same cells stained for flow cytometric analysis in panel C. (E) Cell morphology of exponentially growing wild-type and CDC28E12K cells. Bar = 5 μm. (F) Cell morphology of wild-type, GAL:SWE1, and GAL:SWE1 CDC28E12K strains 4 h after addition of galactose to cells growing in YEPS medium. Bar = 5 μm. The strains used were DLY1 (wild type), JMY1380 (CDC28E12K), DLY2626 (GAL:SWE1), and JMY1386 (CDC28E12K GAL:SWE1). (G) Tyrosine phosphorylation of Cdc28p alleles upon overexpression of Swe1p. GAL:SWE1 strains containing wild-type (DLY2626), Y19F (JMY1390), and E12K (JMY1386) alleles of Cdc28p were induced to overexpress Swe1p by growth for 4 h after addition of galactose to cells growing in YEPS medium. p18Cks1 beads were then used to precipitate the different Cdc28p proteins, which were immunoblotted with anti-phospho-Cdc2 antibody to detect tyrosine-phosphorylated Cdc28p and anti-PSATIRE antibodies to detect total Cdc28p. All cultures were grown at 30°C and, unless otherwise indicated, were grown in YEPD.

Article Snippet: Following electrophoresis, proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.) and stained with anti-phospho-Cdc2 antibody Tyr 15 (used at 1:1,000 dilution according to the manufacturer’s recommendations).

Techniques: Mutagenesis, Over Expression, Sequencing, Isolation, Staining