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. 2014 Jan 2;10(1):e1004004. doi: 10.1371/journal.pgen.1004004

Figure 2. Gwl Thr194 is an essential Cdk1 phosphorylation site.

Figure 2

(A) Multiple amino-acid sequence alignment of Gwl sequence following the DFG motif. (B) IP/kinase assay of transiently expressed Flag-tagged WT and T193A/Thr194A mutant Gwl from nocodazole arrested HEK 293T cells using MBP as a substrate. (TCE total cell extract, IP immuno-precipitate) (C) Quantification of kinase assays as shown in (B). The average of 3 independent experiments was calculated and the error bars show the standard deviation between the different assays. (D) IP/Western of transiently expressed Flag WT and Thr194A mutant Gwl from asynchronous, RO3306, or nocodazole arrested HEK 293T cells. (E) Immunoblot of HeLa cell extracts following a double thymidine release sampled at indicated time points. Cell cycle progression was simultaneously monitored by PI staining and FACS analysis (see Fig. S2B). The cells passed through mitosis between 8 and 10 hours following release as indicated. (F) Gwl phosphorylation by CycA/Cdk2 in vitro. Flag WT and Thr194A Gwl was transiently expressed and purified from asynchronous HEK 293T cells and incubated with recombinant CycA/Cdk2, following treatment with alkaline phosphatase (aPh) in the indicated samples. The proteins were analysed by immuno-blotting with anti-Gwl and Gwl pThr194 antibodies. (G) Centrosome staining of Thr194 phosphorylated Gwl in prophase cells using co-localisation with γ-tubulin as a centrosomal marker. (H) Differential Thr194 phosphorylation of Gwl at spindle poles and centromeres in metaphase and anaphase cells. CenpA staining was used as a centromeric marker. Deconvolved maximum intensity projections are shown.