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Figure 5 | Cell Division

Figure 5

From: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation

Figure 5

Phosphorylation of residue S98 by CDK2 stabilizes Xic2. A. Degradation assay. 35S-labeled Xic2 was incubated in HSS with buffer control (DMSO), 1mM roscovitine (ROSC), GST (10uM), or GST-hp27 (10uM) with (+) or without (-) ΦX174 (ssDNA, 10ng/ul). The mean percentage of Xic2 remaining from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. B. Schematic representation of Xic2 and S/T-P consensus sites. C. Xic2 degradation assay. 35S-labeled Xic2 wildtype (WT) or CDK phosphorylation mutants (T58A, S98A, or S131A) were incubated in LSS with (+) or without (-) XSC (10ng/ul). The mean percentage of remaining Xic2 from three independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. D. Xic2 degradation assay. 35S-labeled Xic2 wildtype (WT) or triple-mutant (T58A, S98A, S131A) was incubated in LSS with (+) or without (-) XSC (10ng/ul) for 0 to 3 hrs as indicated. The mean percentage of remaining Xic2 from three independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. E. Xic2 degradation assay. Top panel: 35S-labeled Xic2 wildtype (WT) or glutamic acid phosphomimetic E mutants (S98E, S131E, or S98E/S131E) were incubated in HSS with (+) or without (-) 10 ng/ul ssDNA (ssDNA) for 0 to 3 hrs as indicated. Bottom panel: The percentage of Xic2 remaining was calculated for each sample where the zero hour time point was normalized to 100%. Error bars (Standard error of the mean) are shown. P values were calculated by student t-test comparing each sample with wildtype Xic2 (WT). 1.5 hr p values: S98E (0.115), S131E (0.310), and S98E/S131E (0.015). 3 hr p values: S98E (0.370), S131E (0.603), and S98E/S131E (0.172). For all figures, the ubiquitinated Xic2 protein bands are indicated as “XIC2-(UB)n”.

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