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. 2008 Mar 10;180(5):915–929. doi: 10.1083/jcb.200707126

Figure 1.

Figure 1.

Identification of SIRT2 as a Cdk substrate. (A) High-density protein arrays (two filters with 37,830 clones preselected for high protein expression by virtue of N-terminal 6xHIS tags [Bussow et al., 1998]) were incubated with recombinant human cyclin E–Cdk2 and γ-[32P]ATP, washed, and exposed to x-ray film. Potential substrates appear as double spots, as indicated by circles. A portion of one filter is displayed. (B) cyclin–Cdk complexes were incubated with or without their respective substrates (Cdk4 complexes with GST-pRb773-928 and Cdk2 and 1 complexes with histone H1) and γ-[32P]ATP. Proteins were resolved by 7–17% SDS-PAGE and visualized by autoradiography (32P, top) or Coomassie blue staining (CB, bottom). Kinase complexes are abbreviated (e.g., D1–K4 for cyclin D1–Cdk4). (C) Bacterially expressed GST-SIRT2 full-length fusion protein was phosphorylated with the indicated kinases as described in B. (D) Schematic comparison of Saccharomyces cerevisiae Sir2 with human SIRT1, 2, 6, and 7. The catalytic domains and the potential Cdk phosphorylation sites are indicated. (E) Bacterially expressed GST-SIRT2 fusion proteins, as indicated, were phosphorylated with 25 fcatal cyclin E–Cdk2. GST and histone H1 served as controls. Protein analysis was performed as in B. (F) The experiment was performed as in E with 3 fcatal of recombinant p35–Cdk5. For a control, 25 μM roscovitine was added to inhibit Cdk5 activity.