Phosphorylation Regulates SIRT1 Function
Figure 6
Threonine 530 and serine 540 of SIRT1 are substrates for cyclinB/Cdk1 in vitro.
A. Western blot analysis of nuclear (N) and cytoplasmic (C) fractions of cells transfected with FLAG-SIRT1 expression vectors encoding wild type (WT) and mutant SIRT1 proteins. Blots were subsequently reacted with antibodies against pericentrin and GAPDH to demonstrate the purity of the nuclear and cytoplasmic fractions, respectively. B. Western blot analysis of WT and mutant proteins in the presence and absence of the proteasome inhibitor MG132. C/D. In vitro kinase assays showing that threonine 530 and serine 540 are substrates for cyclinB/Cdk1 in vitro. The results are shown by autoradiograph (top panels) and Coomassie blue stained gel (bottom panels) in (C) and by the plot of ATP incorporation (as measured by scintillation countering) vs. time in (D).