Extended Data Fig. 10: Acetylation relieves the inhibitory interaction between the KIM and the catalytic kinase domain.
From: SIRT1 deacetylates WEE1 and sensitizes cancer cells to WEE1 inhibition
(a) WEE1 self-associates in an intermolecular fashion. HEK293T cells transfected with the indicated plasmids were irradiated with 40 J/m2 UV and allowed to recover for 4 hr. Cell lysates were then prepared and western blot analysis was carried out as indicated. (b) WEE1 exists primarily as a dimer in non-irradiated cells. U2OS cells were irradiated with 40 J/m2 UV and allowed to recover for the indicated time. Cell lysates were then prepared and western blot analysis was carried out as indicated. (c) Mutation of Asp405 to Ala on WEE1-CK impairs its interaction with WEE1-N. Bacterially purified GST-WEE1-N or GST-WEE1-N-K177Q was incubated with amylose resin conjugated with MBP-WEE1-CK, MBP-WEE1-CK-E402A, MBP-WEE1-CK-D405A, or MBP-WEE1-CK-D426A. Proteins retained on the amylose resin were immunoblotted with anti-GST antibody. (d) Mutation of Asp405 to Ala leads to constitutive activation of WEE1. Bacterially purified MBP-WEE1-WT, MBP-WEE1-K177Q, or MBP-WEE1-D405A was incubated with SFB-CDK1 in a kinase reaction mixture. After reaction, proteins were resolved by SDS-PAGE and subjected to immunoblotting. (e) Bacterially purified MBP-WEE1-N, MBP-WEE1-N-K177R, or MBP-WEE1-N-K177Q was mixed with SFB-WEE1 immunopurified from HEK293T cells that were left untreated or irradiated with 40 J/m2 UV and allowed to recover for 4 hr in a kinase reaction buffer. The mixture was then incubated with SFB-CDK1 in the presence of 0.3 mM ATP. After reaction, proteins were resolved by SDS-PAGE and subjected to immunoblotting. (f) Bacterially purified MBP-WEE1-N, MBP-WEE1-N-K177R, or MBP-WEE1-N-K177Q was mixed with MBP-WEE1-CK in a kinase reaction buffer. The mixture was then incubated with SFB-CDK1 in the presence of 0.3 mM ATP. After reaction, proteins were resolved by SDS-PAGE and subjected to immunoblotting. Data in A-F were independently replicated at least three times, with similar results.
