Ime2 phosphates are completely resistant to Cdc14 activity
(A) A model substrate (Fin1) was phosphorylated by Cdk1 or Ime2 and the rate of phosphate release upon incubation with Cdc14, PP2A-Cdc55 or PP2C was determined by TCA precipitation and scintillation counting of soluble phosphate.
(B) APCCdh1 can be reactivated by Cdc14 after phosphorylation by Cdk1 but not Ime2. A reconstituted APC assay was performed using Cdh1 that was first phosphorylated by either Cdk1 or Ime2 and then treated with increasing amounts of Cdc14 (SDS-PAGE and autoradiography of 35S-securin on the left; quantitation on the right. Data are represented as mean +/- SD).
(C) APCCdh1 is controlled by a complex variety of kinases and phosphatases. The ability of different phosphatases to reactivate Cdh1 following Ime2 or Cdk1 phosphorylation was assessed as in panel B.