Figure 2.

The Gwl-ENSA Pathway Creates a Cdk Activity Threshold of Mitotic Phosphorylation in a Reconstituted System

(A) Simple Cdk:Cyc/PP2A:B55 antagonism. Red and blue represent mitotic and anti-mitotic effects, respectively.

(B) Time-course analysis of probe phosphorylation (Pho’n) shown as in (A), with increasing Cdk1:CycB levels ([PP2A:B55] = 50 nM). A sample without PP2A:B55 was used as 100% control (ctr.; open black squares).

(C) Cdk:Cyc/PP2A:B55 antagonism with the Gwl-ENSA pathway.

(D) Time-course analysis of probe phosphorylation shown as in (C) ([PP2A:B55] = 50 nM, [Gwl] = 20 nM, and [ENSA] = 300 nM). (E) Endpoint analyses of (B) (black) and (D) (red).

(F) Aliquots of (B) and (D) were analyzed for Gwl (top), ENSA-S67 phosphorylation (middle; upper arrowhead indicates S67-phosphorylated form of ENSA), and ENSA-T28 phosphorylation (bottom). Slower and faster migrating forms of Gwl are indicated by square brackets. Samples free from PP2A:B55 are shown in lanes 9 and 18 as fully phosphorylated controls. Representative result of five experiments is shown here.

(G and H) Simulation analyses of probe phosphorylation of (B) and (D).

(I) The steady states of phosphorylated probe in (G) and (H), which can be directly compared with the endpoint analysis panel in (E).

The T50-NCP probe was used in this figure. See also Figures S2 and S3 and Tables S1 and S2.