FIG. 5.

Reduced phosphorylation of M1 and qM on T239. G₁/S-synchronized U2-OS cells were transfected with the indicated Wee1 proteins, synchronized, and used to prepare extracts from cells enriched in late-S and G₂ phases. (A) T239 phosphorylation of Wee1. Anti-P-T239 antibody was used for immunoblotting (IB) of endogenous Wee1 IPs, without or with phosphatase treatment. IB with anti-Wee1 antibody demonstrated a shift in mobility with phosphatase treatment and absence of proteolysis. (B) Reduced T239 phosphorylation in M1 and T239A. Cells expressing wild-type (wt) Wee1, T239A, or M1 were subjected to IB for Wee1 (Myc tag), P-T239, or tubulin (loading control). Note the lack of reactivity of T239A with the P-T239 antibody and reduced reactivity of M1. (C) Reduced T239 phosphorylation in M1 and qM. Extracts from cells expressing wild-type Wee1, M1, or qM were subjected to IB for P-T239. The blot was then stripped and reprobed for Wee1 (Myc tag). The ratios (means ± standard deviations) of P-T239 were as follows: M1 to the wild type, 0.48 ± 0.11 (n = 6), T239A to the wild type, 0.11 ± 0.02 (n = 7), qM to the wild type, 0.40 (range, n = 2). (D) Phosphorylation of S121/123 was unaffected in M1. Extracts from cells transfected with empty vector, the wild type, or M1 were subjected to IB with P-S121/123 or Myc antibody. (E) Cyclin A (CycA) knockdown reduces Wee1 T239 phosphorylation. Extracts of cells transfected with wild-type Wee1 and either scrambled (con) or cyclin A siRNA were subjected to IB for Wee1 (Myc tag), P-T239, cyclin A, or tubulin (loading control). The ratio (mean ± standard deviation) of P-T239 for cyclin A RNAi versus control was 0.52 ± 0.07 (n = 4). (F) M1 and T239A mediate greater Cdk phosphorylated at Y-15 (P-Y15) than the wild type in vivo. Synchronized U2-OS cells were transfected with empty vector (V) and wild-type, M1, T239A, and KD proteins, and cell lysates were prepared from late S/G₂-enriched populations. Cdk P-Y15 was assayed by IB. The mean (± standard deviation) ratios of Cdk P-Y15 observed following transfection were as follows: wild type to vector, 1.8 ± 0.2 (n = 4), M1 to wild type, 1.7 ± 0.2 (n = 7), T239A to wild type, 2.2 ± 0.2 (n = 6), KD to wild type, 0.93 ± 0.03 (range, n = 2). The numbers below the panels present quantitation of the main bands observed relative to the control. tub, tubulin.