FIGURE 5.

Cyclin B-CDK1 participates in phosphorylation of Thr¹⁷⁶ of GATA2. A, evaluation of antibodies against phospho-Thr¹⁷⁶-GATA2 (p-T176-GATA2). WT-GATA2-myc-His, or T176A-GATA2-myc-His were transiently expressed in HEK293 cells. Cell lysates prepared with lysis buffer containing phosphatase inhibitors were subjected to immunoblotting with anti-GATA2 or anti-p-T176-GATA2. B, Thr¹⁷⁶ of endogenous GATA2 protein is phosphorylated in HeLa cells. Endogenous GATA2 protein was depleted by GATA2 siRNA, and lysates were subjected to immunoblotting with the indicated antibodies. C, putative consensus motif for phosphorylation by CDK1 and CDK2 in the CPD motif containing Thr¹⁷⁶ in GATA2. D–F, Thr¹⁷⁶ in GATA2 is efficiently phosphorylated by cyclin B1-CDK1 in vitro. Recombinant GST-WT-GATA2 (D and F) or GST-RB (E) was incubated with the indicated recombinant kinases in reaction buffer with 1 mm ATP for 30 min and then subjected to immunoblotting with the indicated antibodies. G, inhibition of Thr¹⁷⁶ phosphorylation of GATA2 by CDK inhibitors. HEK293 cells were transfected with WT-GATA2-myc-His and then treated with CDK inhibitor (1 μm RO-3306 or 1 μm butyrolactone I) for 5 h. Cell lysates were subjected to immunoblotting with anti-p-T176-GATA2 or anti-GATA2.