FIG 7.

Thr-156 of GATA3 is phosphorylated by CDK2. (A) Transiently expressed GATA3 is phosphorylated at Thr-156 in HEK293 cells. HEK293 cells were transfected with WT or T156A GATA3 and treated with 20 μM MG132 and 20 nM okadaic acid for 5 h to inhibit proteolysis and dephosphorylation of GATA3. Cell lysates were prepared with lysis buffer containing phosphatase inhibitors and protease inhibitors and subjected to immunoblot analysis using phospho-T156-GATA3 or Myc antibodies. (B) CDK2 phosphorylates GATA3 peptide in a Thr-156-dependent manner in vitro. A synthetic peptide corresponding to aa 150 to 161 of WT or T156A GATA3 was incubated with [γ-³²P]ATP and the indicated kinases at 30°C for 30 min (top panel). S11 peptide (KAPLTPKKAK) is efficiently phosphorylated by various CDKs (22). To confirm the activities of the CDKs used in our experiments, we performed in vitro kinase assays using the S11 peptide as a positive control. Wild-type or T156A synthetic GATA3 peptide or S11 peptide was incubated with [γ-³²P]ATP along with the indicated CDKs at 30°C for 30 min (bottom panel). The peptides were trapped on P81 papers and monitored for radioactivity using a liquid scintillation counter. (C and D) Recombinant GATA3 is phosphorylated at Thr-156 by CDK2 in vitro. WT or T156A GST-fused GATA3 was expressed in E. coli and affinity purified using glutathione-Sepharose 4B. GST alone was prepared in parallel as a control. The proteins were incubated with the kinases as indicated in reaction buffer at 30°C for 30 min. Reaction products were subjected to immunoblot analysis with the indicated antibodies.