FIG. 1.

Identification of two residues of ZEBRA phosphorylated by PKC α in vitro. (A) His-tagged wild-type ZEBRA and mutants Z(S186A), Z(T159A), and Z(T159A/S186A) were expressed in E. coli and purified on a nickel column; 250 ng of each protein was phosphorylated by PKC α using [γ-³²P]ATP. The phosphorylated proteins were separated by SDS-10% PAGE, transferred to a nitrocellulose membrane, and autoradiographed. (B) Coomassie blue stain of the gel. (C) The ³²P-labeled bands corresponding to ZEBRA were excised and digested with trypsin. Aliquots of each digest, containing 10,000 cpm, were applied to thin-layer cellulose plates and analyzed by two-dimensional thin-layer cellulose separation. a, wild type ZEBRA; b, Z(S186A); c, Z(T159A); d, mixture of the tryptic digests of Z(S186A) and Z(T159A).