FIG. 1.

Agnoprotein is phosphorylated by PKC in vitro. (A) Deduced amino acid sequence of agnoprotein. Potential phosphorylation sites on agnoprotein for PKC (Ser7, Ser11, and Thr21) are depicted by arrows. (B) PKC phosphorylates agnoprotein in vitro. One microgram of bacterially produced GST alone or GST-agnoprotein (GST-Agno) fusion protein, immobilized on GST-Sepharose beads, was incubated with PKC as described in Materials and Methods. Phosphorylated proteins, which bound to GST-Sepharose beads, were extensively washed with reaction buffer, fractionated by SDS-12% PAGE, and visualized by autoradiography. (C) Analysis of bacterially produced GST and GST-agnoprotein by a SDS-12% PAGE, followed by Coomassie staining. Due to rapid degradation of the agnoprotein, it is difficult to produce it as a single-banded protein in bacteria (34).