Figure 2.

Recombinant SRK autophosphorylates on serine and threonine residues in a membranous environment. (A) Microsomes from uninfected Sf21 cells (C) or from Sf21 cells expressing SRK₃HA (HA), SRK₃His (His) or kinase defective ^mSRK₃His (^mHis) were radiolabeled with [γ-³²P]ATP. In some cases, proteins (lanes 4 and 5) were purified on Ni-NTA agarose beads after radiolabeling. Proteins were separated by SDS/PAGE and detected by autoradiography. (B) Radiolabeled SRK₃His was purified on Ni-NTA agarose beads and hydrolyzed. Free amino acids were separated in two dimensions by chromatography and electrophoresis as indicated, and radiolabeled amino acids were detected by autoradiography. Phosphoamino acids (P-ser, P-thr, and P-tyr for phosphoserine, phosphothreonine, and phosphotyrosine, respectively) were positioned by staining nonradiolabeled phosphoamino acids, added before separation, with nihydrin. Pi indicates inorganic phosphate.