RTKB2 autophosphorylation. (A) RTKB2 kinase was first treated with GST-YOP phosphatase, then incubated for the indicated times in the presence of 0.5 mM ATP. Top panel: the autophosphorylation reaction contained [γ-32P]-ATP, and the reaction was analyzed by SDS-PAGE and autoradiography. Incorporation of 32P into RTKB2 kinase was also measured by scintillation counting, and the stoichiometry (mol phosphate/mol RTKB2) is presented below the gel. Bottom panel: unlabeled ATP was used in the reaction, which was analyzed by SDS-PAGE and Western blotting with anti-pTyr antibody. Also analyzed were samples of RTKB2 kinase before YOP treatment (“pre”) and after YOP treatment but before autophosphorylation (0 min.). (B) The activity of Sf9-purified RTKB2 towards the E4YM4 synthetic peptide was measured either directly (RTKB2), after treatment with YOP tyrosine phosphatase (dephos. RTKB2), or after treatment with YOP followed by an autophosphorylation reaction (30 min at 30°C; dephos. RTKB2 + MgATP). Activity was measured using the phosphocellulose paper binding assay.
Schultheiss et al. BMC Biochemistry 2013 14:4 doi:10.1186/1471-2091-14-4