Figure 5.

The RM2 domain acts as a kinase substrate and SH2-binding site. (A) Left panel: purified RM2-6 (1 μM) was incubated with RTKB2 (250 nM) in kinase assay buffer containing 0.8 mM ATP at 30°C for the indicated times. Aliquots were removed, boiled with SDS-PAGE sample buffer, and analyzed by Western blotting with anti-pTyr antibody. Right panel: similar experiments were carried out with purified MbSrc1 kinase. (B) Purified RM2-6 was phosphorylated by MbSrc1 in the presence of [γ-³²P]-ATP. A sample of the reaction mixture is shown in the left panel. Right panel: the reaction mixture containing phosphorylated RM2 domain was incubated for 30 minutes with 25 μl of immobilized GST or a GST-MbSrc1 SH2 domain fusion protein [20] in binding buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, total volume = 200 μl). In one reaction, an SH2-binding synthetic peptide (Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Lys-Gln; labeled “pY” on the gel) was added at a concentration of 100 μM as a competitor. The glutathione-agarose beads were washed 4 times with 1 ml of binding buffer. Proteins were eluted by boiling with SDS-PAGE sample buffer and visualized by autoradiography.