FIG. 3.

In vitro phosphorylation of SHPS-1 by c-Src. (A) Src phosphorylates SHPS-1 in vitro. Purified GST–cytoSHPS-1 bound to glutathione-agarose beads was phosphorylated by purified recombinant Src in the absence (no ATP, lane 3) or presence of [³²P]ATP for the indicated time periods at 37°C. After incubation, GST–cytoSHPS-1 and bound proteins were recovered by centrifugation. The lane labeled Src alone contains the same amount of Src used in the other lanes in the absence of any exogenous substrate. Coomassie staining and autoradiographs are shown. Note that the entire mass of the GST–SHPS-1 fusion protein shifts to at least two more-slowly migrating forms, implying stoichiometric phosphorylation, and that about 5 to 10% of input Src becomes bound to SHPS-1 in a phosphorylation-dependent manner. (B) SHPS-1 is a better substrate than enolase or FAK. Comparison of Src-catalyzed incorporation of ³²P into GST–cytoSHPS-1, enolase, and GST-FAK-CT (GST-FAK C-term). Saturating amounts of each substrate (3 μg) were phosphorylated with 0.05 μg of purified recombinant Src for 5 min at 30°C in an assay buffer containing 30 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl₂, 10 mM MnCl₂, 7.5 μM unlabeled ATP, and 10 μCi of [³²P]ATP. The kinetics of incorporation were linear over this time period (data not shown). Reactions were terminated with 5× sample buffer, and the proteins were separated by SDS-PAGE. Coomassie staining and autoradiographs are shown. Arrows indicate GST–cyto-SHPS-1. The numbers shown represent incorporation (in counts per minute per microgram of protein) after the bands were cut out and subjected to Cherenkov counting. No incorporation into GST alone was observed (data not shown). (C) Two-dimensional phosphotryptic analysis of the in vitro phosphorylation reaction. Note the stoichiometric phosphorylation on two sites (inferred from mobility shift data; see above) and the high-level substoichiometric phosphorylation on two others. TLC, thin-layer chromatography; TLE, thin-layer electrophoresis.