P21-activated Kinase 1 (Pak1) phosphorylates Myo5 on S357. Crude preparations of wild type and S357A Myo5 motor/lever constructs were mixed with 250 µM ATP including 20 µCi of ATPγP32 in kinase assay buffer (5 mM MOPS pH 7, 2.5 mM β-glycerophosphate, 5 mM MgCl2, 400 µM EDTA, 1 mM EGTA, and 50 µM DTT) in either the presence or absence of Pak1. Reactions were incubated at 25°C for 60 min, then quenched by adding an equal volume of 2× Tris urea sample buffer (125 mM Tris pH 6.8, 6 M urea, 2% SDS, 0.1% bromophenol blue, 10% β-mercaptoethanol) and resolved on a 10% polyacrylamide gel. The gel was stained with Coomassie, then dried onto Whatman paper and exposed to a storage phosphor screen (Amersham). The Coomassie-stained gel was imaged on a standard photo scanner and the phosphor screen on a Typhoon gel imager (Amersham). Note that there are differences in baseline labeling in the absence of added kinase between the two different protein preps, but the addition of Pak1 clearly results in radiolabeling of wild type but not mutant Myo5.
