Figure 2

Creation of AS-ROCK2. A. ROCK2, V-Src, C-Src and rat PKA sequences were aligned using ClustalW. The v-Src ‘gatekeeper’ residue within the ATP-binding pocket is Ile³³⁸ (indicated by arrow) and is primarily responsible for restricting the ability of protein kinases from utilizing N6-substiuted ATP analogues. Ile³³⁸ in v-Src corresponds to Met¹⁶⁰ in ROCK2. B. HEK 293 cells were transfected with various FLAG-ROCK2 constructs where indicated. The following day, cells were lysed and ROCK2 was immunoprecipitated with anti-FLAG conjugated agarose. LIMK peptide was phosphorylated using N₆(Benzyl)ATP, loaded onto streptavidin plates (Pierce), and probed with P-LIMK (Cell Signaling) overnight at room temperature. Secondary decoration with IRDye® 680 anti-rabbit (Li-Cor) was performed at room temperature for 1.5 hours. LIMK peptide phosphorylation was visualized by direct fluorescence scanning with a Li-Cor Odyssey Imager. The relative LIMK phosphorylation signal is shown by the histogram for each ROCK2 mutation. Error bars represent standard deviation of triplicate determinations from three independent experiments. Asterisks indicate significant difference (P < 0.001) with W1161A ROCK2 as using Student’s t-test. C. Kinase activity of W1161A ROCK or W1161A/M160A ROCK2 was determined as in B, using serial dilutions of ATP with concentrations ranging from 1 mM to 12.8 nM. D. As in C, except N₆(Benzyl)ATP was used. For C and D, error bars represent the range of duplicate determinations. Results are representative of three independent experiments.