Figure 4.

ROCK and PAK activate LIMK but not TESK1. Lysates from COS-7 cells transfected with vector alone (mock) or plasmids for Myc-tagged TESK1, TESK1(D170A), or LIMK2 were immunoprecipitated with anti-Myc antibody and incubated with [γ-³²P]ATP and (His)₆-cofilin in the absence (−) or presence (+) of Myc-tagged ROCKΔ3 (A) or PAKΔN (B). Reaction mixtures were run on SDS-PAGE on 9 and 15% gels. In both A and B, the 15% gel was analyzed by autoradiography (top panel) and Amido-black staining for cofilin (second panel). The 9% gel was analyzed by autoradiography to detect phosphorylation of TESK1 and LIMK2 (third panel) and by immunoblotting with anti-Myc antibody (bottom panel). ³²P-incorporation into TESK1 is due to autophosphorylation. Relative kinase activities of TESK1 and LIMK2 treated with or without ROCKΔ3 or PAKΔN are indicated under the top panel, with the activity of wild-type TESK1 and LIMK2 without ROCKΔ3 or PAKΔN taken as 1.0, respectively.