FIG. 4.

PAK5 activates the JNK pathway. 293 cells were transfected with either empty vector or 5 μg of expression vector containing HA-tagged JNK (HA-JNK) either alone or with increasing doses of expression vectors containing Myc-tagged PAK5 (1, 3, and 5 μg) or expression vectors containing Rac2L61 (2.5 μg) or MEKK1Δ (2.5 μg). After transient expression, cells were lysed, and the amount of HA-JNK was normalized by Western blots probed with a mouse anti-HA antibody. Equal amounts of HA-JNK were then immunoprecipitated from whole-cell lysates by using a mouse anti-HA antibody and protein A-Sepharose. The immunoprecipitates were then incubated with recombinant GST–c-Jun in the presence of [γ-³²P]ATP in in vitro kinase buffer. Substrate phosphorylation was analyzed after SDS-PAGE and autoradiography. The phosphorylation of GST–c-Jun is indicated. The numbers indicate the extent (fold) of activation of JNK by PAK5, Rac2L61, and MEKK1Δ as quantitated by phosphorimager analysis.