FIG. 1.

Activation of JNK2 by MKK7 mutants. HEK-293 cells were transiently transfected with 2.5 μg of plasmids encoding Myc-tagged forms of the indicated kinases (pCS3MT-MKK7, pCS3MT-MKK73E, pCS3MT-MKK73A, and pCS3MT-MKK7K149M) and with peVHA-JNK2. Empty pCS3MT vector was added to a total of 7.5 μg of DNA per transfection; 48 h later, cells were lysed in whole-cell lysis buffer. (A) A 100-μg aliquot of protein from each cell extract was separated by SDS-PAGE on a 10% gel, and expression of Myc epitope-tagged MKK7 proteins and HA-JNK2 was analyzed by Western blotting using anti-Myc and anti-HA antibodies, respectively. (B) GST-Jun kinase activity of cell extracts was assayed in vitro, using GST-Jun (amino acids 1 to 135) and [γ-³²P]-ATP as the substrate. GST-Jun was then purified from the reaction mixture and resolved by SDS-PAGE on a 10% gel, and phosphorylation was visualized by autoradiography. See Materials and Methods for details. MKK7wt, wild-type MKK7.