FIG. 8.

PKCβII phosphorylates and activates MEKK-1 in vitro. (A) Purified PKCβII was incubated with GST–MEKK-1 (lane 2) or GST (lane 3). As a control, PKCβII was omitted from the incubation with GST–MEKK-1 (lane 1). Material adsorbed to glutathione-agarose beads was analyzed by immunoblotting (IB) with anti-PKCβII antibody. (B) PKCβII was incubated with kinase-inactive GST–MEKK-1 (E. coli derived) or GST in the presence of [γ-³²P]ATP. As a control, GST–MEKK-1 was incubated with [γ-³²P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. (C) Kinase-active GST–MEKK-1 (yeast derived) bound to glutathione beads was incubated with alkaline phosphatase. After being washed, the beads were incubated in the absence or presence of purified PKCβII and ATP. The GST–MEKK-1-containing beads were washed again and then incubated with SEK1 (K-R) and [γ-³²P]ATP. Chelerythrine chloride (200 μM) was added to the incubation shown in lane 3. The reaction products were analyzed by SDS-PAGE and autoradiography.