FIG. 5.

Activation of MEKK1 by TRAF2 and GCK is not additive; GCK also activates MLK3. (A) TRAF2 activation of MEKK1 in vitro: requirement for the TRAF2 RING domain. HA-MEKK1 (wild-type) was immunoprecipitated from 293 cells as for Fig. 3 and incubated with equimolar concentrations of purified (Fig. 3A) GST-TRAF2, GST-GCK, or GST-TRAF2ΔRING as described for Fig. 3B. Controls were incubated with GST alone. GST-SEK1 was added as for Fig. 3B, and the incubation was continued. Samples were subjected to SDS-PAGE and either autoradiography or immunoblotting as for Fig. 3 and 4. The bar graph shows a phosphorimager quantitation of the kinase assays.