FIG. 7.

Increasing concentrations of TRAF2ΔRING suppresses GCK activation of MEKK1 in vitro. GCK can also activate MLK3 but not MEKK3. (Top, left) Titration of increasing concentrations of purified (Fig. 3A) TRAF2ΔRING suppresses in vitro activation of MEKK1 by GCK. Immobilized MEKK1 was incubated with GCK in the presence or absence of the indicated amounts of TRAF2ΔRING. (Bottom, left) GCK also activates MLK3 but fails to activate MEKK3 in vitro. Immobilized FLAG-MLK3 or Myc-MEKK3 was incubated with the purified GCK or TRAF2 preparation (Fig. 3A) used in the titration experiment; however, samples were run on separate SDS-PAGE gels. The method was as described for Fig. 3 to 5. Activity was measured as phosphorylation of substrate protein [GST-SEK1(K129R)] and autophosphorylation (auto-P). At right is a quantitation of the results.