FIG. 3.

In vitro activation of MEKK1 by purified GCK, GCK CTD, and TRAF2. (A) Purification of GST (lane 1), GST-GCK (lane 2), GST-GCK CTD (lane 3), GST-TRAF2ΔRING (lane 4), and GST-TRAF2 (lane 5). These preparations were used for the in vitro studies described herein (Fig. 3 to 5 and 7). Proteins were purified from transfected 293 cells as described previously (48), and 10% of each preparation was subjected to SDS-PAGE and Coomassie blue staining. (B) GCK stimulates MEKK1 autophosphorylation in vitro but does not itself significantly phosphorylate full-length MEKK1. HA-tagged wild type (wt) or kinase-dead (D1350N) MEKK1 was immunoprecipitated from transfected 293 cells and incubated with soluble GST-GCK and purified as described above, with [γ-³²P]ATP. Incubations in the absence of GCK were performed with GST. Phosphorylation of the MEKK1 polypeptides was analyzed after SDS-PAGE and autoradiography (top). Expression of the MEKK1 polypeptides was determined by immunoblotting with anti-HA (bottom). (C) GCK activates MEKK1 in vitro. The kinase domain of GCK is dispensable for MEKK1 activation. Wild-type or D1350N HA-MEKK1 was immunoprecipitated as for panel B and incubated with an equimolar amount of purified GST-GCK or the GCK CTD (see above) plus [γ-³²P]ATP. Incubations in the absence of GST-GCK or GST-GCK CTD were performed with GST. After a brief incubation, GST-SEK1(K129R) was added. The reaction was stopped with SDS-EDTA, and the samples were subjected to SDS-PAGE and autoradiography (top two panels, left) or immunoblotting with anti-HA. Kinase activity was assessed as MEKK1 autophosphorylation and phosphorylation of GST-SEK1(K129R). The bar graph on the right represent phosphorimager quantitation of the kinase assay results. Quantitation of MEKK1 autophosphorylation for samples not containing MEKK1 represents phosphorimager counts of mock anti-FLAG immunoprecipitations in the region of the gel corresponding to the migration position of MEKK1.