FIG. 5.

Interaction of JIP3 with MAPKKK. (A) Epitope-tagged MAPKKKs and GST-JIP3a were expressed in COS7 cells. Control experiments were performed with empty vector instead of the MAPKKK expression vectors. GST-JIP3a protein was isolated on glutathione-agarose beads. The binding of MAPKKK to JIP3 was examined by immunoblot analysis using an antibody that binds the epitope tag. (B) Epitope-tagged T7-JIP3a and HA-MLK3 were expressed in COS7 cells. Lysates were prepared, and the amount of JIP3a and MLK3 was examined by immunoblot analysis using monoclonal antibodies to the T7 and HA epitopes. HA-MLK3 was immunoprecipitated with the HA antibody, and T7-JIP3a in the immunoprecipitates was detected by immunoblot analysis with an antibody to the T7 epitope tag. (C) Interaction of purified recombinant MLK3 with JIP3a. Epitope-tagged HA-MLK3 was isolated by immunoprecipitation and was eluted by incubation with HA synthetic peptide (20 μg/ml) for 2 h at 4°C. The purified soluble MLK3 was incubated with GST or GST-JIP3a immobilized on glutathione-agarose. The amount of MLK3, GST, and GST-JIP3a was examined by immunoblot analysis using HA or GST antibodies. The agarose beads were washed, and bound MLK3 was detected by immunoblot analysis with an antibody to the HA epitope tag. (D) Deletion analysis of JIP3. To define the MLK3 binding region of JIP3, fragments of JIP3a (residues 1 to 1337, 1 to 815, 1 to 442, 420 to 815, and 800 to 1337) fused to GST were immobilized on glutathione-agarose. Control experiments were performed with immobilized GST. These immobilized proteins were incubated with MLK3 (top) or luciferase (bottom) prepared by in vitro translation in the presence of [³⁵S]methionine. Binding to the immobilized proteins was examined following SDS-PAGE by autoradiography.