Figure 3.

p38 MAPK is a substrate of STEP. (A) Rat brain homogenates were passed through STEP₄₆ C300S affinity column. Eluants were processed for immunoblot analysis using anti-p38 MAPK antibody (SM- starting material, E- eluant). (B) GST, GST-STEP₄₆ or GST-STEP₄₆ C300S fusion proteins were incubated with recombinant p38 MAPK protein. Bound proteins were extracted and processed for immunoblot analysis using anti-p38 MAPK (upper panel) antibody. Blots were re-probed with anti-GST (lower panel) antibody. (C) GST-STEP₄₆ or GST-STEP₄₆ phosphorylated with PKA were incubated with active p38α MAPK (phosphorylated at the regulatory threonine and tyrosine residues) for the specified times. Samples were analyzed using a phospho-specific antibody that recognizes p38 MAPK when it is phosphorylated at the regulatory tyrosine residue (TEY^P-p38). Blots were re-probed with anti-p38 MAPK antibody. (D-E) Hela cells were transfected with (D) V5-STEP₄₆ or one of its mutants (S49A and S49A/C300S); or (E) V5-STEP₄₆ S49A and Flag-tagged p38α, β, γ or δ. STEP was immunoprecipitated with anti-V5 antibody. Samples were analyzed either with anti-p38 (upper panel, D) or -anti Flag antibody (upper panel, E). Blots were re-probed with anti-V5-HRP antibody (lower panel, D-E). In E total lysate was analyzed using anti-Flag antibody to demonstrate expression of all p38 isoforms in Hela cells. (F-G) Untransfected Hela cells (F) and Hela cell transfected with V5-STEP₄₆ or V5-STEP₄₆-S49A (G) were treated with 0.3M sorbitol for 15 min and processed for immunocytochemical staining with anti-phospho p38 (T^PEY^P-p38; F, G) and anti-V5-HRP (G) antibodies.