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. 2005 Apr;25(7):2733–2743. doi: 10.1128/MCB.25.7.2733-2743.2005

FIG. 8.

FIG. 8.

JIP4 is a substrate for JNK and p38 MAP kinase but not ERK2. (A) JIP4 is a substrate for JNK and p38α MAPK but not ERK2. UV-stimulated JNK1 (upper panel) and p38α MAPK (middle panel), together with phorbol myristate acetate (PMA)-stimulated ERK2 (lower panel), were employed in immune complex kinase assays using [γ-32P]ATP and GST-JIP3 or GST-JIP4 as the substrates. Control experiments were performed using known substrates for JNK (c-Jun), p38α MAPK (ATF2), and ERK2 (Elk-1). The phosphorylation of the substrates was examined by SDS-PAGE and autoradiography. (B) Immune complex kinase assays were performed using wild-type JIP3 and a mutated JIP3 [JIP3(mu)] protein in which the three sites of phosphorylation by JNK were replaced with Ala (Thr-266, Thr-276, and Thr-287). These phosphorylation sites are conserved in JIP4 (Thr-52, Thr-61, Thr-96, and Thr-112). These Thr phosphorylation sites were replaced with Ala residues. Wild-type JIP4 and the mutant JIP4 protein [JIP4(mu)] were examined as substrates for JNK and p38α MAP kinase. Control experiments were performed using wild-type and mutant JIP3 proteins for JNK assays (upper panel) and ATF2 for p38α MAP kinase assays (lower panel).