IDENTIFICATION OF MITOGEN-ACTIVATED PROTEIN (MAP) KINASE-ACTIVATED PROTEIN KINASE-3, A NOVEL SUBSTRATE OF CSBP P38 MAP KINASE

CSBP p38 is a mitogen-activated protein kinase that is activated in re sponse to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the b ait in a yeast two-hybrid screen, we have identified and cloned a nove l kinase which shares similar to 70% amino acid identity to mitogen-ac tivated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP ki nase-3 was confirmed in vitro by the precipitation of epitope-tagged C SBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co expressed i n HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSB P2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeL a cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preinc ubation of cells with SB203580, a specific inhibitor of CSBP kinase ac tivity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in v ivo.