HYPOXIA AND HYPOXIA REOXYGENATION ACTIVATE RAF-1, MITOGEN-ACTIVATED PROTEIN-KINASE KINASE, MITOGEN-ACTIVATED PROTEIN-KINASES, AND S6 KINASEIN CULTURED RAT CARDIAC MYOCYTES/

In response to hypoxia and reoxygenation, mammalian cells are known to express a variety of genes to adapt to these external stresses or lea d to further cell damage. We investigated the intracellular signaling cascades in cultured rat cardiac myocytes subjected to hypoxia or hypo xia followed by reoxygenation (hypoxia/reoxygenation). Here, we show t hat both hypoxia and hypoxia/reoxygenation caused rapid activation of the mitogen-activated protein kinase kinase kinase (MAPKKK) activity o f Raf-1. This was followed by the sequential activation of mitogen-act ivated protein kinase kinase (MAPKK), mitogen-activated protein (MAP) kinases, and S6 kinase (p90(rsk)). Furthermore, hypoxia caused hyperph osphorylation of Raf-1. The maximal hyperphosphorylation of Raf-1 appe ared to be accompanied by a significant decrease in MAPKKK activity. T hese results strongly suggest the following: (1) Intracellular signals initiated by both hypoxia and hypoxia/reoxygenation converge on Raf-1 and activate its MAPKKK activity. Then, Raf-1 activates downstream se rine/threonine kinases including MAPKK, MAP kinases, and p90(rsk). (2) Raf-1 is not only located upstream from MAPKK and MAP kinases but als o may be phosphorylated by MAP kinases directly or indirectly, and at least Raf-1 kinase activity may be downregulated by this feedback mech anism.