Hypoxia and hypoxia/reoxygenation activate Raf-1, mitogen-activated protein kinase kinase, mitogen-activated protein kinases, and S6 kinase in 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 lead to further cell damage. We investigated the intracellular signaling cascades in cultured rat cardiac myocytes subjected to hypoxia followed by reoxygenation (hypoxia/reoxygenation). Here, we show that both hypoxia and hypoxia/reoxygenation caused rapid activation of the mitogen-activated protein kinase kinase kinase (MAPKKK), activity of Raf-1. This was followed by the sequential activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein (MAP) kinases, and S6 kinase (p90rsk). Furthermore, hypoxia caused hyperphosphorylation of Raf-1. The maximal hyperphosphorylation of Raf-1 appeared to be accompanied by a significant decrease in MAPKKK activity. These 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, Raf1 activates downstream serine/threonine kinases including MAPKK, MAP kinases and p90rsk. (2) Raf-1 is not only located upstream from MAPKK and MAP kinases but also may be phosphorylated by MAP kinases directly or indirectly, and at least Raf-1 kinase activity may be downregulated by this feedback mechanism.
Seko
Y
Y
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Tobe
K
K
Ueki
K
K
Kadowaki
T
T
Yazaki
Y
Y
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004789:Enzyme Activation; D006321:Heart / Q000503:physiopathology*; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D009206:Myocardium / Q000201:enzymology*; D010084:Oxidation-Reduction; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D017208:Rats, Wistar; D019893:Ribosomal Protein S6 Kinases
D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases