Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells.
In the renal medulla during antidiuresis, the extracellular fluid becomes hyperosmotic. Madin-Darby canine kidney (MDCK) epithelial cells adapt in hyperosmotic conditions and serve as a useful tissue culture model for cellular responses to hyperosmolality. We demonstrate that hyperosmolality stimulates phospholipase C, Raf-1 kinase mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase activities and that it increases phosphorylation of Raf-1 kinase, and p42 MAP kinase in MDCK cells. Stimulation of these kinases is osmolality-dependent (from 300 to 600 mosm/kg H2O). The time course of activation is sequential; the peak stimulation for Raf-1 kinase is at 5 min, at 10 min for MAP kinase kinase and MAP kinase, and at 20 min for S6 kinase. The activation of Raf-1 kinase and MAP kinase is inhibited by phorbol 12-myristate 13-acetate pretreatment in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein, herbimycin) do not significantly suppress hyperosmolality-induced MAP kinase activity. The increase of Ins-1,4,5-P3 levels by hyperosmolality suggests that activation of these kinases is mediated at least partially via activation of phospholipase C. Thus, hyperosmolality stimulates the serine/threonine kinases, Raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase, via predominantly protein kinase C-dependent, tyrosine kinase-independent pathways in MDCK cells.
Terada
Y
Y
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Tomita
K
K
Homma
M K
MK
Nonoguchi
H
H
Yang
T
T
Yamada
T
T
Yuasa
Y
Y
Krebs
E G
EG
Sasaki
S
S
Marumo
F
F
D000595:Amino Acid Sequence; D000818:Animals; D016227:Benzoquinones; D002478:Cells, Cultured; D004285:Dogs; D004789:Enzyme Activation; D019833:Genistein; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology; D007679:Kidney Medulla / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D047029:Lactams, Macrocyclic; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D009281:Naphthalenes; D009994:Osmolar Concentration; D010766:Phosphorylation; D011083:Polycyclic Compounds / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011809:Quinones / Q000494:pharmacology; D019893:Ribosomal Protein S6 Kinases; D017828:Rifabutin / Q000031:analogs & derivatives; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D016227:Benzoquinones; D007529:Isoflavones; D047029:Lactams, Macrocyclic; D009281:Naphthalenes; D011083:Polycyclic Compounds; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011809:Quinones; D017828:Rifabutin; C020754:herbimycin; D015544:Inositol 1,4,5-Trisphosphate; D019833:Genistein; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019893:Ribosomal Protein S6 Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate