MEK1 is required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes.
We tested whether activation of mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase-1 (MEK1) is required and sufficient for extracellular signal-regulated kinase (ERK) activation in airway smooth muscle cells. First, we transiently cotransfected bovine tracheal myocytes with an epitope-tagged ERK2 and a dominant-negative or a constitutively active form of the gene encoding MEK1 and assessed ERK2 activation by in vitro phosphorylation assay. Expression of the dominant-negative MEK1 inhibited platelet-derived growth factor (PDGF)-induced ERK2 activation, whereas expression of the constitutively active MEK1 induced ERK2 activation, suggesting that MEK1 is required and sufficient for ERK activation in these cells. Next, we assessed the effect of PD-98059, a synthetic MEK inhibitor, on PDGF-induced MEK1 and ERK activation. PD-98059 (10 microM) inhibited MEK1 and ERK activation, confirming that MEK1 is required for ERK activation in bovine tracheal myocytes. PD-98059 had no effect on Src or Raf-1 activity, evidence that PD-98059 is a specific inhibitor of MEK in this system. Finally, PD-98059 reduced PDGF-induced [(3)H]thymidine incorporation in a concentration-dependent manner, suggesting that catalytic activation of MEK1 and ERKs is required for DNA synthesis. We conclude that MEK1 is required for PDGF-induced ERK activation in bovine tracheal myocytes and that MEK1 and ERKs are required for PDGF-induced DNA synthesis in these cells.
Karpova
A Y
AY
Department of Pediatrics, University of Chicago, Illinois 60637, USA.
Abe
M K
MK
Li
J
J
Liu
P T
PT
Rhee
J M
JM
Kuo
W L
WL
Hershenson
M B
MB
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009130:Muscle, Smooth / Q000166:cytology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D015398:Signal Transduction; D014132:Trachea / Q000166:cytology / Q000378:metabolism*; D014162:Transfection
D004791:Enzyme Inhibitors; D005419:Flavonoids; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one