Identification and characterization of a nerve growth factor-stimulated mitogen-activated protein kinase activator in PC12 cells.
Nerve growth factor treatment of PC12 cells results in the rapid activation of MAP kinases. These enzymes are activated through interaction with a protein "activator." The mitogen-activated protein (MAP) kinase activator has been partially purified by ion exchange and gel filtration chromatography. The activator has an apparent molecular mass of 50-60 kDa. The MAP kinase activator is rapidly generated in response to nerve growth factor (NGF) and can be detected within 30 s of exposure, reaching maximal levels within 2 min and then declining to near basal levels by 15-20 min. The activation of MAP kinase is dependent upon the time of incubation with the activator and on activator concentration. The MAP kinase activator is itself a protein kinase that phosphorylates MAP kinases and mediates their activation. The NGF-stimulated MAP kinase activator phosphorylates MAP kinase on serine, threonine, and tyrosine residues, establishing this enzyme as dual specific kinase. The MAP kinase activator is itself a phosphoprotein whose phosphorylation on tyrosine residues is stimulated upon NGF treatment of the cells. The enzyme activity of MAP kinase activator is abolished by treatment with both the tyrosine-specific phosphatase PTP-1 and the serine/threonine-specific phosphatase PP2A. The activator is produced in response to NGF, epidermal growth factor, and fibroblast growth factor. The protein kinase inhibitor K252a selectively inhibits the ability of NGF to generate MAP kinase activator activity. These data suggest that the upstream events governing MAP kinase activation involve the regulated phosphorylation of dual specificity MAP kinase activator as an immediate consequence of receptor activation.
Department of Neurology and Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
R K
RK
Jaiswal
M B
MB
Murphy
G E
GE
Landreth
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D007700:Kinetics; D020929:Mitogen-Activated Protein Kinase Kinases; D009414:Nerve Growth Factors / Q000502:physiology*; D016716:PC12 Cells; D010749:Phosphoprotein Phosphatases / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000494:pharmacology; D013379:Substrate Specificity
D009414:Nerve Growth Factors; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases