Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2.
Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.
Miyasaka
T
T
Laboratory of Molecular Oncology, Rockefeller University, New York, New York.
Chao
M V
MV
Sherline
P
P
Saltiel
A R
AR
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004789:Enzyme Activation / Q000187:drug effects; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000494:pharmacology*; D010673:Pheochromocytoma; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012969:Sodium Fluoride / Q000494:pharmacology; D019311:Staurosporine; D013552:Swine; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D008869:Microtubule-Associated Proteins; D009414:Nerve Growth Factors; D047428:Protein Kinase Inhibitors; D012969:Sodium Fluoride; D006493:Heparin; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D019311:Staurosporine; D002118:Calcium