Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells.
A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.
Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
H
H
Tsao
J M
JM
Aletta
L A
LA
Greene
D000310:Adrenal Gland Neoplasms; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000494:pharmacology*; D010673:Pheochromocytoma; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D008869:Microtubule-Associated Proteins; D009414:Nerve Growth Factors; D005346:Fibroblast Growth Factors; D006493:Heparin; D011494:Protein Kinases