Characterization of a nerve growth factor-stimulated protein kinase in PC12 cells which phosphorylates microtubule-associated protein 2 and pp250.
Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.
Landreth
G E
GE
Department of Neurology, Medical University of South Carolina, Charleston.
Smith
D S
DS
McCabe
C
C
Gittinger
C
C
D000310:Adrenal Gland Neoplasms / Q000201:enzymology*; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000494:pharmacology*; D010673:Pheochromocytoma / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D003598:Cytoskeletal Proteins; D008869:Microtubule-Associated Proteins; D009414:Nerve Growth Factors; D008345:Manganese; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases