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Characterization of a Nerve Growth Factor‐Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule‐Associated Protein 2 and pp250
Author(s) -
Landreth Gary E.,
Smith Deanna S.,
McCabe Craig,
Gittinger Cynthia
Publication year - 1990
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1990.tb04165.x
Subject(s) - protein kinase a , nerve growth factor , map2k7 , microbiology and biotechnology , biochemistry , phosphorylation , kinase , c raf , protein kinase c , phosphatase , casein kinase 2 , mitogen activated protein kinase kinase , casein kinase 2, alpha 1 , cyclin dependent kinase 2 , biology , chemistry , receptor
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 M r of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pl of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in V max for MAP2. The K m with respect to the MAP2 substrate was approximately 50 n M and was not altered by NGF treatment. A novel feature of the NGF‐stimulated enzyme was its sharp dependence on Mn 2+ 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.