Nerve growth factor (NGF) responses by non-neuronal cells: detection by assay of a novel NGF-activated protein kinase.
Past work described the partial purification and characterization of a novel serine protein kinase activity designated protein kinase N (PKN) that is activated by nerve growth factor (NGF) in cultured PC12 cells [Rowland et al. (1987) J. Biol. Chem. 262; 7504-7513]. We have now devised a rapid, sensitive technique for partially purifying and assaying PKN activity in cell extracts. This methodology was applied to the IARC-EW-1 osteosarcoma and several additional non-neuronal cell lines that possess NGF receptors but that lack both morphological and a variety of additional biochemical responses to NGF. In each case, NGF significantly elevated PKN activity. The assay also revealed activation of PKN activity in IARC-EW-1 cells by additional agents, including epidermal growth factor, fibroblast growth factor, phorbol ester, and a cAMP analog. Also tested were an NGF-receptor-deficient PC12 cell variant and sublines thereof into which human NGF receptors had been introduced [Hempstead et al. (1989) Science 243; 373-375]. Acquisition of the NGF receptors resulted in NGF-activatable PKN activity. These findings indicate that detection of PKN activity may serve as a sensitive means to test NGF responsiveness in cells lacking macroscopic responses to the factor and that non-neuronal cells may be useful for studying primary signaling events in the NGF mechanism of action.
Volonté
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Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Greene
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LA
D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D006801:Humans; D009414:Nerve Growth Factors / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000502:physiology; D017475:Receptors, Nerve Growth Factor; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000201:enzymology
D009414:Nerve Growth Factors; D047428:Protein Kinase Inhibitors; D011687:Purines; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D011494:Protein Kinases; C061570:protein kinase N; D011493:Protein Kinase C