Nerve growth factor-activated protein kinase N. Characterization and rapid near homogeneity purification by nucleotide affinity-exchange chromatography.
Protein kinase N (PKN) is a protein kinase rapidly activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma and additional cell types. PKN is selectively inhibited by purine analogs, and this property has served both as a diagnostic for PKN activity and to establish its apparent involvement in certain pathways of the NGF mechanism of action. The present work has focused on further characterization, identification, and purification of NGF-activated PKN. We show here that PKN can be substantially enriched by elution from ion exchange resins with ATP. We exploited this novel technique (nucleotide affinity exchange chromatography) to devise two alternative isolation schemes for PKN. One utilizes sequential chromatographic steps and provides a preparation that is apparently 60% homogeneous for PKN and represents a total enrichment of approximately 10,000-fold. The other is a single column procedure and includes prewashes with NAD. This method yields material that is about 5-10% homogeneous for PKN, requires about 1 h, and can be applied to multiple samples in parallel. The ATP elution technique furthermore distinguishes NGF-regulated from basal PKN activity and thereby suggests the presence of distinct PKN isoforms. The applications of sucrose gradient centrifugation, gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/silver staining, affinity labeling with 8-azido-ATP/SDS-PAGE, and autophosphorylation (after SDS-PAGE, blotting and renaturation) all indicate that PKN has an apparent molecular mass of 45-47 kDa and is mainly monomeric in solution. These and additional properties appear to distinguish PKN from many previously described protein kinases.
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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D000255:Adenosine Triphosphate / Q000737:chemistry; D000345:Affinity Labels; D000818:Animals; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D009243:NAD / Q000737:chemistry; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016716:PC12 Cells; D011493:Protein Kinase C; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D016622:Silver Staining
D000345:Affinity Labels; D009414:Nerve Growth Factors; D009243:NAD; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C061570:protein kinase N; D011493:Protein Kinase C