Identification of an inducible 85-kDa nuclear protein kinase

Abstract

To identify inducible protein kinases localized exclusively in the nucleus, nuclear and cytosolic extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to an Immobilon-P membranes were incubated in phosphorylation buffer containing [gamma-32P]ATP. Autoradiographs of the membranes revealed an 85-kDa 32P-labeled band; the intensity of this band was transiently increased in nuclear but not in cytosolic extracts from interleukin-1 alpha-treated cells. Incorporation of 32P label into a blotted protein band suggested the presence of an interleukin-1 alpha-responsive 85-kDa nuclear protein kinase. Fractionation of nuclear extracts by Mono Q failed to separate the kinase activity from the substrate, indicating that the 85-kDa band identified on the Immobilon-P membrane represents a protein kinase that undergoes autophosphorylation. Phosphoamino acid analysis of the 85-kDa band showed that this enzyme is a serine/threonine kinase. Purified pp90RSK could not be identified by the denaturation-renaturation method, indicating that the 85-kDa kinase identified here is not pp90RSK. This observation, nuclear but not cytoplasmic localization, and the fact that antibodies to known protein kinases kinase failed to recognize it suggest that the enzyme identified here is a novel protein kinase.