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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.

We have previously shown that interleukin-1 alpha (IL-1 alpha) and interferon-gamma (IFN-gamma) induce surface IgM expression, stimulate Na+/H+ exchange, and activate protein kinase C in the murine pre-B lymphocyte cell line, 70Z/3. Because the two structurally different lymphokines induce similar effects, in this study we set out to compare the properties of the IL-1 and IFN-gamma surface receptors. In contrast to their similar cellular effects, we found that IL-1 alpha and IFN-gamma receptors have different properties. 70Z/3 have high (100 sites/cell) and low (900 sites/cell) affinity IL-1 receptors with dissociation constants (KD) 6 x 10(-11) and 10(-9) M, respectively. In contrast, IFN-gamma receptors are of one class with a KD of 3 x 10(-10) M and are at a higher number, 8000 sites/cell. After binding to their receptors both IL-1 alpha and IFN-gamma are internalized and intracellularly degraded, but the rate of internalization of IFN-gamma is greater than IL-1 alpha. The effective median concentrations (EC50) of IL-1 alpha- or IFN-gamma-induced surface IgM expression are similar (4-5 x 10(-12) M). However, at this concentration 10-fold more of IFN-gamma than IL-1 alpha molecules are bound per cell. Our studies indicate that structurally different lymphokines can induce similar biological events even though their signaling is mediated by surface receptors whose properties are different.