Interferon a induces protein kinase C - E ( PKC - E ) gene expression and a 4 . 7 - kb PKC - E - related transcript

Protein kinases play key roles in the induction by human interferon a (IFN-a) of specific gene expression and biological activity in various human cell lines. We now report that IFN-a increased the 7-kb transcript for the E isotype of protein kinase C (PKC-e) and the cellular content ofPKC-e 24 and 48 hr after IFN-a addition (a 2-fold and 6-fold increase, respectively). Furthermore, IFN-a markedly induced a 4.7-kb transcript that hybridized to a PKC-e-specific, but not to a PKC-i-specific, cDNA probe. The induction of the 4.7-kb PKC-e-related mRNA by IFN-a had the following properties reported for the classical IFN-a-stimulated genes: rapid kinetics of induction, high maintained levels in IFN-a-sensitive but not in IFN-a-resistant cell lines, protein synthesis-independent induction, and high sensitivity to inhibitors of protein tyrosine kinase activity. These results show that the regulation of gene expression by IFN-a include not only the classical IFN-astimulated genes but also the coordinated regulation of two PKC-e-related transcripts that appeared to be highly relevant to the biological actions of IFN-a. Interferon (IFN) is an important biologic response modifier that affects cell growth and differentiation, the immune response, and the ability of cells to support virus replication (1). IFN treatment of cells leads to distinctive alterations in gene expression. An early and physiologically significant event induced by IFNs is the rapid transcriptional activation of a series of genes called the IFN-a-stimulated genes (ISGs) (2). Pfeffer and coworkers (3-5) have shown that the signal generated by IFN-a binding to surface receptors involves the activation of protein kinases, including the e isotype of protein kinase C (PKC-e). PKC is a family of related serine/threonine kinases with pivotal roles in cell signaling and in a variety of cellular responses (6). Cloning studies have revealed at least 10 PKC isotypes belonging to three major groups: conventional PKCs (a, fI, ,311, and y), nonconventional PKCs (8, e, q, and 6), and atypical PKCs (rand A). The individual PKC isotypes exhibit distinct requirements for cofactors (Ca2+ and phospholipids) and activators (phorbol esters), tissue-specific distributions, and enzymological properties including substrate specificity (7-11). These differences suggest that individual PKC isotypes have unique functions in cell signaling. Sequence analysis of the PKC isotypes demonstrate that, although there are highly conserved regions, these proteins are distinguished by highly variable regions within their regulatory and protein kinase domains. We now report that IFN-a increased the cellular content of the 90-kDa PKC-e probably by inducing the expression of the 7-kb PKC-E mRNA. However, the predominant PKC-erelated transcript induced by IFN-a was a 4.7-kb mRNA. The regulation of this 4.7-kb mRNA had characteristics similar to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. those of the classical ISGs, ISG15 and ISG54, including rapid induction by low concentrations ofIFN-a, protein-synthesisindependent induction, and sensitivity to protein-tyrosine kinase inhibitors. Although, a 4.7-kb PKC-e-related transcript in rat lung has been identified as PKC-,q, we showed that the 4.7-kb IFN-a-inducible transcript was unrelated to PKC--q, as determined with a cDNA probe and antisera specific for PKC-7q. MATERIALS AND METHODS Materials. Recombinant IFN-a (1 x 109 units/mg of protein), designated IFNaConl, was provided by L. Blatt (AMGEN) for these studies. IFN-a activity was expressed in international reference units/ml as assayed by antiviral protection against vesicular stomatitis virus on human fibroblasts, using the National Institutes ofHealth IFN-a standard for reference. Cycloheximide and nucleotides were purchased from Sigma. Bio-Gel P-60 was purchased from BioRad. Genistein, NACS.37 resin, DNA polymerase I, DNase I, and restriction enzymes were purchased from GIBCO/ BRL. Oligo(dT)-cellulose was purchased from Pharmacia. Herbimycin was purchased from Kamiya Biomedical (Thousand Oaks, CA). Plasmids. The ISG15 plasmid contains a 400-nt genomic Taq I second exon fragment and the ISG54 plasmid contains a 250-nt EcoRI-Taq I second exon fragment (12). The y-actin plasmid contains the entire untranslated and coding regions up to aa 144 (13). The PKC-e-specific plasmid (provided by J. Knopf, Genetics Institute, Cambridge, MA) contains 1% bp ofPKC-e sequence from rat brain and encodes aa 346-406 (V2/V3 regulatory domain region) (14). The PKC-n-specific plasmid contains 900 bp of the V2/V3 regulatory domain of PKC-'q from rat lung (15). Cell Culture. Human Daudi lymphoblastoid cells, ML-2 monocytic cells, U937 histiocytic lymphoma cells, and K-562 erythroleukemia cells were grown in suspension cultures at 2-15 x 105 cells per ml in RPMI 1640 medium supplemented with 10% (vol/vol) defined calf serum (HyClone). HeLa S3 cells were grown in suspension culture at 1-10 x 105 cells per ml in Eagle's minimal essential medium modified for spinner culture and supplemented with 8% defined calf serum. For experiments, cells were suspended at 5-10 x 106 cells per ml in medium prior to the addition of IFN-a or other agents. Measurement ofRNA Levels. Total RNA was isolated from cells by the guanidinium thiocyanate/cesium chloride method and purified by phenol/chloroform extraction or by a single-step method (16, 17). In some experiments, poly(A)+ RNA was prepared from total RNA by affinity chromatography on oligo(dT)-cellulose columns (Pharmacia). Total RNA (40 Mg) or poly(A)+ RNA (10 ,ug) was denatured, electrophoresed, and transferred onto nitrocellulose (18). Abbreviations: IFN, interferon; PKC, protein kinase C; ISG, IFNstimulated gene.