NUCLEAR MULTICATALYTIC PROTEINASE ALPHA SUBUNIT RRC3 - DIFFERENTIAL SIZE, TYROSINE PHOSPHORYLATION, AND SUSCEPTIBILITY TO ANTISENSE OLIGONUCLEOTIDE TREATMENT

Multicatalytic proteinases (MCPs) are macromolecular structures involved in intracellular degradation of many types of proteins. MCPs are composed of a 20S ''core'' which consists of both structural (alpha) and presumed catalytic (beta) subunits in association with complexes of accessory proteins. Immunohistochemical studies have shown MCP subunits to be largely cytoplasmic, although nuclear localization is also observed. Reverse transcription/polymerase chain reaction amplifications were performed with redundant primers to conserved regions within known subunits, in an attempt both to identify potential new subunits and to define the repertoire of subunits expressed in hepatocytes, No new subunits were identified, and we found that RRC3, an alpha subunit of MCPs which contains a putative nuclear localization signal (NLS), was the predominant alpha subunit expressed in hepatocytes and hepatocyte-derived cell lines, Antibodies were developed against a unique C-terminal peptide region of RRC3. Immunohistochemical studies using affinity-purified antibodies showed that RRC3 has both cytoplasmic and nuclear localizations. Immunoprecipitation/immunoblot analyses showed that a significant proportion of nuclear RRC3 was associated with the nuclear scaffold (NS). NS RRC3 showed a significantly smaller M(r) (24 000) than the cytoplasmic form (M(r) 28 000), and only the nuclear form contained phosphotyrosine. In metabolic labeling experiments with [P-32]orthophosphate, the major nuclear and NS form observed showed an M(r) of 24 000, whereas no labeling of cytosolic RRC3 was observed. A minor P-32-labeled band of M(r) 28 000 was also observed in nuclei, and this M(r) 28 000 form was found in the soluble nuclear extract within MCP complexes. These results suggest that tyrosine phosphorylation of the cytosolic form (M(r) 28 000) rapidly triggers nuclear import, which is in turn quickly followed by conversion to the major M(r) 24 000 form associated with NS. Treatment with antisense oligonucleotides targeted to the initiation site of RRC3 reduced the growth of a hepatocyte-derived cell line by 95% and produced a marked morphological change (in the absence of overt toxicity). Under these treatment conditions, RRC3 mRNA was dramatically reduced. RRC3 protein was also dramatically reduced in the NS, but showed only a small reduction in cytosol, suggesting that the nuclear RRC3 may be important in cell growth and differentiation.