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

Multicatalytic proteinases (MCPs) are macromolecular structures involv ed in intracellular degradation of many types of proteins. MCPs are co mposed of a 20S ''core'' which consists of both structural (alpha) and presumed catalytic (beta) subunits in association with complexes of a ccessory proteins. Immunohistochemical studies have shown MCP subunits to be largely cytoplasmic, although nuclear localization is also obse rved. Reverse transcription/polymerase chain reaction amplifications w ere performed with redundant primers to conserved regions within known subunits, in an attempt both to identify potential new subunits and t o define the repertoire of subunits expressed in hepatocytes, No new s ubunits were identified, and we found that RRC3, an alpha subunit of M CPs which contains a putative nuclear localization signal (NLS), was t he predominant alpha subunit expressed in hepatocytes and hepatocyte-d erived cell lines, Antibodies were developed against a unique C-termin al 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 s ignificant proportion of nuclear RRC3 was associated with the nuclear scaffold (NS). NS RRC3 showed a significantly smaller M(r) (24 000) th an the cytoplasmic form (M(r) 28 000), and only the nuclear form conta ined phosphotyrosine. In metabolic labeling experiments with [P-32]ort hophosphate, 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 co mplexes. These results suggest that tyrosine phosphorylation of the cy tosolic form (M(r) 28 000) rapidly triggers nuclear import, which is i n turn quickly followed by conversion to the major M(r) 24 000 form as sociated with NS. Treatment with antisense oligonucleotides targeted t o the initiation site of RRC3 reduced the growth of a hepatocyte-deriv ed cell line by 95% and produced a marked morphological change (in the absence of overt toxicity). Under these treatment conditions, RRC3 mR NA was dramatically reduced. RRC3 protein was also dramatically reduce d in the NS, but showed only a small reduction in cytosol, suggesting that the nuclear RRC3 may be important in cell growth and differentiat ion.