NUCLEAR MULTICATALYTIC PROTEINASE SUBUNIT RRC3 IS IMPORTANT FOR GROWTH-REGULATION IN HEPATOCYTES

Multicatalytic proteinases (MCPs) are macromolecular structures involv ed in the intracellular degradation of many types of proteins. MCPs ar e composed of a 205 ''core'' of both structural (alpha) and presumed c atalytic (beta) subunits, in association with regulatory proteins. The y are characteristically found in bath the nucleus and cytoplasm of ce lls, although mechanisms governing the subcellular distribution of MCP s are not known. RRC3, an alpha subunit of rat MCPs, contains bath a p utative nuclear localization signal (NLS) and a potential tyrosine pho sphorylation site which could play a role in nuclear import, and the n uclear form of RRC3 appears to be involved in the regulation of cell g rowth. Here we have generated a variety of RRC3 expression constructs to study features of RRC3 important in nuclear localization and cell g rowth, PCR was utilized to develop constructs containing point mutatio ns in either the putative NLS (K-51 mutated to A) or at a potential ty rosine phosphorylation site (Y-121 mutated to F), and an epitope from influenza hemagglutinin (HA) was added in triplicate to the C-terminus of the constructs as a means of identification. RRC3 constructs were then made in which the nucleotide sequence near the translation initia tion site of RRC3 was modified in such a way that the amino acid seque nce of the protein translated from die constructs is unchanged from th at of normal RRC3, thus allowing differential modulation of endogenous RRC3 with antisense oligonucleotide treatment. These N-terminally mod ified constructs art: designated mC3, mC3(NLS), and mC3(y). In vitro t ranscription/translation reactions with these constructs produced the expected products, which were immunoprecipitated with a mouse monoclon al anti-HA antibody. Immunohistochemical studies with hepatocyte cell lines transiently transfected with either mC3(NLS) or mC3(y) showed on ly cytoplasmic staining, whereas cells transfected with mC3 had a stai ning pattern typical of endogenous RRC3 (both cytoplasmic and nuclear) with strong staining of the nuclear perimeter, Immunoblot analyses of subcellular fractions from stably transfected CWSV1 cells showed mC3 product in both the cytosol and nucleus of cells, whereas mC3(NLS) or mC3(y) products were restricted to the cytosol. CWSV1 cells stably tra nsfected with the pTet-Splice vector containing no insert (as a contro l) were markedly inhibited (80C/c) in cell growth and showed altered m orphology when treated with antisense oligonucleotides targeted to end ogenous RRC3, reproducing previous studies. Similarly, CWSV1 cells sta bly transfected with either mC3(NLS) or mC3(y) constructs showed analo gous growth inhibition and morphologic alteration upon antisense treat ment. In contrast, CWSV1 cells stably transfected with the mC3 constru ct showed normal growth and morphology following antisense oligonucleo tide treatment, demonstrating that replenishment of nuclear RRC3 was n ecessary and sufficient to relieve growth inhibition. In P-32-metaboli c labeling studies, mC3 was tyrosine-phospharylated in cytosol as the full-length protein (M,36 000). mC3(NLS) was also phosphorylated in cy tosol, whereas mC3y was not. Nuclear mC3 showed phosphorylation of a M , 27 000 processed form while neither mC3(NLS) nor mC3y showed any pho sphorylated nuclear products. Our results show that nuclear RRC3 is im portant in control of cell growth and that both the NLS and Y-121 are important in nuclear localization of RRC3. Control of nuclear import b y tyrosine phosphorylation may represent a novel regulatory mechanism, and our results further suggest that RRC3 may travel as a maverick su bunit.