Multicatalytic proteinases (MCPs) are macromolecular structures involved in the intracellular degradation of many types of proteins. MCPs are composed of a 20S 'core' of both structural (α) and presumed catalytic (β) subunits, in association with regulatory proteins. They are characteristically found in both the nucleus and cytoplasm of cells, although mechanisms governing the subcellular distribution of MCPs are not known. RRC3, an α subunit of rat MCPs, contains both a putative nuclear localization signal (NLS) and a potential tyrosine phosphorylation site which could play a role in nuclear import, and the nuclear form of RRC3 appears to be involved in the regulation of cell growth. Here we have generated a variety of RRC3 expression constructs to study features of RRC3 important in nuclear localization and cell growth. PCR was utilized to develop constructs containing point mutations in either the putative NLS (K51 mutated to A) or at a potential tyrosine phosphorylation site (Y1 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 initiation site of RRC3 was modified in such a way that the amino acid sequence of the protein translated from the constructs is unchanged from that of normal RRC3, thus allowing differential modulation of endogenous RRC3 with antisense oligonucleotide treatment. These N-terminally modified constructs are designated inC3, mC3(SLS) and mC3(Y). In vitro transcription/translation reactions with these constructs produced the expected products, which were immunoprecipitated with a mouse monoclonal anti-HA antibody. Immunohistochemical studies with hepatocyte cell lines transiently transfected with either mC3(SLS) or mC3(Y) showed only cytoplasmic staining, whereas cells transfected with mC3 had a staining 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 transfected with the pTet-Splice vector containing on insert (as a control) were markedly inhibited (80%) in cell growth and showed altered morphology when treated with antisense oligonucleotides targeted to endogenous RRC3, reproducing previous studies. Similarly, CWSV1 cells stably transfected with either mC3(NLS) or mC3(Y) constructs showed analogous growth inhibition and morphologic alteration upon antisense treatment. In contrast, CWSV1 cells stably transfected with the mC3 construct showed normal growth and morphology following antisense oligonucleotide treatment, demonstrating that replenishment of nuclear RRC3 was necessary and sufficient to relieve growth inhibition. In 32P-metabohc labeling studies, mC3 was tyrosine- phosphorylated in cytosol as the full-length protein (M, 36 000). mC3(NLS) was also phosphorylated in cytosol, whereas mC3(Y) was not. Nuclear mC3 showed phosphorylation of a M(r) 27 000 processed form while neither mC3(NLS) nor mC3(Y) showed any phosphorylated nuclear products. Our results show that nuclear RRC3 is important in control of cell growth and that both the NLS and Y121 are important in nuclear localization of RRC3. Control of nuclear import by tyrosine phosphorylation may represent a novel regulatory mechanism, and our results further suggest that RRC3 may travel as a maverick subunit.
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