Nuclear Multicatalytic Proteinase α Subunit RRC3: Differential Size, Tyrosine Phosphorylation, and Susceptibility to Antisense Oligonucleotide Treatment

Catharine M. Benedict, Ling Ren, Gary Clawson

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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 (α) and presumed catalytic (β) 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 a subunit of MCPs which contains a putative nuclear localization signal (NLS), was the predominant a subunit expressed in hepatocytes and hepatocytederived 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 Mr (24 000) than the cytoplasmic form (Mr 28 000), and only the nuclear form contained phosphotyrosine. In metabolic labeling experiments with [32P]orthophosphate, the major nuclear and NS form observed showed an MT of 24 000, whereas no labeling of cytosolic RRC3 was observed. A minor 32P-labeled band of Mr 28 000 was also observed in nuclei, and this Mr 28 000 form was found in the soluble nuclear extract within MCP complexes. These results suggest that tyrosine phosphorylation of the cytosolic form (Mr 28 000) rapidly triggers nuclear import, which is in turn quickly followed by conversion to the major Mr 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.

Original languageEnglish (US)
Pages (from-to)9587-9598
Number of pages12
JournalBiochemistry
Volume34
Issue number29
DOIs
StatePublished - Jan 1 1995

Fingerprint

Nuclear Matrix
Phosphorylation
Antisense Oligonucleotides
Proteasome Endopeptidase Complex
Scaffolds
Tyrosine
Hepatocytes
Forms (concrete)
Labeling
Cells
Cell Line
Nuclear Localization Signals
Phosphotyrosine
Proteins
Antibody Affinity
Cell Nucleus Active Transport
Antibodies
Polymerase chain reaction
Accessories
Cell growth

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{8f48b6ecfe474846a8d49dee23adc550,
title = "Nuclear Multicatalytic Proteinase α Subunit RRC3: Differential Size, Tyrosine Phosphorylation, and Susceptibility to Antisense Oligonucleotide Treatment",
abstract = "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 (α) and presumed catalytic (β) 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 a subunit of MCPs which contains a putative nuclear localization signal (NLS), was the predominant a subunit expressed in hepatocytes and hepatocytederived 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 Mr (24 000) than the cytoplasmic form (Mr 28 000), and only the nuclear form contained phosphotyrosine. In metabolic labeling experiments with [32P]orthophosphate, the major nuclear and NS form observed showed an MT of 24 000, whereas no labeling of cytosolic RRC3 was observed. A minor 32P-labeled band of Mr 28 000 was also observed in nuclei, and this Mr 28 000 form was found in the soluble nuclear extract within MCP complexes. These results suggest that tyrosine phosphorylation of the cytosolic form (Mr 28 000) rapidly triggers nuclear import, which is in turn quickly followed by conversion to the major Mr 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.",
author = "Benedict, {Catharine M.} and Ling Ren and Gary Clawson",
year = "1995",
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Nuclear Multicatalytic Proteinase α Subunit RRC3 : Differential Size, Tyrosine Phosphorylation, and Susceptibility to Antisense Oligonucleotide Treatment. / Benedict, Catharine M.; Ren, Ling; Clawson, Gary.

In: Biochemistry, Vol. 34, No. 29, 01.01.1995, p. 9587-9598.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Nuclear Multicatalytic Proteinase α Subunit RRC3

T2 - Differential Size, Tyrosine Phosphorylation, and Susceptibility to Antisense Oligonucleotide Treatment

AU - Benedict, Catharine M.

AU - Ren, Ling

AU - Clawson, Gary

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N2 - 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 (α) and presumed catalytic (β) 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 a subunit of MCPs which contains a putative nuclear localization signal (NLS), was the predominant a subunit expressed in hepatocytes and hepatocytederived 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 Mr (24 000) than the cytoplasmic form (Mr 28 000), and only the nuclear form contained phosphotyrosine. In metabolic labeling experiments with [32P]orthophosphate, the major nuclear and NS form observed showed an MT of 24 000, whereas no labeling of cytosolic RRC3 was observed. A minor 32P-labeled band of Mr 28 000 was also observed in nuclei, and this Mr 28 000 form was found in the soluble nuclear extract within MCP complexes. These results suggest that tyrosine phosphorylation of the cytosolic form (Mr 28 000) rapidly triggers nuclear import, which is in turn quickly followed by conversion to the major Mr 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.

AB - 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 (α) and presumed catalytic (β) 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 a subunit of MCPs which contains a putative nuclear localization signal (NLS), was the predominant a subunit expressed in hepatocytes and hepatocytederived 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 Mr (24 000) than the cytoplasmic form (Mr 28 000), and only the nuclear form contained phosphotyrosine. In metabolic labeling experiments with [32P]orthophosphate, the major nuclear and NS form observed showed an MT of 24 000, whereas no labeling of cytosolic RRC3 was observed. A minor 32P-labeled band of Mr 28 000 was also observed in nuclei, and this Mr 28 000 form was found in the soluble nuclear extract within MCP complexes. These results suggest that tyrosine phosphorylation of the cytosolic form (Mr 28 000) rapidly triggers nuclear import, which is in turn quickly followed by conversion to the major Mr 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.

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