Nuclear multicatalytic proteinase alpha subunit RRC3: differential size, tyrosine phosphorylation, and susceptibility to antisense oligonucleotide treatment.
Nuclear multicatalytic proteinase alpha subunit RRC3: differential size, tyrosine phosphorylation, and susceptibility to antisense oligonucleotide treatment.
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核多催化蛋白酶α亚基RRC3：差异大小、酪氨酸磷酸化和对反义寡核苷酸处理的敏感性。
DOI：
10.1021/bi00029a036
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Clawson,GA
中科院分区：
文献类型：
--
作者：
Benedict,CM;Ren,L;Clawson,GA
摘要：多解性蛋白水解酶(MCPs)是参与多种蛋白质细胞内降解的大分子结构。MCP由一个20S的“核心”组成，它由结构(A)和推测的催化(J3)亚基组成，并伴随着附属蛋白的复合体。免疫组织化学研究表明，MCP亚单位主要位于胞浆内，但也可观察到细胞核的定位。逆转录/聚合酶链式反应扩增已知亚基内的保守区，试图识别潜在的新亚基，并确定在肝细胞中表达的亚基。我们没有发现新的亚基，我们发现RRC3是MCPs的一个α亚基，它包含一个可能的核定位信号(NLS)，是在肝细胞和肝细胞来源的细胞系中表达的主要α亚基。抗体是针对RRC3的一个独特的C-末端多肽区域而开发的。使用亲和纯化抗体的免疫组织化学研究表明，RRC3既有细胞质定位，也有核定位。免疫沉淀/免疫印迹分析表明，很大比例的核RRC3与核支架(NS)有关。NS RRC3的MR(24000)显著小于胞浆形式(MR 28000)，并且只有核形式含有磷酸酪氨酸。在[~(32)P]正磷酸盐的代谢标记实验中，观察到的主要核型和NS形式的MT为24000，而胞质RRC3未观察到标记。在细胞核中还观察到一条较小的~(32)P标记的MT 28000条带，这种MR 28000形式存在于MCP复合体的可溶核提取液中。这些结果表明，胞浆形式(MR 28000)的酪氨酸磷酸化迅速触发核进口，进而迅速转化为与NS相关的主要MR 24000形式。针对RRC3起始点的反义寡核苷酸处理使肝细胞来源的细胞系的生长减少了95%，并产生了显著的形态变化(没有明显的毒性)。在此处理条件下，RRC3基因的表达水平显著降低。RRC3蛋白在NS中也显著减少，但在胞浆中仅有少量减少，提示核RRC3可能在细胞生长和分化中起重要作用。
Revised Manuscript Received April 25, 1995® 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 (a) and presumed catalytic (J3) subunits in association with complexes of accessory proteins. Immunohistochemical studies have shown MCP subunitsto 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 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 Mr (24 000) than the cytoplasmicform (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 MT 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 byconversion to the major Mr 24 000 form associated with NS. Treatment with antisense oligonucleotides targetedto 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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DOI：
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发表时间：
1990
期刊：
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research
影响因子：
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DOI：
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发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Hoffman,L;Rechsteiner,M
通讯作者：
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影响因子：
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