MARCH9介导磷酸酶RPTPα的泛素化降解

doi:10.3969/j.issn.1674-8115.2025.08.003

摘要/Abstract

摘要：

目的·探讨E3泛素连接酶膜相关锌指蛋白9（membrane-associated RING-CH 9，MARCH9）调控受体型蛋白质酪氨酸磷酸酶α（receptor protein tyrosine phosphatase alpha，RPTPα）泛素化修饰的分子机制及其生物学功能。方法·采用Western blotting鉴定RPTPα的泛素化修饰类型及MARCH9对其泛素化水平的调控作用；比较MARCH9野生型、MARCH9酶活突变体（MARCH9 S198A或MARCH9-HC/CC），以及shRNA介导的内源MARCH9敲低对RPTPα蛋白稳定性的影响；利用蛋白酶体抑制剂MG132、自噬抑制剂3-MA和溶酶体抑制剂氯喹（chloroquine，CQ）鉴定MARCH9介导的RPTPα泛素化降解类型；探究43 ℃热激促进RPTPα蛋白降解的可能机制；利用慢病毒载体构建MARCH9单敲低（H1299-shMARCH9）及MARCH9/RPTPα双敲低（H1299-shMARCH9-shRPTPα）的肺癌稳转细胞株，通过CCK-8细胞增殖、平板克隆形成和软琼脂克隆形成实验，评估MARCH9或RPTPα对肺癌细胞增殖与集落形成能力的影响；通过血管拟态形成实验和划痕实验检测MARCH9或RPTPα对肺癌细胞侵袭、迁移能力的影响；建立裸鼠皮下移植瘤模型评价MARCH9或RPTPα对肺癌细胞体内成瘤能力的影响；通过生物信息学方法比较MARCH9和RPTPα在肺癌患者中的表达差异与预后相关性。结果·RPTPα主要发生K63连接型多聚泛素化修饰，E3泛素连接酶MARCH9过表达显著增强其泛素化水平；过表达野生型MARCH9而非酶活突变体能显著降低RPTPα蛋白稳定性，而shRNA敲低内源MARCH9后RPTPα蛋白水平又会明显上升；CQ而非MG132或3-MA处理能增加RPTPα蛋白稳定性，提示MARCH9主要介导RPTPα的泛素化-溶酶体降解途径；43 ℃热激可以特异增强MARCH9-RPTPα相互作用并促进RPTPα蛋白降解。功能实验显示：与对照H1299细胞相比，H1299-shMARCH9细胞的RPTPα蛋白水平升高，细胞增殖速率加快，克隆形成和细胞侵袭能力均明显增强，裸鼠中成瘤能力也明显增强，而双敲低MARCH9/RPTPα的稳转细胞株又能逆转上述表型（均P<0.05）。生物信息学分析肺癌患者相关数据，显示RPTPα高表达与肺癌患者不良预后及肿瘤转移呈正相关，而MARCH9则为负相关。结论·MARCH9介导了磷酸酶RPTPα的K63型泛素化依赖的溶酶体降解过程。这一发现为开发靶向RPTPα的肿瘤治疗策略提供了新的依据。

关键词: 蛋白质酪氨酸磷酸酶RPTPα, E3泛素连接酶MARCH9, 泛素化, 热激, 肺癌

Abstract:

Objective ·To investigate the molecular mechanisms and biological functions of the E3 ubiquitin ligase membrane-associated RING-CH 9 (MARCH9) in regulating the ubiquitination of receptor protein tyrosine phosphatase alpha (RPTPα). Methods ·Western blotting was employed to identify the ubiquitination type of RPTPα and to evaluate the regulatory effect of MARCH9 on its ubiquitination level; Comparative analysis of RPTPα protein stability was performed among wild-type MARCH9, catalytically inactive MARCH9 mutants (MARCH9 S198A or MARCH9-HC/CC), and endogenous MARCH9 knockdown via shRNA. Proteasome inhibitor MG132, autophagy inhibitor 3-MA, and lysosomal inhibitor chloroquine (CQ) were used to determine the degradation pathway of MARCH9-mediated RPTPα ubiquitination. The mechanism underlying 43 ℃ heat shock-induced RPTPα degradation was explored. Stable lung cancer cell lines with MARCH9 single-knockdown (H1299-shMARCH9) and MARCH9/RPTPα double-knockdown (H1299-shMARCH9-shRPTPα) were established using lentiviral vectors. CCK-8 proliferation assay, colony formation assay, and soft agar assay were conducted to evaluate the effects of MARCH9 or RPTPα on lung cancer cell proliferation and clonogenicity. Vasculogenic mimicry formation assay and scratch wound healing assay were performed to assess the impacts on tumor cell invasion and migration. Subcutaneous xenograft models in nude mice were established to examine in vivo tumorigenicity. Bioinformatics analysis was used to compare the expression differences and prognostic correlations of MARCH9 and RPTPα in lung cancer patients. Results ·RPTPα predominantly underwent K63-linked poly-ubiquitination, which was significantly enhanced by MARCH9 overexpression. Wild-type MARCH9, but not its catalytic mutants, markedly reduced RPTPα protein stability, while endogenous MARCH9 knockdown increased RPTPα levels. CQ, not MG132 or 3-MA, restored RPTPα stability, indicating that MARCH9 mediated lysosomal degradation of RPTPα through ubiquitination. Heat shock at 43 ℃ specifically enhanced MARCH9-RPTPα interaction, promoting RPTPα degradation. Functional assays revealed that, compared to control H1299 cells, MARCH9-knockdown cells exhibited elevated RPTPα levels, accelerated proliferation, enhanced clonogenicity and invasive capacity, and increased tumorigenicity in nude mice. These phenotypes could be reversed by double knockdown of MARCH9/RPTPα. Bioinformatics analysis demonstrated that high RPTPα expression correlated with poor prognosis and tumor metastasis in lung cancer patients, while MARCH9 showed inverse correlations. Conclusion ·MARCH9 mediates K63-linked ubiquitination-dependent lysosomal degradation of phosphatase RPTPα, providing new insights into developing RPTPα-targeted cancer therapeutic strategies.

Key words: protein tyrosine phosphatase RPTPα, E3 ubiquitin ligase MARCH9, ubiquitination, heat shock, lung cancer

中图分类号:

R734.2

张宇琴, 依力夏提·艾合买提, 王艳丽, 阳志, 黄建. MARCH9介导磷酸酶RPTPα的泛素化降解[J]. 上海交通大学学报（医学版）, 2025, 45(8): 957-968.

ZHANG Yuqin, AIHEMAITI Yilixiati, WANG Yanli, YANG Zhi, HUANG Jian. Ubiquitination and degradation of RPTPα mediated by MARCH9[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(8): 957-968.

图/表 8

表1 PCR引物序列

Tab 1 Primers sequences for PCR

Primer	Forword (5′→3′)	Reverse (5′→3′)
MARCH9-HA	CGCGGATCCGCCACCATGCTCAAGTCTCG GCTCCGCA	CCGGAATTCTCAAGCGTAGTCTGGGACGTCG TATGGGTAGACTGTAGTGACCCTCATGACA
MARCH9-H136A/C139S	TCAGCCCAGCCTCATCCGCTGGAT	GCCGTGCAGCGCACTGAGCCGT
MARCH9-CC152/155SS	TCAGCTACTTCAAGTACCAGGTCCTGGC	GCTCACTGCTCCAGGAGCCCCTCTCG
MARCH9-S198A	GCCATCTCCTGGCTCATCTGGTCCT	GGCAACCAGGAAGAGCGAGCCCAGA

表2 shRNA序列

Tab 2 Sequence of shRNA

Name	Sequence (5'→3')
MARCH9 shRNA1	CCGGGTCCAGATTGCTGCCATAGTTCTCGAGAACTATGGCAGCAATCTGGACTTTTTG
MARCH9 shRNA2	CCGGGCAGTGGAAGGTCCTAAATTACTCGAGTAATTTAGGACCTTCCACTGCTTTTTG
MARCH9 shRNA3	CCGGCCCTGGAGAGAACCGTCTTTACTCGAGTAAAGACGGTTCTCTCCAGGGTTTTTG

图1 MARCH9 介导的磷酸酶RPTPα K63连接型泛素化修饰Note： A. 293T cells were co-transfected with RPTPα-HA and single-point K/R Myc-Ub plasmid as indicated. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting analysis with anti-Myc antibody. B. 293T cells were co-transfected with RPTPα-Myc and K-only HA-Ub plasmid as indicated. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by Western blotting analysis with anti-HA antibody. C. H1299-RPTPα-Myc stable cells were transfected with HA-Ub or HA-Ub KR as indicated, along with MARCH9-HA. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by Western blotting analysis with anti-HA antibody. D. H1299-RPTPα-Myc stable cells were transfected with Flag-Ub alone, together with MARCH9-HA or MARCH9 HC-CC-HA. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by Western blotting analysis with anti-Flag antibody. E. 293T cells were transfected with MARCH9-HA. Cell lysates were immunoprecipitated with anti-RPTPα antibody, followed by Western blotting analysis with anti-HA antibody.

Fig 1 MARCH9-mediated K63-linked ubiquitination of phosphatase RPTPα

图2 MARCH9 介导的RPTPα蛋白质降解Note： A. 293T cells were transfected with RPTPα-Myc alone or co-transfected with MARCH9-HA or MARCH9 S198A-HA for 48 h. Cells were lysed and the ectopic RPTPα protein level was detected by Western blotting. B. 293T cells were transfected with MARCH9-HA or MARCH9 HC/CC-HA for 48 h. Cells were lysed and the endogenous RPTPα protein level was detected by Western blotting. C. H1299-RPTPα-GFP stable cells were transfected with MARCH9-HA for the indicated times. The ectopic RPTPα-GFP protein level was detected by Western blotting. D. MARCH9-knockdown H1299 stable cells were generated by polyclonal lenti-viral infections with the pLKO.1-shRNA. The endogenous RPTPα and MARCH9 protein levels were detected by Western blotting. E. H1299-RPTPα-Myc stable cells were transfected with MARCH9-HA for 24 h, followed by CHX (100 μg/mL) treatment for the indicated times. RPTPα protein stability was detected by Western blotting. Tubulin was used as a loading control. F. H1299-RPTPα-Myc stable cells were transfected with MARCH9-HA for 24 h, followed by treatment with CQ, MG132 or 3-MA for 6 h. Then the cells were lysed and the RPTPα protein level was detected by Western blotting. G/H. H1299-RPTPα-Myc stable cells were treated with CQ (50 μmol/L) or MG132 (10 μmol/L) for the indicated times. RPTPα protein stability was detected by Western blotting.

Fig 2 MARCH9-mediated degradation of RPTPα protein

图3 热激-MARCH9 调控的RPTPα泛素化降解Note： A. H1299-RPTPα-GFP stable cells were transfected with Flag-Ub for 24 h. Cells were cultured at 43 ℃ for the indicated times, and lysed for immunoprecipitation with anti-RPTPα antibody, followed by Western blotting analysis with anti-Flag antibody. B. H1299-RPTPα-GFP stable cells were cultured at 43 ℃ for the indicated times. Then cells were lysed and the RPTPα protein level was analyzed by immunoblotting with anti-GFP antibody. C. 293T cells were transfected with MARCH9-HA for 48 h. Cells were cultured at 43 ℃ for 4 h alone, or followed by recovery at 37 ℃ for 4 h. Cell lysates were immunoprecipitated with anti-RPTPα antibody, followed by Western blotting analysis with anti-HA antibody. D. MARCH9-shRNA H1299 stable cells and control H1299 cells were cultured at 43 ℃ for 4 h. The endogenous RPTPα and MARCH9 protein levels were detected by Western blotting.

Fig 3 Heat shock and MARCH9-mediated ubiquitination degradation of RPTPα

图4 MARCH9 介导的RPTPα降解抑制肿瘤细胞的恶性转化Note： A. MARCH9-shRNA or MARCH9-shRNA/RPTPα-shRNA H1299 stable cell lines were generated by a lenti-viral system. Endogenous levels of Src pY527, RPTPα and MARCH9 proteins were detected by Western blotting. B. Growth curves of H1299 stable cells expressing vector, MARCH9-shRNA, or MARCH9-shRNA/RPTPα-shRNA were measured using the CCK-8 assay. Data are presented as x±s. Statistical significance was determined by Student's t-test (n=6). C. Soft-agar colony formation assays of H1299 stable cells expressing vector, MARCH9-shRNA, or MARCH9-shRNA/RPTPα-shRNA. Representative images of six replicates are shown, and colony numbers were quantified (n=6). Data are presented as x±s. The significance was determined by Student's t-test. D. H1299 stable cells expressing vector, MARCH9-shRNA, or MARCH9-shRNA/RPTPα-shRNA were seeded at 300 cells well and cultured for 15 d. The number of colonies was scored and representative images are shown. Quantification of clones for each group (n=3) is presented as x±s. The significance was determined by Student's t-test. E. Vasculogenic mimicry assays of H1299 stable cells expressing vector, MARCH9-shRNA, or MARCH9-shRNA/RPTPα-shRNA. The representative photographs of cell morphology were taken. F. Wound healing assay of H1299 stable cells expressing vector, MARCH9-shRNA, or MARCH9-shRNA/RPTPα-shRNA. Representative images were taken at 0 h and 14 h time points. G. Tumor images (left) of male BALB/c nude mice subcutaneously injected with suspension H1299 stable cells expressing vector, MARCH9-shRNA, or MARCH9-shRNA/RPTPα-shRNA (3×106 cells/mouse). Xenografted tumors were taken out, dissected, and weighed after 4 weeks. Quantification of tumors for each group (n=5) is shown (right). Data are presented as x¯±s. The significance was determined by Student's t-test. ①P=0.006, ②P<0.001, ③P=0.010, ④P=0.003.

Fig 4 MARCH9-mediated degradation of RPTPα suppresses the oncogenic potential of tumor cells

图5 RPTPα 、 MARCH9 与肺癌相关性的生物信息学分析Note： A.Correlation between RPTPα mRNA level and overall survival in human lung cancer. B. Correlation between MARCH9 mRNA level and overall survival in human lung cancer. C. Correlation between RPTPα mRNA level and lung cancer metastasis. D. Correlation between MARCH9 mRNA level and lung cancer invasion.

Fig 5 Bioinformatics analysis of RPTPα, MARCH9, and their association with lung cancer

图6 MARCH9 介导RPTPα泛素化降解的工作模型Note： A schematic model. RPTPα promotes tumor progression by dephosphorylating Src pY527 and activating Src kinase. Heat shock promotes the binding of the ubiquitin E3 ligase MARCH9 to RPTPα and enhances its K63-linked ubiquitination and degradation, thereby antagonizing the RPTPα-Src signaling pathway.

Fig 6 Working model of MARCH9-mediated ubiquitination and degradation of RPTPα

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