{"title":"CRISPR-Cas9介导的RALA敲除和重构。RALA S194磷酸化在ras依赖性和ras非依赖性癌症中的检测和作用。","authors":"Mayuresh Vishwas Konde, Siddhi Inchanalkar, Tushar Manik Sherkhane, Nilesh Deshpande, Mishika Virmani, Kajal Singh, Nagaraj Balasubramanian","doi":"10.1242/bio.061884","DOIUrl":null,"url":null,"abstract":"<p><p>Downstream of oncogenic RAS, RALA is critical for cancer tumorigenesis, possibly regulated by phosphorylation of its Serine194 residue. We made CRISPR-Cas9 RALA knockout (RALA KO) in three RAS-dependent and two RAS-independent cancer cells. Detection of RALA S194 phosphorylation using the commercial anti-phospho-RALA antibody lacks specificity in all three RAS-dependent cancers. siRNA knockdown of RALA and AURKA inhibition by MLN8237 (VMLN) also did not affect pS194RALA detection in these cancers. RALA KO MiaPaCa2 (RAS-dependent) and MCF7 (RAS-independent) cells, stably reconstituted with WT-RALA and S194A-RALA mutants, showed no effect on RALA activation. Tumor growth was, however, restored partly by WT-RALA, but not S194A-RALA mutant. Thus, RALA S194 phosphorylation is needed for tumor formation, not affecting its activation, but possibly through its localization.</p>","PeriodicalId":9216,"journal":{"name":"Biology Open","volume":" ","pages":""},"PeriodicalIF":1.7000,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12320973/pdf/","citationCount":"0","resultStr":"{\"title\":\"CRISPR-Cas9 mediated RALA knockout and reconstitution: insights into the detection and role of RALA S194 phosphorylation in Ras-dependent and Ras-independent cancers.\",\"authors\":\"Mayuresh Vishwas Konde, Siddhi Inchanalkar, Tushar Manik Sherkhane, Nilesh Deshpande, Mishika Virmani, Kajal Singh, Nagaraj Balasubramanian\",\"doi\":\"10.1242/bio.061884\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Downstream of oncogenic RAS, RALA is critical for cancer tumorigenesis, possibly regulated by phosphorylation of its Serine194 residue. We made CRISPR-Cas9 RALA knockout (RALA KO) in three RAS-dependent and two RAS-independent cancer cells. Detection of RALA S194 phosphorylation using the commercial anti-phospho-RALA antibody lacks specificity in all three RAS-dependent cancers. siRNA knockdown of RALA and AURKA inhibition by MLN8237 (VMLN) also did not affect pS194RALA detection in these cancers. RALA KO MiaPaCa2 (RAS-dependent) and MCF7 (RAS-independent) cells, stably reconstituted with WT-RALA and S194A-RALA mutants, showed no effect on RALA activation. Tumor growth was, however, restored partly by WT-RALA, but not S194A-RALA mutant. Thus, RALA S194 phosphorylation is needed for tumor formation, not affecting its activation, but possibly through its localization.</p>\",\"PeriodicalId\":9216,\"journal\":{\"name\":\"Biology Open\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":1.7000,\"publicationDate\":\"2025-07-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12320973/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biology Open\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1242/bio.061884\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/7/21 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biology Open","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1242/bio.061884","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/7/21 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
在致癌RAS的下游，RALA对癌症的发生至关重要，可能受其Serine194残基磷酸化的调节。我们在3个ras依赖型和2个ras非依赖型癌细胞中进行了CRISPR-Cas9 RALA敲除（RALA KO）。使用商用抗磷酸化RALA抗体检测RALA S194磷酸化在所有三种ras依赖性癌症中缺乏特异性。siRNA敲低RALA和MLN8237 （VMLN）抑制AURKA也不影响这些癌症中pS194RALA的检测。用WT-RALA和S194A-RALA突变体稳定重建的RALA KO MiaPaCa2 （ras依赖）和MCF7 （ras独立）细胞对RALA的激活没有影响。然而，WT-RALA可以部分恢复肿瘤生长，而S194A-RALA突变体则不能。因此，RALA S194的磷酸化是肿瘤形成所必需的，不影响其激活，但可能通过其定位。
CRISPR-Cas9 mediated RALA knockout and reconstitution: insights into the detection and role of RALA S194 phosphorylation in Ras-dependent and Ras-independent cancers.
Downstream of oncogenic RAS, RALA is critical for cancer tumorigenesis, possibly regulated by phosphorylation of its Serine194 residue. We made CRISPR-Cas9 RALA knockout (RALA KO) in three RAS-dependent and two RAS-independent cancer cells. Detection of RALA S194 phosphorylation using the commercial anti-phospho-RALA antibody lacks specificity in all three RAS-dependent cancers. siRNA knockdown of RALA and AURKA inhibition by MLN8237 (VMLN) also did not affect pS194RALA detection in these cancers. RALA KO MiaPaCa2 (RAS-dependent) and MCF7 (RAS-independent) cells, stably reconstituted with WT-RALA and S194A-RALA mutants, showed no effect on RALA activation. Tumor growth was, however, restored partly by WT-RALA, but not S194A-RALA mutant. Thus, RALA S194 phosphorylation is needed for tumor formation, not affecting its activation, but possibly through its localization.
期刊介绍：
Biology Open (BiO) is an online Open Access journal that publishes peer-reviewed original research across all aspects of the biological sciences. BiO aims to provide rapid publication for scientifically sound observations and valid conclusions, without a requirement for perceived impact.
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