CRISPR-Cas9介导的RALA基因敲除与重建：对RALA S194磷酸化在Ras依赖性和Ras非依赖性癌症中的检测及作用的见解
CRISPR-Cas9 mediated RALA knockout and reconstitution: insights into the detection and role of RALA S194 phosphorylation in Ras-dependent and Ras-independent cancers.
作者信息
Konde Mayuresh Vishwas, Inchanalkar Siddhi, Sherkhane Tushar Manik, Deshpande Nilesh, Virmani Mishika, Singh Kajal, Balasubramanian Nagaraj
机构信息
IISER Pune: Indian Institute of Science Education Research Pune, India.
University of Massachusetts Amherst, India.
出版信息
Biol Open. 2025 Jul 15;14(7). doi: 10.1242/bio.061884. Epub 2025 Jul 21.
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.
在致癌性RAS的下游，RALA对癌症肿瘤发生至关重要，其丝氨酸194残基的磷酸化可能对其起到调控作用。我们在三种RAS依赖型和两种RAS非依赖型癌细胞中构建了CRISPR-Cas9介导的RALA基因敲除（RALA KO）。使用商业化的抗磷酸化RALA抗体检测RALA S194磷酸化，在所有三种RAS依赖型癌症中均缺乏特异性。在这些癌症中，通过小干扰RNA（siRNA）敲低RALA以及使用MLN8237（VMLN）抑制极光激酶A（AURKA），也不会影响pS194RALA的检测。用野生型RALA（WT-RALA）和S194A-RALA突变体稳定重建的RALA基因敲除的 MiaPaCa2细胞（RAS依赖型）和MCF7细胞（RAS非依赖型），对RALA激活无影响。然而，野生型RALA可部分恢复肿瘤生长，而S194A-RALA突变体则不能。因此，RALA S194磷酸化是肿瘤形成所必需的，它不影响RALA的激活，但可能通过其定位发挥作用。
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