CRISPR-Cas9 RALA knockout and reconstitution. 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 (V MLN ) 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. Tumour 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.