# CRISPR-Cas9 mediated RALA knockout and reconstitution: insights into the detection and role of RALA S194 phosphorylation in Ras-dependent and Ras-independent cancers

## TL;DR

This study explores how RALA S194 phosphorylation affects tumor formation in cancer cells, finding it influences localization rather than activation.

## Contribution

The study reveals a novel role of RALA S194 phosphorylation in tumor formation through localization rather than activation.

## Key findings

- RALA S194 phosphorylation is essential for tumor formation in RAS-dependent and RAS-independent cancers.
- Phosphorylation at S194 does not affect RALA activation but may influence its localization.
- Commercial anti-phospho-RALA antibody lacks specificity in RAS-dependent cancers.

## Abstract

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.

Summary: This study gives insight into the novel role of RALA S194 phosphorylation in regulating tumor formation in cancer cells. It also shows that this regulation of tumor formation comes from its role in RALA localization rather than RALA activation.