Regulation of RAS function by active site autophosphorylation

• Published in: The FASEB journal 2021-05, Vol.35 (S1), p.n/a, Article fasebj.2021.35.S1.01476
• Main Authors: Johnson, Christian, Seo, Hyuk‐Soo, Terrell, Elizabeth, Geffken, Ezekiel, Lakhani, Jimit, Song, Kijun, Hagel, Kimberly, Popow, Olesja, Mattos, Carla, Morrison, Deborah, Dhe‐Paganon, Sirano, Haigis, Kevin
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2021.35.S1.01476

A unifying feature of the RAS superfamily is a functionally conserved cycle that small GTPases use to transition between active and inactive states. Here, we demonstrate that active site autophosphorylation of the oncogenic mutant K‐RASA59T is an irreversible and intrinsic regulatory mechanism that alters nucleotide exchange, effector interaction, and downstream signaling. We used x‐ray crystallography, enzymology, molecular dynamics and cell assays to explore the mechanism of autophosphorylation and its impact on RAS function. Our crystal structures of H‐RASA59T support a zero‐order and intramolecular mechanism of autophosphorylation that involves direct attack of threonine 59 on GTP. Further, we find that post‐phosphorylation, intrinsic nucleotide exchange is enhanced and favored over GEF mediated nucleotide exchange. Our crystal structures of H‐RASA59E and K‐RASA59E support this difference in biochemical activity. The impact of active site reorganization extends beyond stabilization of the nucleotide bound state to RAS effector interactions and utilization. Using effector binding assays, as well as classic assays of K‐RAS cell transformation, our data demonstrate that K‐RASA59T and K‐RASA59E do not depend on RAF kinases for cell transformation. Finally, we used structural analysis and molecular dynamics to show that other small GTPases with a conserved threonine or serine at residue 59 likely undergo autophosphorylation. Many of these small GTPases have similar biochemical features to K‐RASA59T, including overall stabilization of the GTP versus GDP bound state. We speculate that autophosphorylation is an unexplored means of regulation for these atypical small GTPases.