Regulation of GTPase function by autophosphorylation

• Published in: Molecular cell Vol. 82; no. 5; pp. 950 - 968.e14
• Main Authors: Johnson, Christian W., Seo, Hyuk-Soo, Terrell, Elizabeth M., Yang, Moon-Hee, KleinJan, Fenneke, Gebregiworgis, Teklab, Gasmi-Seabrook, Genevieve M.C., Geffken, Ezekiel A., Lakhani, Jimit, Song, Kijun, Bashyal, Puspalata, Popow, Olesja, Paulo, Joao A., Liu, Andrea, Mattos, Carla, Marshall, Christopher B., Ikura, Mitsuhiko, Morrison, Deborah K., Dhe-Paganon, Sirano, Haigis, Kevin M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.03.2022
• Subjects: Animals; autophosphorylation; Crystallography, X-Ray; GTP Phosphohydrolases - genetics; GTP Phosphohydrolases - metabolism; GTPase; Guanosine Triphosphate - metabolism; kinase; Mammals - metabolism; molecular dynamics; NMR; Nucleotides; phosphoryl transfer; protein crystallography; Proteins; RAF; RAS; RASSF; Signal Transduction; protein crystallography; kinase; NMR; RAS; autophosphorylation; RASSF; RAF; phosphoryl transfer; molecular dynamics; GTPase
• ISSN: 1097-2765; 1097-4164; 1097-4164
• DOI: 10.1016/j.molcel.2022.02.011

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts. [Display omitted] •Small GTPases undergo autophosphorylation through active site Ser/Thr substitution•K-RAS autophosphorylation inhibits GTP hydrolysis and promotes nucleotide exchange•K-RAS autophosphorylation promotes cellular transformation•Autophosphorylation inhibits K-RAS binding to effectors Johnson et al. identify a group of GTPases that undergo autophosphorylation via a conserved active site substitution. Using RASA59T as a prototypical autophosphorylating GTPase, they show that autophosphorylation is a stable post-translational modification that inhibits GTP hydrolysis and enhances nucleotide exchange. Despite promoting cell transformation, autophosphorylation inhibits K-RAS effector interactions.