title: Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1).
abstract: RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Zhenhao
authors.initials: Z
authors.lastname: Fang
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada cmarshal@uhnres.utoronto.ca.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Marshall
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Jiani C
authors.initials: JC
authors.lastname: Yin
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Mohammad T
authors.initials: MT
authors.lastname: Mazhab-Jafari
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Geneviève M C
authors.initials: GM
authors.lastname: Gasmi-Seabrook
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Smith
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Tadateru
authors.initials: T
authors.lastname: Nishikawa
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Neel
authors.affiliation: From the Department of Medical Biophysics, Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada mikura@uhnres.utoronto.ca.
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism; D000077192:Adenocarcinoma of Lung; D019943:Amino Acid Substitution; D002460:Cell Line; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009634:Noonan Syndrome / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D014571:Urologic Neoplasms / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D009363:Neoplasm Proteins; D006160:Guanosine Triphosphate; C486776:RIT1 protein, human; D018631:ras Proteins
keywords: GTP hydrolysis; Noonan Syndrome; RIT1; Ras protein; cancer; nuclear magnetic resonance (NMR); nucleotide exchange; small GTPase; structure-function