Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1)

• Published in: The Journal of biological chemistry Vol. 291; no. 30; pp. 15641 - 15652
• Main Authors: Fang, Zhenhao, Marshall, Christopher B., Yin, Jiani C., Mazhab-Jafari, Mohammad T., Gasmi-Seabrook, Geneviève M.C., Smith, Matthew J., Nishikawa, Tadateru, Xu, Yang, Neel, Benjamin G., Ikura, Mitsuhiko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.07.2016; American Society for Biochemistry and Molecular Biology
• Subjects: Adenocarcinoma - genetics; Adenocarcinoma - metabolism; Adenocarcinoma of Lung; Amino Acid Substitution; cancer; Cell Line; GTP hydrolysis; Guanosine Triphosphate - chemistry; Humans; Hydrolysis; Lung Neoplasms - genetics; Lung Neoplasms - metabolism; Molecular Bases of Disease; Mutation, Missense; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Noonan Syndrome; Noonan Syndrome - genetics; Noonan Syndrome - metabolism; nuclear magnetic resonance (NMR); nucleotide exchange; Protein Domains; Ras protein; ras Proteins - chemistry; ras Proteins - genetics; ras Proteins - metabolism; RIT1; small GTPase; structure-function; Urologic Neoplasms - genetics; Urologic Neoplasms - metabolism; RIT1; GTP hydrolysis; nuclear magnetic resonance (NMR); structure-function; Noonan Syndrome; nucleotide exchange; cancer; Ras protein; small GTPase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.714196

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.