Development of a versatile HPLC-based method to evaluate the activation status of small GTPases
| Autor: | Meguri Ohta, Kenji Kontani, Makoto Araki, Kaho Yoshimoto, Toshiaki Katada |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
dox
doxycycline GTPase-activating protein Cytoskeleton organization Mutation Missense tuberous sclerosis complex GTPase Biochemistry RHEB Ras homolog enriched in brain Humans Small GTPase HRAS high-performance liquid chromatography Molecular Biology Chromatography High Pressure Liquid mammalian target of rapamycin GDI guanine-nucleotide dissociation inhibitor S6K S6 kinase biology RHEB IP-RP-HPLC ion-pair reversed-phase HPLC Chemistry Mental Disorders Cell Biology TBA-B tetrabutylammonium bromide Cell biology Enzyme Activation HEK293 Cells Amino Acid Substitution small GTPase mTORC1 mechanistic target of rapamycin complex 1 biology.protein BSA bovine serum albumin Ras Homolog Enriched in Brain Protein Signal transduction TSC2 GAP GTPase-activating proteins Ras protein signal transduction Research Article HeLa Cells |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/j.jbc.2021.101428 |
| Popis: | Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable with those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, is involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood. |
| Databáze: | OpenAIRE |
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