title: Binding of calcium ions to Ras promotes Ras guanine nucleotide exchange under emulated physiological conditions.
abstract: Both Ras protein and calcium play significant roles in various cellular processes via complex signaling transduction networks. However, it is not well understood whether and how Ca(2+) can directly regulate Ras function. Here we demonstrate by isothermal titration calorimetry that Ca(2+) directly binds to the H-Ras.GDP.Mg(2+) complex with moderate affinity at the first binding site followed by two weak binding events. The results from limited proteinase degradation show that Ca(2+) protects the fragments of H-Ras from being further degraded by trypsin and by proteinase K. HPLC studies together with fluorescence spectroscopic measurements indicate that binding of Ca(2+) to the H-Ras.GDP.Mg(2+) complex remarkably promotes guanine nucleotide exchange on H-Ras under emulated physiological Ca(2+) concentration conditions. Addition of high concentrations of either of two macromolecular crowding agents, Ficoll 70 and dextran 70, dramatically enhances H-Ras guanine nucleotide exchange extent in the presence of Ca(2+) at emulated physiological concentrations, and the nucleotide exchange extent increases significantly with the concentrations of crowding agents. Together, these results indicate that binding of calcium ions to H-Ras remarkably promotes H-Ras guanine nucleotide exchange under emulated physiological conditions. We thus propose that Ca(2+) may activate Ras signaling pathway by interaction with Ras, providing clues to understand the role of calcium in regulating Ras function in physiological environments.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Jun-Ming
authors.initials: JM
authors.lastname: Liao
authors.forename: Zhong-Ying
authors.initials: ZY
authors.lastname: Mo
authors.forename: Ling-Jia
authors.initials: LJ
authors.lastname: Wu
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Yi
authors.initials: Y
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D006150:Guanine Nucleotides / Q000378:metabolism*; D006801:Humans; D007477:Ions / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology; D018631:ras Proteins / Q000378:metabolism* / Q000502:physiology*
chemical_list: D006150:Guanine Nucleotides; D007477:Ions; D018631:ras Proteins; D002118:Calcium