Photo-Regulation of the Interaction between Ras and Ralgds using GTP Analogues Composed of Photochromic Molecules

Abstract

Small GTPases of the Ras family have significant roles of signal transduction in eukaryotic cells. The G-proteins transmit the signals from a number of receptors to a variety of effectors. RalGDS is a guanine nucleotide dissociation stimulator for Ral, which is a member of the Ras GTPase superfamily that regulates cellular proliferation, differentiation and transformation by mediating multiple signal transduction pathways. RalGDS can specifically promote the conversion from an inactive GDP-bound state to an active GTP-bound state for Ral. Previously we have demonstrated that the GTPase activity of K-Ras modified with thiol reactive azobenzene derivatives at the functional sites were photo-reversibly controlled upon ultraviolet and visible light irradiation. Aim of this study is to control the interaction of Ras with RalDGS photo-reversibly using photochromic molecules. Recently we found that a fluorescent GTP analogue, 2′(3′)-O-[6-(N-(7- nitrobenz-2- oxa-1,3-diazol-4-yl)amino)hexanoic]-GTP (NBD-GTP) which has a fluorescent probe at 2’ or 3’ position of ribose induced binding of Ras to RalGDS more significantly than regular GTP. NBD-GDP did not induce binding of Ras to RalGDS. The fluorophore NBD might affect as an enhancer of the interaction between Ras and RalGDS. In this study, we synthesized novel photo-responsive GTP analogues that have photochromic molecules at 2’ or 3’ position of ribose, 2′(3′) -O-Azobenzene-EDA-GTP (AB-EDA-GTP), 2′(3′) -O-Spiropyran-EDA-GTP (SP-EDA-GTP). Trans-isomer of AB-EDA-GTP induced more significant binding of Ras to RalGDS than Cis-isomer. On the other hand, SP-EDA-GTP did not show the significant difference between the spiro and merocyanine isomers.