Photo-Regulation of Small G Protein RhoA Signal Cascade used by Photochromic Molecules

Abstract

Small G protein, known as biomolecule switch protein, has a critical role of intracellular signal transduction using GDP-GTP exchange cycles. RhoA is one of the small G-protein Rho families, which plays important roles of regulation of cellular processes such as cell motility, adhesion, and apoptosis. It has been shown that GTP bound active state of RhoA induced enzymatic reaction through downstream cascade such as Rho-associate kinase (ROCK). In this study, we performed to control the function of RhoA using photochromic molecules. We prepared the RhoA mutant Q63L that exhibits constitutive active state. Subsequently cysteine residues were introduced into the functional sites of switch 1 and 3 regions of the mutant. The mutants were modified with thiol group reactive photochromic compound 4-phenylazophenyl maleimide (PAM). Photo-regulations of the GDP-GTP exchange on the PAM modified RhoA mutants were monitored with fluorescently labeled GTP derivate Mant-GTP. Cis-PAM-mutants showed faster GDP-GTP exchange than Trans-PAM-mutants in the absence Mg. As another way, we designed the photochromic RhoA binding domain peptide, which compete with ROCK in order to control the RhoA function photo-reversibly. The peptide that has a similar amino acid sequence to the RhoA binding coiled-coil region of ROCK and a single cysteine residue at N-terminal was synthesized. The peptides were cross-linked with bifunctional azobenzene derivatives, 4,4’-azobenzene-dimaleimide (ABDM) resulting in dimerization. Interaction of the photochromic peptide with RhoA was examined by the method of pull down assay and fluorescence depolarization. Cis-ABDM peptide showed slightly more effective inhibition of RhoA and ROCK interaction than Trans-ABDM peptide.