Leucine-301 residue in a conserved carboxy-terminal motif of Rassf1A, SARAH, is required for interacting with MST1

Abstract

The tight coordination of cell proliferation and death is strongly required to determine the final number of cells in an organ or organism. Until now, many oncogenes and tumor suppressors have been identified and they have roles for regulating either cell growth or death. Recently, a subset of genes controlling both cell growth and death simultaneously was identified using Drosophila eye perfect for studying about proliferation and apoptosis. They are warts, salvador and hippo whose human relatives are LATS, WW45 and MST. Because MST1 binds to Rassf1A in mammalian cell there may exist a binding partner of hippo in Drosophila. Here, by sequence homology search, we identified RA(Ras-associaion) domain, a functional domain of Rass1A, containing protein in Drosophila, and named it dRassf. Using UAS-GAL4 based overexpression system, we overexpressed dRassf in eye, but the phenotype was normal. Also the eye containing many homozygous mitotic clones for dRassf deletion mutant allele had absolutely normal phenotype. However, the functional C-terminal domain of dRassf is still expressed in homozygous dRassf deletion mutant, and the possible interaction between dRassf and Hippo was proposed by the existence of the conserved C-terminal domain, SARAH. In addition, we generated a point mutant of human Rassf1A protein which cannot bind to MST1 in vivo, whereas wild type Rassf1A strongly binds to MST1. Although the physiological meaning of the loss of interaction ability has not been characterized, from the previous report about the regulation of apoptosis by Rass1A/MST1 in mammalian cells and the existence of Salvador/Warts/Hippo complex in Drosophila, we propose that Rassf1A containing complex has a role for regulating cell growth and apoptosis in human cases and that Leucine-301 residue in the conserved SARAH domain located to carboxy-terminal end of Rassf1A has a crucial role to construct the complex.