Ras activation by hydrostatic pressure involves GDP release and is enhanced by GAP and GEF in vitro.

Abstract

Hydrostatic pressure (HP) is a necessary stimulus for cell differentiation and growth in cultured chondrocytes. We hypothesized that Ras activation is involved in HP-induced cellular reactions and examined whether Ras, with or without its regulators, has HP sensitivity by using an in vitro system to measure Ras activity under HP. This in vitro system included mRaichu, a FRET-based Ras activity probe. We found that HP of 28 MPa activated Ras activity by 10.7% in the absence of the GAP and GEF domains. HP also induced rapid dissociation of a fraction of mant-GDP from Ras. HP-induced dissociation of GDP from Ras in the presence of GTP would explain the HP-induced Ras activation. A low concentration of GAP domain derived from p120GAP enhanced the HP-induced Ras activation to 15.3% by decreasing the Ras activity under atmospheric pressure (AP). In contrast, high concentrations of the GAP domain removed the HP activation by reducing the Ras activity to very low levels under both HP and AP conditions. Moreover, a broad concentration range (1-1000 nM) of GEF domain derived from hSOS-1 enhanced the HP-induced Ras activation. HP also increased Ras activity under conditions containing GEF and GAP domains to mimic cellular Ras activity. Based on these results, we propose that the HP-induced Ras activation revealed in this study is involved in the differentiation and growth stimulation of chondrocytes subjected to HP.