Abstract
Recently, we have discovered a novel phosphatase-like gene from ascidian and named as CiVSP (Ciona intestinalis voltage sensor-containing phoshatase). This protein consists of two domains; the putative voltage sensor domain and the phosphatase domain. The voltage sensor domain showed high similarity to the transmembrane region of voltage-gated channels. Indeed, the Xenopus oocyte injected with CiVSP cRNA generated gating current in response to membrane depolarization by two-electrode voltage clamp. The phosphatase domain showed high similarity to PTEN (phosphatase and tensin homolog deleted from chromosome ten), a phosphatase which dephosphorylate the phosphate at the 3'-position of phosphatidylinositol 3,4,5-trisphosphate (PIP3). In order to examine whether CiVSP also dephosphorylates PIP3 like PTEN, we performed in vitro phosphoinositide phosphatase assay by colorimetric method using malachite green. We found that the CiVSP possessed the phosphatase activity to PIP3 like PTEN. The substitution of cystein residue at the position 363, which corresponds to the active site of PTEN, to serine resulted in a complete loss of phosphatase activity. In order to know the substrate specificity of CiVSP for various phosphoinositides, we expressed the phosphatase domain fused to glutathione S-transferase (GST) in E.coli , purified and performed in vitro phosphatase assay. Further, the phosphatase activity of CiVSP expressed in mammalian cells was also investigated. [Jpn J Physiol 54 Suppl:S130 (2004)]
