Science Topics - 79

3′ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP)
Tatsuki Kurokawa (Yasushi Okamura)*

Voltage-sensing phosphatase (VSP) consists of the two domains: voltage-sensor domain and the cytoplasmic region with phosphoinositide-phosphatase activities. The phosphatase region exhibits remarkable sequence similarity to PTEN, a tumor suppressor phosphatase. VSPs dephosphorylate the 5position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI (4,5)P2] upon voltage depolarization. However, it is unclear whether VSPs also have 3phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities with radiolabeled PI(3,4,5)P3. TLC assay showed that the 3phosphate of PI(3,4,5)P3 was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] was removed by VSPs. Monitoring of PI(3,4)P2 levels with the pleckstrin homology (PH) domain from tandem PH domain containing protein (TAPP1) fused with GFP (PHTAPP1-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5phosphatase activity of VSP toward PI(3,4,5)P3. However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P3 is dephosphorylated at the 5position, PI(3,4)P2 is then dephosphorylated at the 3position. These results suggest that substrate specificity of the VSP changes with membrane potential.



Kurokawa T, Takasuga S, Sakata S, Yamaguchi S, Horie S, Homma KJ, Sasaki T, Okamura Y, PNAS, 109: 10089-10094 (2012)