The Use of Engineered Voltage-Sensing Phosphatases to Study the Determinants of Substrate Specificity of Phosphoinositide Phosphatases

Abstract

Ciona intestinalis voltage sensing phosphatase (Ci-VSP) is the founding member of a new class of voltage-sensing proteins, and exhibits PI(3,4,5)P3 and PI(4,5)P2 5’phosphatase activity upon membrane depolarisation (Murata and Okamura, 2007; Iwasaki et al., 2008; Halaszovich et al., 2009). Ci-VSP consists of a N-terminal voltage sensing domain (VSD) and a C-terminal phosphatase domain (PD) that is highly homologous to the phosphoinositide (PI) phosphatase PTEN (Murata et al., 2005). Recently, we reported on a voltage-sensitive chimera of the Ci-VSP voltage-sensor and the tumour suppressor PTEN (Ci-VSPTEN) in agreement with PTEN's 3’phosphatase activity (Lacroix et al., 2011). Here we extend this knowledge to a novel chimera of the VSD of Ci-VSP and the PTEN homologue TPIPα (also known as TPTE2), termed Ci-VSP/TPIP (Walker et al., 2001).Using whole cell voltage-clamp and total internal reflection microscopy with genetically-encoded phosphoinositide sensors, we show that Ci-VSP/TPIP is a voltage-sensitive PI(3,4,5)P3 and PI(4,5)P2 5’phosphatase in contrast to previous reports of TPIPα as a 3’phosphatase (Walker et al., 2001). The voltage dependence of Ci-VSP/TPIP was shifted to hyperpolarized potentials compared to Ci-VSP. Functional comparisons of Ci-VSP, Ci-VSPTEN and Ci-VSP/TPIP identified alanin/glycine 365 as the critical determinant of PTEN's substrate specificity, since Ci-VSPTEN(A365G) produced robust voltage-dependent PI(3,4,5)P3 5’ phosphatase activity, in contrast to the 3’phosphatase activity of wild-type PTEN.In conclusions, our data characterise the 5’phosphatase Ci-VSP/TPIP and reveal that engineered voltage-sensing phosphatases can be used to study the activity and substrate specificity of the important tumour suppressor PTEN in vivo.This work was supported by a Research Grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to C.R.H and by Deutsche Forschungsgemeinschaft through SFB 593 TP A12 to D.O.