A Ci-VSP/TPIPα chimera exhibiting voltage-dependent PI(4,5)P2/PIP3-5’-phosphatase activity
Carlos A. Villalba-Galea: 0000-0002-6489-4651
Conference Title/Conference Publication
Biophysical Society 55th Annual Meeting
March 5-9, 2011
Date of Presentation
3, Supplement 1
Phosphoinositide (PI) concentrations in cell membranes play an important role in many cellular processes. PI phosphatases are crucial for the regulation of these concentrations. Among these phosphatases are the voltage-sensitive phosphatases (VSPs) like the prototypical Ci-VSP, which provide a new paradigm for the control of enzymatic activity. Recently, we showed that voltage sensitivity can be conferred to the cytoplasmic phosphatase PTEN: by fusing the voltage sensor domain (VSD) of Ci-VSP to the catalytic domain (CD) of PTEN, we generated an engineered chimeric VSP, Ci-VSPTEN.
Here we extend this work and report on a chimera created by fusing Ci-VSP's VSD to the CD of the putative PI-phosphatase TPIPα. We demonstrate that this Ci-VSP/TPIP chimera exhibits voltage dependent enzymatic activity. The substrate and position specificity of Ci-VSP/TPIP is analyzed using genetically encoded PI-specific fluorescence labeled probes, the membrane binding of which is assessed using total internal reflection fluorescence (TIRF) microscopy. Control over the membrane voltage is achieved by whole-cell patch clamping.
We find that upon depolarization, the membrane binding of the PIP3 specific probe, Btk-PH, and the PI(4,5)P2 specific probe, PLCδ1-PH, decreases, when co-expressed with Ci-VSP/TPIP. In contrast, membrane binding of the PI(3,4)P2 specific TAPP1-PH and the PI(4)P specific OSBP-PH probes increases upon depolarization.
These findings identify the Ci-VSP/TPIP chimera as a voltage-sensitive PI(4,5)P2/PIP3-5’-phosphatase. We conclude that TPIPα is a 5’-phosphatase in-vivo, in contrast to PIP3-3’-phosphatase activity in-vitro reported previously (Walker et al., Biochem. J. (2001) 360, 277 −283).
These data will help to further the understanding of the mechanism by which voltage control is exerted in VSPs. Additionally, our experiments demonstrate the usefulness of engineered VSPs as a novel tool for the analysis of PI-phosphatases in-vivo, an obligatory complement to in-vitro characterization.
Supported by Deutsche Forschungsgemeinschaft (SFB593 TPA12 to D.O.)
Halaszovich, Christian R.; Frezza, Ludivine; Schreiber, Daniela N.; Leitner, Michael G.; Villalba-Galea, Carlos A.; and Oliver, Dominik, "A Ci-VSP/TPIPα chimera exhibiting voltage-dependent PI(4,5)P2/PIP3-5’-phosphatase activity" (2011). School of Pharmacy and Health Sciences Faculty Presentations. 422.