A human phospholipid phosphatase activated by a transmembrane control module.

The recent discovery of voltage-sensitive phosphatases (VSPs) (1) established a novel molecular principle of electrochemical coupling: VSPs directly mediate the degradation of phosphoinositides in response to depolarization of the membrane potential (2, 3). These so-far-unique electro-enzymes consist of a transmembrane voltage sensor domain (VSD) homologous to the voltage sensors of voltage-gated ion channels and an intracellular C-terminal catalytic domain (CD) with high similarity to the tumor suppressor lipid phosphatase, PTEN (Fig. 1A) (1). The enzymatic activity of the CD is controlled by the VSD via an intramolecular conformational switch (4, 5). At typical negative resting voltages, the CD is inactive and is rapidly activated at more-positive (depolarized) voltages. The VSP homologs characterized to date are phosphoinositide 5-phosphatases that degrade the major signaling phospholipids PI(4,5)P2 and PI(3,4,5)P3 (2). Both phosphoinositides have key signaling roles in many cellular processes, including cell proliferation and differentiation, cytoskeletal dynamics, membrane trafficking, and control of ion channels (6, 7). Although little is known to date about the biological functions of VSPs, the ubiquitous roles of phosphoinositides and of electrical signaling suggest a potential impact on a large spectrum of cellular processes. Fig. 1. Generation of a functional hVSP1 chimera targeted to the plasma membrane. A: Domain structure of Ci-VSP and human VSP1 (full-length variant) and construction of the chimera hVSP1CiV. Inset shows catalytic CX5R motif. S1-S4, transmembrane segments 1–4; ... The principle of operation of VSPs was initially demonstrated for Ci-VSP, the prototypic VSP from the invertebrate chordate Ciona intestinalis. Subsequently, functional vertebrate VSPs have been identified in fishes and amphibia (8, 9). The VSP gene is also conserved in mammals (10). In general, there appears to be one VSP homolog in mammalian genomes; in the human genome, however, there are two expressed homologs, TPTE and TPTE2 (also termed TPIP) and additional pseudo-genes (10, 11). TPTE conforms to the architecture of VSPs, but it lacks phosphatase activity due to amino acid exchanges in the catalytic CX5R motif in the P-loop of the phosphatase domain (12). In contrast, the intracellular domain of TPTE2/TPIP has phosphoinositide phosphatase activity in vitro (11). According to its conserved catalytic center, it is apparently the bona-fide ortholog of nonmammalian VSPs and the VSP homologs in other mammals [e.g., mouse PTEN2 (13), also termed mTpte (14)]. For consistent nomenclature, we will thus rename both homologs hVSP1 (for TPIP/TPTE2) and hVSP2 (for human TPTE). Various splice variants of hVSP1 have been reported that mainly differ in the VSD-homologous transmembrane domain. Because all splice variants examined so far in expression systems lacked localization to the plasma membrane, it is unknown whether enzymatic activity is controlled by the VSD-homologous domain and, specifically, whether it is sensitive to membrane potential. Consequently the prevalence of VSP-mediated signaling in mammals has remained unknown. In human and mouse, VSPs are expressed mainly in the testis (10, 13) but also in brain and stomach (11). Cellular expression has been detected in secondary spermatocytes and early spermatids in human and mouse (10, 13), whereas in the tunicate, Ciona intestinalis, Ci-VSP, is also found in mature sperm (1). Thus, a role of VSPs in phosphoinositide signaling in mammalian spermatogenesis or sperm physiology is an exciting possibility. Here, we address the enzymatic properties and the molecular mechanism of activation of hVSP1 using fluorescent sensors for phosphoinositides in living cells. Because hVSP1 localized predominantly to intracellular compartments when transfected into culture cells, we generated a chimeric hVSP1 containing the N-terminus from Ci-VSP that robustly targets to the plasma membrane. Functional assays showed that hVSP1 is a phosphoinositide 5-phosphatase that can be activated via the N-terminal VSD. These findings support a role for VSP-mediated phosphoinositide signaling in mammalian spermatocyte differentiation or sperm function.