Simple scheme of lipid enzyme can explain complex lives of phosphoinositides

Abstract

Phospholipids in biological membranes play multiple roles: They not only allow electrical signaling but also serve as precursors of second messengers in morphogenesis and cancer progression. Voltage-sensing phosphatases (VSPs) (1, 2) are unique membrane proteins that link these two functions. The VSP contains both a cytoplasmic lipid phosphatase and a transmembrane voltage sensor that is similar to the voltage sensor of voltage-gated ion channels. The voltage sensor domain is connected to a downstream cytoplasmic region with remarkable similarity to a tumor suppressor enzyme, phosphatase and tensin homolog (PTEN). PTEN dephosphorylates two species of phosphoinositides (PIs), PI(3,4,5)P 3 and PI(3,4)P 2 , in plasma membranes, antagonizing PI-3 kinase activity. Changes of PTEN's phosphatase activities are known to be associated with many human diseases including cancer, diabetes, and autism spectrum disorders (3). In the VSP, membrane depolarization activates the voltage sensor domain, leading to activation of PI phosphatase activity (4). Despite remarkable structural similarity to PTEN, VSP substrate preference toward PIs differs from substrate preference of PTEN; in particular, VSP dephosphorylates PI(4,5)P 2 , whereas PTEN does not. To understand the physiological roles of VSP, it is important to know the kinetic profiles of the various PIs that can be dephosphorylated by VSP upon membrane potential change. In PNAS, Keum et al. (5) precisely characterize and model the kinetics and voltage dependence of VSP activity toward different PI substrates.Detailed properties of enzymatic activities toward diverse PIs have previously been studied by in vitro enzyme assays using color detection of released phosphate with malachite green or separation of radiolabeled PIs by TLC (6). However, these methods have some limitations because they are difficult to apply to the full-length protein of VSP harboring the transmembrane segments. Several PI-binding motifs, such as the plextrin homology domain, can selectively recognize the headgroup of each species of PI, and fusions of such motifs with fluorescent polypeptides can report dynamic change of each species of PI in living cells. These probes have been used by many laboratories to study multiple types of PIs [PI(3,4,5)P 3 , PI(4,5)P 2 , and PI(3,4)P 2 ] in VSP-expressing cells under different membrane potentials (4, 6-9). One puzzling phenomenon in some of these detailed studies is that upon membrane depolarization, PI(3,4)P 2 initially increases, but it later decreases. These changes depend on the dual-enzyme activity of VSP toward PI(3,4,5)P 3 and PI(3,4)P 2 : PI(3,4,5)P 3 is turned into PI(3,4)P 2 through 5′-phosphatase activity, and PI(3,4)P 2 is then dephosphorylated by 3′-phosphatase activity. Such dynamic regulation of PI(3,4)P 2 by VSP activity might be physiologically relevant, because chick fibroblasts overexpressing VSP form remarkable fine processes that depend on increased PI(3,4)P 2 (10). Interestingly, an increase of PI(3,4)P 2 is observed at a lower voltage th...