Clustered phosphatidylinositol 4,5 bisphosphate accumulation and ezrin phosphorylation in response to CLIC5A

Abstract

CLIC5A (encoded by CLIC5) is a component of the ezrin-NHERF2-podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation without changing ezrin abundance, increased the association of ezrin with the cytoskeletal fraction and enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] reporter RFP-PH-PLC to translocate from the cytosol to discrete plasma membrane clusters at the cell surface, where it colocalized with CLIC5A. Transiently expressed HA-PIP5Ka colocalized with GFP-CLIC5A and was pulled from cell lysates by GST-CLIC5A, and silencing of endogenous PIP5Ka abrogated CLIC5A-dependent ERM phosphorylation. N-and C-terminal deletion mutants of CLIC5A, which failed to associate with the plasma membrane, failed to colocalize with PIP5Ka, did not alter the abundance of PI(4,5)P 2 plasma membrane clusters and failed to enhance ezrin phosphorylation. Relative to wild-type mice, in CLIC5-deficient mice, the phosphorylation of glomerular ezrin was diminished and the cytoskeletal association of both ezrin and NHERF2 was reduced. Therefore, the mechanism of CLIC5A action involves clustered plasma membrane PI(4,5)P 2 accumulation through an interaction of CLIC5A with PI(4,5)P 2 -generating kinases, in turn facilitating ezrin activation and actin-dependent cell surface remodeling.

Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterol nanoclusters

“…For example, cytoplasmic proteins such as ezrin4142, syntaxin-143 have also been described to induce or interact with PIP 2 nanoclusters. As a general mechanism, proteins with basic interfaces can recruit acidic lipids that, in turn, can facilitate recruitment and clustering of these proteins into nanodomains4445.…”

Three Decades of Chloride Intracellular Channel Proteins: From Organelle to Organ Physiology

“…In bone marrow-derived macrophages, CLIC4 is observed in the nucleus and its expression increases with lipopolysaccharide (LPS) stimulation (Domingo-Fernandez, Coll, Kearney, Breit, & O'Neill, 2017). Hepatocellular cancer (HCC) cell lines (HepG2, Huh7, and SNU387) have native CLIC5 in the nucleus (Flores-Tellez, Lopez, Vasquez Garzon, & Villa-Trevino, 2015) and ectopic expression in HeLa cells localizes CLIC5 to the nucleus (Al-Momany, Li, Alexander, & Ballermann, 2014). These findings indicate that CLIC1 is a native nuclear membrane channel and that other CLICs can translocate to nucleus under certain conditions or when overexpressed.…”

Emerging biological roles of Cl− intracellular channel proteins

“…In a COS7-cell overexpression system, CLIC5A promotes the clustering of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] at the plasma membrane through its interaction with PI5P4K isoforms, which is thought to facilitate actin-dependent membrane remodeling (Al-Momany et al, 2014). Whether these interactions are direct and whether and how CLIC4 and CLIC5 are capable of regulating PIPK activity remains unclear.…”

“…Biochemical and colocalization studies suggest that, upon recruitment to the plasma membrane, both CLIC4 and CLIC5 function in a complex with ERM and/or actin-binding scaffold proteins, such as Na + /H + exchange regulatory factor 2 (NHERF2, officially known as SLC9A3R2) and the above-mentioned ERM proteins (Al-Momany et al, 2014;Berryman and Bretscher, 2000;Berryman et al, 2004;Ponsioen et al, 2009;Salles et al, 2014;Tavasoli et al, 2016a;Viswanatha et al, 2013). The association of CLIC4 and CLIC5 with the actin cytoskeleton and the finding that CLICs traffic between distinct subcellular compartments upon receptor stimulation (see below) strongly suggest a role for CLIC proteins in actin-dependent membrane trafficking.…”

“…(2) CLIC5 recruits PIP kinases (PIPKs) to influence local phosphoinositide levels at the plasma membrane (Al-Momany et al, 2014). (3) CLIC5 stabilizes membrane-actin linkages at hair cell stereocilia in complex with ERM proteins and taperin (T) (Salles et al, 2014).…”