Role of Phosphorylation in Moesin Interactions with PIP2-Containing Biomimetic Membranes

Abstract

Moesin, a protein of the ezrin, radixin, and moesin family, which links the plasma membrane to the cytoskeleton, is involved in multiple physiological and pathological processes, including viral budding and infection. Its interaction with the plasma membrane occurs via a key phosphoinositide, the phosphatidyl(4,5)inositol-bisphosphate (PIP), and phosphorylation of residue T558, which has been shown to contribute, in cellulo, to a conformationally open protein. We study the impact of a double phosphomimetic mutation of moesin (T235D, T558D), which mimics the phosphorylation state of the protein, on protein/PIP/microtubule interactions. Analytical ultracentrifugation in the micromolar range showed moesin in the monomer and dimer forms, with wild-type (WT) moesin containing a slightly larger fraction (∼30%) of dimers than DD moesin (10-20%). Only DD moesin was responsive to PIP in its micellar form. Quantitative cosedimentation assays using large unilamellar vesicles and quartz crystal microbalance on supported lipid bilayers containing PIP reveal a specific cooperative interaction for DD moesin with an ability to bind two PIP molecules simultaneously, whereas WT moesin was able to bind only one. In addition, DD moesin could subsequently interact with microtubules, whereas WT moesin was unable to do so. Altogether, our results point to an important role of these two phosphorylation sites in the opening of moesin: since DD moesin is intrinsically in a more open conformation than WT moesin, this intermolecular interaction is reinforced by its binding to PIP. We also highlight important differences between moesin and ezrin, which appear to be finely regulated and to exhibit distinct molecular behaviors.

Sequential binding of ezrin and moesin to L-selectin regulates monocyte protrusive behaviour during transendothelial migration

“…However, these results provide no understanding of their subcellular localisation during TEM. Numerous studies have shown that PIP 2 binding of moesin precedes phosphorylation of ERM proteins ( Ben-Aissa et al, 2012 ; Lubart et al, 2018 ). To address the impact of PIP 2 binding on moesin activation during TEM, a series of lysine (K) to asparagine (N) mutations at positions 253, 254, 262 and 263 (K253N, K254N, K262N and K263N), which are known to be important for PIP 2 binding ( Barret et al, 2000 ), were engineered into the moesin–GFP FERM domain (denoted 4N) and stably reconstituted into cells lacking endogenous moesin.…”

Two Sides of the Coin: Ezrin/Radixin/Moesin and Merlin Control Membrane Structure and Contact Inhibition

“…Indeed, Förster Resonance Energy Transfer (FRET) and co-immunoprecipitation experiments have shown that ezrin oligomers form in cells [77,90], and dimeric ezrin has been purified from placental tissue [91,92]. The interaction of the FERM and CTD is strong with little exchange between monomers and dimers occurring [92,93,94,95]. Small-angle X-ray scattering (SAXS) studies of purified ezrin dimer have shown that it largely adopts a domain-swapped dumbbell structure (Figure 4b) [56].…”

Charge-dependent interactions of monomeric and filamentous actin with lipid bilayers

“…The linkage between the cortex and the membrane is thus realized by linker proteins, for example, the ERM proteins Ezrin, Radixin, and Moesin (24,25), which can simultaneously bind to the membrane and the actin filaments. The mechanism of this binding is rather complex and depends on posttranslational alterations of the binding protein (26,27) as well as the composition of the membrane (28)(29)(30)(31). Divalent ions are ubiquitous in cells.…”