Year: 2011
Type: article
Abstract: Loss of the functional NF2 gene product, merlin, gives rise to the tumor predisposition syndrome neurofibromatosis type 2 (NF2).The NF2 tumor suppressor protein, merlin, is related to the ERM (Ezrin, Radixin, and Moesin) family of plasma membrane-actin cytoskeleton linker proteins.Binding of phosphatidylinositol 4,5bisphosphate (PIP 2 ) to the amino-terminal FERM (Four-point one, Ezrin, Radixin and Moesin) domain of ezrin is required for the conformational activation, proper subcellular localization and function of ezrin, however, the role of phosphoinositide binding in merlin function is unclear.Despite strong evidence indicating that the association of merlin with the plasma membrane is critical for its growth suppressive function, the mechanisms by which merlin associates with the plasma membrane are not well understood.Moreover, a significant proportion of merlin resides in cholesteroldependent membrane rafts, although its mode of attachment to rafts is unknown.Here we report that merlin FERM domain binds phosphoinositides including PIP 2 .We further show that mutating the conserved amino acids in the PIP 2 binding motif (K79, K80, K269, E270, K278 and K279) to asparagines (merlin 6N), drastically reduces phosphoinositide binding without affecting the folding of merlin.Abolition of FERM domain-mediated phosphoinositide binding results in merlin 6N being predominantly cytosolic and significantly more mobile than membrane-bound merlin.Specifically, we also demonstrate that FERM domain phosphoinositide binding is required for the association of merlin with membrane rafts.We thus identify phosphoinositide binding as a mechanism that targets merlin to the membrane and specifically to the membrane rafts.iii Based on similarity to ERM proteins, it is predicted that FERM-CTD (carboxy terminal domain) interaction mediates the closed conformation of merlin, which can be disrupted by S518 phosphorylation and/or PIP 2 binding; however this has not been directly tested.Notably, our data demonstrate that the central α-helical domain primarily mediates the closed conformation of merlin.Unexpectedly, unlike in ERM proteins, we find that neither PIP 2 binding nor S518 phosphorylation cause a significant change in merlin conformation as detected by Förster resonance energy transfer.Therefore, contrary to the current conformational model for merlin, our data demonstrate that merlin exists as a monomer in a closed conformation and does not undergo large-scale folding or unfolding under conditions of PIP 2 binding or S818 phosphorylation.Additionally, FERM domain phosphoinositide binding does not regulate merlin phosphorylation at S518.Importantly, we demonstrate that the merlin 6N mutant is significantly compromised in its ability to suppress cell proliferation, indicating that FERM domain phosphoinositide binding is necessary for the growth suppressive function of merlin.Furthermore, we rule-out that these differences between merlin 6N and merlin wild-type are due to a folding defect caused by the introduction of 6N mutations.Most interestingly, retargeting the merlin 6N mutant into membrane rafts is sufficient to restore its growth suppressive function.In summary, our results demonstrate that FERM domain-mediated phosphoinositide binding and membrane raft association are critical for the growth inhibitory function of merlin.Further studies are needed to identify therapeutic strategies based on the association of merlin with membrane rafts that might be beneficial in the treatment of NF2.
Author: Timmy Mani
Institution: University of Cincinnati
Language: English
Subfield: Clinical Neurology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2283217038