ABSTRACT
Mutations in the neurofibromatosis 2 tumor suppressor gene (NF2) encoding merlin (moesin-ezrin-radixin like-protein) induce tumors of the nervous system. Merlin localizes to the cell membrane where it links the actin cytoskeleton to membrane proteins. Cell proliferation is regulated by merlin in many cell types, but merlin's tumor suppressor function still remains unclear. Phosphorylation has been suggested to regulate merlin's activity. The C-terminal serine 518 is phosphorylated both by p21-activated kinases (PAKs) and protein kinase A (PKA). In this work, we identify a novel PKA phosphorylation site, serine 10, in the N terminus of merlin. We show that a non-phosphorylatable form of serine 10 (S10A) affects cellular morphology. Regulation of this site also influences actin cytoskeleton organization and dynamics in vivo, as merlin S10A reduces the amount of cellular F-actin and merlin S10D stabilizes F-actin filaments. By using a wound-healing assay and live cell imaging, we demonstrate that dephosphorylation of serine 10 leads to defects in migration, possibly through altered ability of the cells to form lamellipodia. This study suggests a role for merlin in mediating PKA-induced changes of the actin cytoskeleton.
Subject(s)
Actin Cytoskeleton/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoskeleton/metabolism , Neurofibromin 2/metabolism , Neurofibromin 2/physiology , Actins/metabolism , Animals , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , COS Cells , Cell Movement/drug effects , Cells, Cultured , Chlorocebus aethiops , Cyclic AMP-Dependent Protein Kinases/physiology , Cytoskeletal Proteins/metabolism , Humans , Models, Biological , Mutagenesis, Site-Directed , Neurofibromin 2/chemistry , Neurofibromin 2/genetics , Phosphorylation , Polymers/metabolism , Protein Structure, Tertiary , Pseudopodia/drug effects , Pseudopodia/metabolism , Serine/genetics , Serine/metabolism , Thiazolidines/pharmacology , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/physiologyABSTRACT
Merlin and ezrin are homologous proteins with opposite effects on neoplastic growth. Merlin is a tumor suppressor inactivated in the neurofibromatosis 2 disease, whereas upregulated ezrin expression is associated with increased malignancy. Merlin's tumor suppressor mechanism is not known, although participation in cell cycle regulation has been suggested. To characterize merlin's biological activities, we screened for molecules that would interact with merlin but not ezrin. We identified the cyclin B-binding protein and cell cycle regulator HEI10 as a novel merlin-binding partner. The interaction is mediated by the alpha-helical domain in merlin and the coiled-coil domain in HEI10 and requires conformational opening of merlin. The two proteins show partial subcellular colocalization, which depends on cell cycle stage and cell adhesion. Comparison of Schwann cells and schwannoma cultures demonstrated that the distribution of HEI10 depends on merlin expression. In transfected cells, a constitutively open merlin construct affected HEI10 protein integrity. These results link merlin to the cell cycle control machinery and may help to understand its tumor suppressor function.
