ABSTRACT
Merlin, a tumor suppressor encoded by the neurofibromatosis type 2 gene, has been shown to suppress tumorigenesis by inhibiting the Cullin 4-RING E3 ubiquitin ligase CRL4(DCAF) (1) in the nucleus. This inhibition is mediated by direct binding of merlin to DDB1-and-Cullin 4-associated Factor 1 (DCAF1), yet the binding mode of merlin to DCAF1 is not well defined. Here, we report structural and biophysical studies of the merlin binding to DCAF1 and its interference with CD44 binding. The crystal structure of the merlin FERM domain bound to the DCAF1 C-terminal acidic tail reveals that the hydrophobic IILXLN motif located at the C-terminal end of DCAF1 binds subdomain C of the FERM domain by forming a ß-strand. The binding site and mode resemble that of merlin binding to the CD44 cytoplasmic tail. Competition binding assay showed that CD44 and DCAF1 compete for binding to the merlin FERM domain in solution. The CD44 cytoplasmic tail is known to be cleaved for nuclear translocation by regulated intra-membrane proteolysis (RIP). Our structure implies that, in the nucleus, the CD44 cytoplasmic tail cleaved by RIP could release DCAF1 from merlin by competing for binding to the merlin FERM domain, which results in the inhibition of merlin-mediated suppression of tumorigenesis.
Subject(s)
Carcinogenesis/pathology , Carrier Proteins/chemistry , Hyaluronan Receptors/chemistry , Neurofibromin 2/chemistry , Amino Acid Sequence , Animals , Carcinogenesis/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Crystallography, X-Ray , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Humans , Hyaluronan Receptors/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Neurofibromin 2/genetics , Neurofibromin 2/metabolism , Protein Serine-Threonine Kinases , Protein Structure, Secondary , Protein Structure, Tertiary , Ubiquitin-Protein LigasesABSTRACT
Grb2-associated regulator of Erk/MAPK1 (GAREM) is an adaptor molecule in the EGF-mediated signaling pathway. GAREM is expressed ubiquitously in human organs and cultured cells. Two GAREM homologues are encoded by the human genome. Therefore, previously identified GAREM is named GAREM1. Here we characterized a new subtype of GAREM, GAREM2, that is specifically expressed in the mouse, rat, and human brain. Three GAREM2 tyrosines (Tyr-102, Tyr-429, and Tyr-551) are phosphorylated upon EGF stimulation and are necessary for binding to Grb2. Furthermore, GAREM2 and Shp2 regulate Erk activity in EGF-stimulated cells. These characteristics are similar to those of GAREM1. GAREM2 is expressed in some neuroblastoma cell lines and is also tyrosine-phosphorylated and bound to Grb2 after treatment with EGF. Eventually, GAREM2 regulates Erk activation in the presence of EGF or insulin like growth factor 1. GAREM2 also regulates insulin-like growth factor 1-induced neuronal differentiation of the SH-SY5Y neuroblastoma cell line. Although the structure and function of both GAREM subtypes are similar, GAREM1 is recruited into the nucleus and GAREM2 is not. Nuclear localization of GAREM1 might be controlled by a GAREM1-specific nuclear localization sequence and 14-3-3ε binding. The N-terminal 20 amino acids of GAREM1 make up its nuclear localization sequence that is also a 14-3-3ε binding site. The GAREM family is a new class of adaptor molecules with subtype-specific biological functions.
