title: Interactions between kinase scaffold MP1/p14 and its endosomal anchoring protein p18.
abstract: Scaffold and adaptor proteins provide means for the spatial organization of signaling cascades. MP1 is a scaffold protein in the RAF/MEK/ERK pathway and together with p14 forms a heterodimer that was shown to be responsible for localization of MEK to the late endosomal compartment. However, the mechanism by which MP1/p14 tethers MEK to the endosomal membrane was not resolved. Recently, an adaptor protein p18 was identified as a binding partner of MP1/p14. p18 is attached to the endosomal surface by myristoyl and palmitoyl groups located at the N-terminus of the protein and tethers the signaling complex to the cytoplasmic surface of late endosomes. p18 expressed in E. coli is retained in inclusion bodies, and we developed a protocol to refold it from the denatured state. Coexpression of p18 with MP1/p14 leads to a soluble protein complex. We examined the interaction of p18 with the MP1/p14 constitutive heterodimer. We cloned various constructs of p18 and characterized their behavior and interactions with MP1/p14 in vitro using SEC and pull-down assays. We determined that the refolded p18 is a monomer in solution with molten globule characteristics. Its binding to MP1/p14 promotes folding and ordering. We also identified a proteolytically stable fragment of p18 and showed that it binds to MP1/p14 with similar affinity to the full-length construct and determined an apparent dissociation constant in the low micromolar range for the interaction. Finally, we show that the ∼60 C-terminal residues of p18 are not required for in vitro interaction with MP1/p14 heterodimer, in contrast to previously reported findings showing that truncation of 41 C-terminal residues of p18 prevents endosomal localization of MP1/p14.
authors: Biotechnology Research Institute, NRCC, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada.
authors: James
authors: J
authors: Magee
authors: Miroslaw
authors: M
authors: Cygler
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D017353:Gene Deletion; D051379:Mice; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C426009:LAMTOR2 protein, mouse; C114559:Lamtor3 protein, mouse; D011506:Proteins; C539361:lipid raft adaptor p18 protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases