Interactions between kinase scaffold MP1/p14 and its endosomal anchoring protein p18

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Journal titleBiochemistry
Pages36963705; # of pages: 10
AbstractScaffold 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.
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AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedYes
NRC number53140
NPARC number18178003
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Record identifier1d1184ef-9bb4-451c-8750-90a377cdd8c4
Record created2011-07-28
Record modified2017-03-23
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