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Homo- and heterotypic association regulates signalling by the SgK269/PEAK1 and SgK223 pseudokinases


Liu, L; Phua, YW; Lee, RS; Ma, X; Jenkins, Y; Novy, K; Humphrey, ES; Chan, H; Shearer, R; Ong, PC; Dai, W; Saunders, DN; Lucet, IS; Daly, RJ
2016-08-16
Journal of Biological Chemistry
Journal Article
291
41
21571-21583
SgK269/PEAK1 is a pseudokinase and scaffolding protein that plays a critical role in regulating growth factor receptor signal output and is implicated in the progression of several cancers, including those of the breast, colon and pancreas. SgK269 is structurally related to SgK223, a human pseudokinase that also functions as a scaffold, but recruits a distinct repertoire of signaling proteins compared to SgK269. Structural similarities between SgK269 and SgK223 include a predicted alpha-helical region (designated CH) immediately preceding the conserved C-terminal pseudokinase (PK) domain. Structure-function analyses of SgK269 in MCF-10A mammary epithelial cells demonstrated a critical role for the CH and PK regions in promoting cell migration and Stat3 activation. Characterization of the SgK269 'interactome' by mass spectrometry-based proteomics identified SgK223 as a novel binding partner, and association of SgK269 with SgK223 in cells was dependent on the presence of the CH and PK domains of both pseudokinases. Homotypic association of SgK269 and SgK223 was also demonstrated and exhibited the same structural requirements. Further analysis using pull-downs and size-exclusion chromatography underscored the critical role of the CH region in SgK269/SgK223 association. Importantly, while SgK269 bridged SgK223 to Grb2, it was unable to activate Stat3 or efficiently enhance migration in SgK223 knock-out cells generated by CRISPR/Cas9. These results reveal previously unrecognized interplay between two oncogenic scaffolds and demonstrate a novel signalling mechanism for pseudokinases whereby homotypic and heterotypic association is used to assemble scaffolding complexes with distinct binding properties and hence qualitatively regulate signal output.
ASBMB
Chemical Biology
10.1074/jbc.M116.748897
27531744
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