Homo- and Heterotypic Association Regulates Signaling by the SgK269/PEAK1 and SgK223 Pseudokinases

Ling Liu,Yu Wei Phua,Rachel S Lee,Xiuquan Ma,Yiping Jenkins,Karel Novy,Emily S Humphrey,Howard Chan,Robert Shearer,Poh Chee Ong,Weiwen Dai,Darren N Saunders,Isabelle S Lucet,Roger J Daly

doi:10.1074/jbc.m116.748897

Abstract

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 with SgK269. Structural similarities between SgK269 and SgK223 include a predicted α-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 pulldowns and size-exclusion chromatography underscored the critical role of the CH region in SgK269/SgK223 association. Importantly, although 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 signaling mechanism for pseudokinases whereby homotypic and heterotypic association is used to assemble scaffolding complexes with distinct binding properties and hence qualitatively regulate signal output.

Highlights

Oncogenic scaffolds and demonstrate a novel signaling mechanism for pseudokinases whereby homotypic and heterotypic association is used to assemble scaffolding complexes with distinct binding properties and qualitatively regulate signal output
Because each member of this family possesses a distinct complement of recruitment sites for Src homology (SH)9 2 and phosphotyrosine binding (PTB) domain-containing signaling proteins, receptor dimerization allows for diversification of signaling outcomes through use of contrasting receptor combinations [7]
Overexpression of WT SgK269 significantly enhanced cell migration compared with control cells (Fig. 2e), and deletion of the N-terminal region had no discernable effect on activity in this assay, deletion of either the CH region or PK domain abolished the pro-migratory effect of SgK269 (Fig. 2e)

Summary

Introduction

Oncogenic scaffolds and demonstrate a novel signaling mechanism for pseudokinases whereby homotypic and heterotypic association is used to assemble scaffolding complexes with distinct binding properties and qualitatively regulate signal output. An equivalent result was obtained when this approach was applied to SgK269 or SgK223 homotypic interaction (Fig. 6, b and c), highlighting a key role for the CH region in direct homo- or heterotypic association of these pseudokinases.

Results

Conclusion