title: Homo- and Heterotypic Association Regulates Signaling by the SgK269/PEAK1 and SgK223 Pseudokinases.
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.
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800.
authors.forename: Ling
authors.initials: L
authors.lastname: Liu
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800.
authors.forename: Yu Wei
authors.initials: YW
authors.lastname: Phua
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800.
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Lee
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800.
authors.forename: Xiuquan
authors.initials: X
authors.lastname: Ma
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Jenkins
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800.
authors.forename: Karel
authors.initials: K
authors.lastname: Novy
authors.affiliation: the Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010.
authors.forename: Emily S
authors.initials: ES
authors.lastname: Humphrey
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800.
authors.forename: Howard
authors.initials: H
authors.lastname: Chan
authors.affiliation: the Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010.
authors.forename: Robert
authors.initials: R
authors.lastname: Shearer
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800.
authors.forename: Poh Chee
authors.initials: PC
authors.lastname: Ong
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052.|the Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, and.
authors.forename: Weiwen
authors.initials: W
authors.lastname: Dai
authors.affiliation: the Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010.|the School of Medical Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia.
authors.forename: Darren N
authors.initials: DN
authors.lastname: Saunders
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052.|the Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, and.
authors.forename: Isabelle S
authors.initials: IS
authors.lastname: Lucet
authors.affiliation: From the Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, Clayton, Victoria 3800, roger.daly@monash.edu.
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Daly
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D000072417:Protein Domains; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002352:Carrier Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; C000607534:PRAG1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C573947:PEAK1 protein, human; D011505:Protein-Tyrosine Kinases
keywords: breast cancer; oncogene; protein kinase; scaffold protein; signal transduction