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

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Pages (from-to)21571-21583
Number of pages13
JournalJournal of Biological Chemistry
Volume291
Issue number41
DOIs
Publication statusPublished - 7 Oct 2016

Keywords

  • breast cancer
  • oncogene
  • protein kinase
  • scaffold protein
  • signal transduction

Cite this

Liu, Ling ; Phua, Yu Wei ; Lee, Rachel S. ; Ma, Xiuquan ; Jenkins, Yiping ; Novy, Karel ; Humphrey, Emily S. ; Chan, Howard ; Shearer, Robert ; Ong, Poh Chee ; Dai, Weiwen ; Saunders, Darren N. ; Lucet, Isabelle S. ; Daly, Roger J. / Homo- and heterotypic association regulates signaling by the SgK269/PEAK1 and SgK223 pseudokinases. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 41. pp. 21571-21583.
@article{e156954214494bf990dcf4477495894c,
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.",
keywords = "breast cancer, oncogene, protein kinase, scaffold protein, signal transduction",
author = "Ling Liu and Phua, {Yu Wei} and Lee, {Rachel S.} and Xiuquan Ma and Yiping Jenkins and Karel Novy and Humphrey, {Emily S.} and Howard Chan and Robert Shearer and Ong, {Poh Chee} and Weiwen Dai and Saunders, {Darren N.} and Lucet, {Isabelle S.} and Daly, {Roger J.}",
year = "2016",
month = "10",
day = "7",
doi = "10.1074/jbc.M116.748897",
language = "English",
volume = "291",
pages = "21571--21583",
journal = "Journal of Biological Chemistry",
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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, 'Homo- and heterotypic association regulates signaling by the SgK269/PEAK1 and SgK223 pseudokinases' Journal of Biological Chemistry, vol. 291, no. 41, pp. 21571-21583. https://doi.org/10.1074/jbc.M116.748897

Homo- and heterotypic association regulates signaling by the SgK269/PEAK1 and SgK223 pseudokinases. / Liu, Ling; Phua, Yu Wei; Lee, Rachel S.; Ma, Xiuquan; Jenkins, Yiping; Novy, Karel; Humphrey, Emily S.; Chan, Howard; Shearer, Robert; Ong, Poh Chee; Dai, Weiwen; Saunders, Darren N.; Lucet, Isabelle S.; Daly, Roger J.

In: Journal of Biological Chemistry, Vol. 291, No. 41, 07.10.2016, p. 21571-21583.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Homo- and heterotypic association regulates signaling by the SgK269/PEAK1 and SgK223 pseudokinases

AU - Liu, Ling

AU - Phua, Yu Wei

AU - Lee, Rachel S.

AU - Ma, Xiuquan

AU - Jenkins, Yiping

AU - Novy, Karel

AU - Humphrey, Emily S.

AU - Chan, Howard

AU - Shearer, Robert

AU - Ong, Poh Chee

AU - Dai, Weiwen

AU - Saunders, Darren N.

AU - Lucet, Isabelle S.

AU - Daly, Roger J.

PY - 2016/10/7

Y1 - 2016/10/7

N2 - 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.

AB - 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.

KW - breast cancer

KW - oncogene

KW - protein kinase

KW - scaffold protein

KW - signal transduction

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U2 - 10.1074/jbc.M116.748897

DO - 10.1074/jbc.M116.748897

M3 - Article

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