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

https://doi.org/10.1074/jbc.M116.748897Get rights and content
Under a Creative Commons license
open access

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.

breast cancer
oncogene
protein kinase
scaffold protein
signal transduction

Cited by (0)

The mass spectrometric raw data and spectral libraries associated with this manuscript are available from ProteomeXchange with the accession number PXD004250.

*

This work was supported in part by Project Grant 1084621 from the National Health and Medical Research Council of Australia. The authors declare that they have no conflicts of interest with the contents of this article.

This article contains supplemental dataset 1 and Table S1.

1

Supported by National Health and Medical Research Council ECR Fellowship 1054497.

2

Recipient of Research Scholar Award 10/RSA/1-28 from the Cancer Institute of New South Wales and an Australian Postgraduate Award from the University of New South Wales.

3

Supported by a Baxter Family Scholarship.

4

Supported by the Australian Cancer Research Foundation.

5

Supported by National Health and Medical Research Council Grant 1052963.

6

Supported by Australian Cancer Research Foundation and by the Walter and Eliza Hall Institute.

7

Both authors are co-senior authors.