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 ␣-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.

Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation

“…Our structural analysis also reveals a unique dimerization motif with no structural similarity with other known helical bundles. This motif regulates Pragmin self-association in human cells, which is consistent with a previous study showing a role for the pseudo-kinase Nterminal extension in protein dimerization (Liu et al, 2016). Interestingly, the dimerization interface is huge, suggesting high stability in solution.…”

“…We reproducibly obtained more than 82 specific interactors with a mean ratio >4 and found 2 protein kinases that were prominently associated with Pragmin (mean ratio >12) including the Ser/Thr kinases AMPK and the TK CSK ( Supplementary Table S2). Previous Pragmin interactors such as Src (Leroy et al, 2009) and SgK269/PEAK1 (Liu et al, 2016) were however not recovered in our SILAC analysis. The prominent interaction of Pragmin with endogenous AMPK and CSK was next confirmed by co-immunoprecipitation experiments, thus validating our SILAC analysis (Supplementary Fig S4).…”

“…Like Pragmin, protein dimerization may regulate the activity of the associated protein kinase. Finally, the dimerization modules may also induce heterotypic association as experimentally observed for Pragmin and SgK269/PEAK1 (Liu et al, 2016). The level of sequence conservation at the interface within the family would also predict heterotypic association with C19Orf35.…”

Emerging concepts in pseudoenzyme classification, evolution, and signaling

“…Pseudokinase domains of guanylyl cyclase-A and guanylyl cyclase-B regulate activity of the tandem guanylyl cyclase domains (81) Molecular switch Phosphorylation of the MLKL pseudokinase domain triggers exposure of the executioner four-helix bundle domain and cell death (47,72) Protein interaction domain MLKL pseudokinase domain is regulated by binding to the RIPK3 kinase domain and HSP90:Cdc37 co-chaperones (73,82) Scaffold for assembly of signaling complexes TRIB pseudokinases nucleate assembly of a complex between a substrate (such as C/EBP) and the E3 ubiquitin ligase COP1, whose intrasubcellular localization is controlled by Tribbles 1 TRIB1 (74)(75)(76)83) (73,84) SgK223 (Pragmin)/SgK269 (PEAK1) forms higher-order signaling assemblies that include Src-family kinases (73,(84)(85)(86)(87)(88) Fundamental metabolic regulators of isoprenoid lipid production UbiB pseudoenzyme family adopts an (inactive?) atypical protein kinase-like fold found in bacteria, archaea, and eukaryotes; human mitochondrial ADCK3 pseudokinase binds nucleotides such as ADP but can be re-engineered into an ATP-dependent autophosphorylating enzyme; also relevant to the yeast Coq8p ATPase (69,89) Pseudo-histidine kinase Protein interaction domain The first of these mechanisms, for which an increasing number of examples are available in the protein kinase, phosphatase, and ubiquitin signaling literature, all retain clear "enzyme-like" overall folds.…”

The pseudokinases SgK269 and SgK223: A novel oncogenic alliance in human cancer

“…1). 8 Amongst the key conserved motifs that characterise bona fide kinases, the aspartate of the DFG motif that is required for Mg 2+ -ATP binding is substituted by an asparagine residue in SgK269 and SgK223. Additionally, the conserved glycine-rich loop that also contributes to ATP binding is absent ( Fig.…”