ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration

Joanne Chia,Frederic A Bard,Emilie Anne Bard-Chapeau,Keit Min Tham,David James Gill

doi:10.7554/elife.01828

Joanne Chia, Frederic A Bard + Show 3 more

Open Access

https://doi.org/10.7554/elife.01828

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Abstract

ER O-glycosylation can be induced through relocalisation GalNAc-Transferases from the Golgi. This process markedly stimulates cell migration and is constitutively activated in more than 60% of breast carcinomas. How this activation is achieved remains unclear. Here, we screened 948 signalling genes using RNAi and imaging. We identified 12 negative regulators of O-glycosylation that all control GalNAc-T sub-cellular localisation. ERK8, an atypical MAPK with high basal kinase activity, is a strong hit and is partially localised at the Golgi. Its inhibition induces the relocation of GalNAc-Ts, but not of KDEL receptors, revealing the existence of two separate COPI-dependent pathways. ERK8 down-regulation, in turn, activates cell motility. In human breast and lung carcinomas, ERK8 expression is reduced while ER O-glycosylation initiation is hyperactivated. In sum, ERK8 appears as a constitutive brake on GalNAc-T relocalisation, and the loss of its expression could drive cancer aggressivity through increased cell motility. DOI: http://dx.doi.org/10.7554/eLife.01828.001.

Highlights

GalNAc-type O-linked glycans are polysaccharides present on secreted and membrane-inserted proteins (Tran and Ten Hagen, 2013)
Earlier work demonstrated that carcinomas stain prominently with antibodies and lectins such as the Helix Pomatia Lectin (HPL), which binds to Tn antigens (Springer, 1983)
In a pilot screen using HeLa cells, which targeted 63 known players of membrane traffic (Chia et al, 2012), we identified that knockdown of the SNARE gene Syntaxin 5 (STX5) reproducibly induced a 6–7-fold increase in Tn levels relative to a non-targeting (NT) control (GFP siRNA) (Figure 1A)

Summary

Introduction

GalNAc-type O-linked glycans are polysaccharides present on secreted and membrane-inserted proteins (Tran and Ten Hagen, 2013). Associated with mucin-like proteins, recent advances in mass spectrometric analysis have revealed O-glycosylation on hundreds of different proteins (Steentoft et al, 2013). Recent results have highlighted specific functional roles for O-glycans; for instance, in regulating the secretion of the phosphatemia regulator FGF23 (Kato et al, 2006) and in processing of the angiopoietin-like factor ANGPTL2 (Schjoldager et al, 2010). O-glycans are synthesised through the step-wise action of various glycosylation enzymes, starting with the UDP-N-Acetyl-Alpha-D-Galactosamine:Polypeptide N-Acetyl-galactosaminyltransferases (GalNAc-Ts), a large family of 20 different isoforms that catalyses the addition of N-Acetylgalactosamine (GalNAc) onto serine or threonine residues (Bennett et al, 2012). The high prevalence and specificity of this cancer glycophenotype is remarkable, with matching normal tissues and benign tumours expressing much lower levels

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