Thesis Title: : Genetics of Golgi apparatus regulation in mammalian cells
Protein glycosylation modifies many different proteins and thus, controls numerous biological processes. While it is known that glycans are variable in different physiological settings, their regulatory mechanisms remain poorly understood. Most glycans are synthesized in a series of sequential biosynthetic reaction in the Golgi apparatus and hence, depend intimately on its compartmentalized organization. To investigate the regulatory mechanisms of Golgi organization, I have performed image-based RNAi screen against kinases and phosphatases and identified 159 genes whose depletion strongly affected Golgi morphology. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades control glycosylation through Golgi remodeling. Collectively, these results provide a genetic overview of the signaling pathways that control Golgi organization and functions in human cells. I focused on the regulation of O-GalNAc glycosylation initiation where 12 negative regulators were identified to control the subcellular localization of glycosyltransferases GalNAc-Ts. Among these, atypical MAPK ERK8 is a potent regulator and is partially localized at the Golgi. Its inhibition induces COPI-dependent GalNAc-T traffic from Golgi to the endoplasmic reticulum (ER), but not of KDEL receptors, revealing the existence of different COPI transport pathways. ERK8 was also found to negatively regulate cell motility which is dependent on O-glycosylation. In human breast and lung carcinomas, ERK8 expression is reduced while ER localised 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.
1. Chia J., Tham K.M., Gill D.J., Bard-Chapeau E., Bard F. (2014)
ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration.
Elife. 2014 3:e01828.
2. Chia J., Goh G., Racine V., Ng S., Kumar P., Bard F. (2012)
RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells.
Mol Syst Biol. 2012; 8:629
Figure legend: A model illustrating signaling regulation of O-glycosylation initiation at the Golgi. 12 negative regulators control the subcellular localisation of GalNAc-Ts. In particular, ERK8 specifically inhibits the formation of COPI carriers containing GalNAc-Ts, and not KDEL receptors, that traffic from the Golgi to ER. This process, in turn, influences cell motility and has potential impact on cancer invasiveness.
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