Src activates retrograde membrane traffic through phosphorylation of GBF1

Abstract

The Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP Exchange factor (GEF) GBF1 and small GTPase Arf1. Here we show that Src induces the formation of tubular transport carriers containing GALNTs. The kinase phosphorylates GBF1 on 10 tyrosine residues; two of them, Y876 and Y898 are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes GBF1 binding to the GTPase; molecular modeling suggests partial melting of the Sec7 domain and intramolecular rearrangement. GBF1 mutants defective for these rearrangements prevent binding, carrier formation and GALNTs relocation, while phosphomimetic GBF1 mutants induce tubules. In sum, Src promotes GALNTs relocation by promoting GBF1 binding to Arf1. Based on residue conservation, similar regulation of GEF-Arf complexes by tyrosine phosphorylation could be a conserved and wide-spread mechanism.

Endogenous and Exogenous Regulatory Signaling in the Secretory Pathway: Role of Golgi Signaling Molecules in Cancer

“…In normal cells, the interaction of GBF1 with Arf is limited and retrograde traffic rate is moderate. Conversely, in tumor cells GBF1 is phosphorylated upon Src activation, which results in increased affinity for Arf-GDP on Golgi membranes ( Chia et al, 2021 ). These reactions lead to the formation of tubules containing GALNTs and the enzymes’ relocation to the ER, wherein they glycosylate resident and neo-synthesized substrates.…”

“…Hyper-activation of Src causes ER-localization of GALNTs, thus increasing their activity ( Gill et al, 2010 ). Recently, Src has been shown to increase the rate of GALNT retrograde traffic through the phosphorylation of GBF1, thus regulating the GBF1-Arf1 interaction ( Chia et al, 2021 ). GBF1 acts as GTP exchange factor (GEF) in the early cis -Golgi and ER-Golgi intermediate compartments (ERGIC) to regulate retrograde traffic from the Golgi to the ER ( Kawamoto et al, 2002 ; Zhao et al, 2002 ; Zhao et al, 2006 ).…”

Critical Determinants in ER-Golgi Trafficking of Enzymes Involved in Glycosylation

“…The direct binding with GRASP55 promoted the correct subcellular localization of GCS by preventing GCS from entering in the retrograde transportation [ 115 ]. The GTP exchange factor GBF1 facilitated the phosphorylation of Arf1-GDP, and the Src tyrosine kinase (Src) played an essential role in the ARF GTP formation [ 116 ]. Src phosphorylated the Y876 and Y898 in the GEF domain C-terminus of GBF1, further increasing the binding between GBF1 and Arf1 and the GALNT relocation [ 116 ].…”

“…The GTP exchange factor GBF1 facilitated the phosphorylation of Arf1-GDP, and the Src tyrosine kinase (Src) played an essential role in the ARF GTP formation [ 116 ]. Src phosphorylated the Y876 and Y898 in the GEF domain C-terminus of GBF1, further increasing the binding between GBF1 and Arf1 and the GALNT relocation [ 116 ].…”

Role of the Mosaic Cisternal Maturation Machinery in Glycan Synthesis and Oncogenesis

“…Once recruited, Arf1 recruits COPI coatomer by binding directly to the β- and γ-COP subunits ( Zhao et al, 1997 ; Zhao et al, 1999 ). Studies have demonstrated that growth factor mediated activation of the well-known oncogene Src kinase phosphorylates GBF1 which activates it to promote Arf1 mediated COPI recruitment and enhanced retrograde transport from Golgi to ER ( Chia et al, 2021 ). This enhanced transport promotes the translocation of GALNT1 and GALNT2 to the ER via COPI recycling vesicles.…”