Src activates retrograde membrane traffic through phosphorylation of GBF1.
Joanne Chia1, Shyi-Chyi Wang1,2, Sheena Wee1
1Institute of Molecular and Cell Biology, Singapore, Singapore.
Elife
|December 6, 2021
Summary
Src tyrosine kinase controls cancer-critical protein glycosylation by relocating GALNTs enzymes. It phosphorylates GBF1, promoting its binding to Arf1, which drives this essential transport event.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Src tyrosine kinase regulates protein glycosylation, a process critical for cancer progression.
- The relocation of GALNTs (GalNAc-transferases) from the Golgi to the ER is essential for this glycosylation control.
- The underlying mechanism by which Src induces this specific Golgi-to-ER trafficking remains elusive.
Purpose of the Study:
- To elucidate the molecular mechanism by which Src tyrosine kinase mediates the relocation of GALNTs enzymes.
- To investigate the role of the Guanine nucleotide Exchange Factor (GEF) GBF1 and small GTPase Arf1 in Src-induced protein trafficking.
Main Methods:
- Utilized Src kinase activity assays and phosphoproteomic analysis to identify phosphorylation sites on GBF1.
- Employed molecular modeling to predict the structural impact of phosphorylation on GBF1-Arf1 interactions.
- Generated and tested GBF1 mutants (defective and phosphomimetic) to assess their role in transport carrier formation and GALNTs relocation.
Main Results:
- Src induces the formation of tubular transport carriers containing GALNTs enzymes.
- Src phosphorylates GBF1 on 10 tyrosine residues, including Y876 and Y898 within the Sec7 GEF domain.
- Phosphorylation at Y876 and Y898 promotes GBF1 binding to Arf1, leading to carrier formation and GALNTs relocation.
- Mutant and phosphomimetic GBF1 analyses confirmed the functional importance of these phosphorylation-dependent rearrangements.
Conclusions:
- Src tyrosine kinase promotes GALNTs relocation by enhancing the binding of GBF1 to Arf1 through specific tyrosine phosphorylations.
- This phosphorylation-dependent regulation of GEF-GTPase interaction represents a potential conserved mechanism for controlling intracellular transport.
- Understanding this pathway offers new insights into the regulation of protein glycosylation in cancer.
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