The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.
- MeSH
- 1-fosfatidylinositol-4-kinasa antagonisté a inhibitory chemie metabolismus MeSH
- biologický transport MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- endozomy účinky léků metabolismus MeSH
- Golgiho aparát účinky léků metabolismus MeSH
- HEK293 buňky MeSH
- inhibitory enzymů metabolismus farmakologie MeSH
- konformace proteinů MeSH
- lidé MeSH
- preklinické hodnocení léčiv MeSH
- rychlé screeningové testy * MeSH
- simulace molekulového dockingu MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Membrane traffic at the trans-Golgi network (TGN) is crucial for correctly distributing various membrane proteins to their destination. Polarly localized auxin efflux proteins, including PIN-FORMED1 (PIN1), are dynamically transported between the endosomes and the plasma membrane (PM) in the plant cells. The intracellular trafficking of PIN1 protein is sensitive to the fungal toxin brefeldin A (BFA), which is known to inhibit guanine nucleotide exchange factors for ADP ribosylation factors (ARF GEFs) such as GNOM. However, the molecular details of the BFA-sensitive trafficking pathway have not been fully revealed. In a previous study, we identified an Arabidopsis mutant BFA-visualized endocytic trafficking defective 3 (ben3) which exhibited reduced sensitivity to BFA in terms of BFA-induced intracellular PIN1 agglomeration. Here, we show that BEN3 encodes a member of BIG family ARF GEFs, BIG2. BEN3/BIG2 tagged with fluorescent proteins co-localized with markers for the TGN/early endosome (EE). Inspection of conditionally induced de novo synthesized PIN1 confirmed that its secretion to the PM is BFA sensitive, and established BEN3/BIG2 as a crucial component of this BFA action at the level of the TGN/EE. Furthermore, ben3 mutation alleviated BFA-induced agglomeration of another TGN-localized ARF GEF, BEN1/MIN7. Taken together, our results suggest that BEN3/BIG2 is an ARF GEF component, which confers BFA sensitivity to the TGN/EE in Arabidopsis.
- MeSH
- ADP-ribosylační faktory genetika metabolismus MeSH
- alely MeSH
- Arabidopsis účinky léků metabolismus MeSH
- brefeldin A farmakologie MeSH
- buněčná membrána účinky léků metabolismus MeSH
- endozomy účinky léků metabolismus MeSH
- fenotyp MeSH
- klonování DNA MeSH
- kompartmentace buňky MeSH
- nesmyslný kodon genetika MeSH
- proteiny huseníčku genetika metabolismus MeSH
- semenáček účinky léků růst a vývoj MeSH
- trans-Golgiho síť účinky léků metabolismus MeSH
- transport proteinů účinky léků MeSH
- výměnné faktory guaninnukleotidů metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
