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Potential Role for Protein Kinases in Regulation of Bidirectional Endoplasmic Reticulum-to-Golgi Transport Revealed by Protein Kinase Inhibitor H89
- Source :
- Molecular Biology of the Cell. 11:2577-2590
- Publication Year :
- 2000
- Publisher :
- American Society for Cell Biology (ASCB), 2000.
-
Abstract
- Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective protein kinase A inhibitor, blocked both ER export and hypo-osmotic-, brefeldin A-, or nocodazole-induced Golgi-to-ER transport. In contrast, H89 did not block the constitutive ER Golgi-intermediate compartment (ERGIC)-to-ER and Golgi-to-ER traffic that underlies redistribution of ERGIC and Golgi proteins into the ER after ER export arrest. Surprisingly, other protein kinase A inhibitors, KT5720 and H8, as well as a set of protein kinase C inhibitors, had no effect on these transport processes. To test whether H89 might act at the level of either the coatomer protein (COP)I or the COPII coat protein complex we examined the localization of βCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but βCOP localization to the Golgi was unaffected. To further investigate the effect of H89 on COPII we developed a COPII recruitment assay with permeabilized cells and found that H89 potently inhibited binding of exogenous Sec13 to ER export sites. This block occurred in the presence of guanosine-5′-O-(3-thio)triphosphate, suggesting that Sec13 recruitment is inhibited at a step independent of the activation of the GTPase Sar1. These results identify a requirement for an H89-sensitive factor(s), potentially a novel protein kinase, in recruitment of COPII to ER export sites, as well as in stimulated but not constitutive Golgi-to-ER transport.
- Subjects :
- Saccharomyces cerevisiae Proteins
medicine.drug_class
Vesicular Transport Proteins
Golgi Apparatus
Biology
Endoplasmic Reticulum
Autoantigens
Article
chemistry.chemical_compound
medicine
Humans
ASK1
Enzyme Inhibitors
Protein kinase A
Protein Kinase Inhibitors
Molecular Biology
COPII
Sulfonamides
Brefeldin A
Membrane Glycoproteins
Nocodazole
Endoplasmic reticulum
Membrane Proteins
Biological Transport
Cell Biology
COPI
Protein kinase inhibitor
Isoquinolines
Phosphoproteins
Protein Kinase A Inhibitor
Cell biology
Nuclear Pore Complex Proteins
Hypotonic Solutions
Microscopy, Fluorescence
chemistry
Guanosine 5'-O-(3-Thiotriphosphate)
Protein Kinases
HeLa Cells
Subjects
Details
- ISSN :
- 19394586 and 10591524
- Volume :
- 11
- Database :
- OpenAIRE
- Journal :
- Molecular Biology of the Cell
- Accession number :
- edsair.doi.dedup.....e4e87f84901cbcdaa6ae88be531e5227