Potential Role for Protein Kinases in Regulation of Bidirectional Endoplasmic Reticulum-to-Golgi Transport Revealed by Protein Kinase Inhibitor H89

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 betaCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but betaCOP 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.

A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis

“…1C). These results indicate that H89 treatment inhibits mitotic Golgi breakdown, as it does BFA-induced Golgi breakdown (10), and this occurs without preventing cells from entering and progressing through mitosis.…”

“…Golgi disassembly in BFA-treated, interphase cells can be blocked by the small molecule H89 (10). Given this effect, we asked whether H89 could also block the disassembly of the Golgi in mitosis.…”

“…Recently, Golgi disassembly caused by Arf1 inactivation in BFA-treated cells was shown to be blocked by pretreatment of cells with the small molecule H89, an inhibitor of PKA, and other kinases (10). This finding prompted us in this study to investigate whether H89 could also inhibit mitotic Golgi disassembly and, if it could, to determine whether this inhibition was mediated through effects on Arf1.…”

Bap31 Is an Itinerant Protein That Moves between the Peripheral Endoplasmic Reticulum (ER) and a Juxtanuclear Compartment Related to ER-associated Degradation

“…To explore this possibility, we investigated whether chemicals that perturb microtubule-dependent membrane transport from the ER affect Bap31 localization. H89 blocks protein export out of the ER (Jamora et al, 1999) perhaps by mildly inhibiting the binding of COPII proteins to ER exit sites (Aridor and Balch, 2000; Lee and Linstedt, 2000), where COPII-coated vesicles involved in ER-to-Golgi transport are formed (Gurkan et al, 2006). It also blocks Golgi disassembly occurring during mitosis, likely by keeping Arf1, a small GTPase that regulates the recruitment of COPI to membranes (LippincottSchwartz and Liu, 2006), in an active state (Altan-Bonnet et al, 2003).…”

“…We could not test H89 at concentrations higher than 100 M because COS-7 cells were detached from coverslips, even those coated with poly-l-lysine. It seemed that the effect of H89 on COPII localization is weaker in COS-7 cells than HeLa cells (Lee and Linstedt, 2000;Puri and Linstedt, 2003;Jiang et al, 2006). Next, we examined whether Bap31, upon H89 treatment, moves to the juxtanuclear region through ER exit sites, as in the case of secretory cargos.…”