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.

Figure 1

H89 blocks hypotonically induced redistribution of GM130 to the ER. HeLa cells grown to 50% confluence on 12-mm glass coverslips were placed in 1 ml of normal medium (A), normal medium containing 50 μM H89 (B), hypotonic (210 mOsm) medium (C), or hypotonic medium containing 50 μM H89 (D) for 20 min at 37°C.

Figure 2

H89 blocks hypotonically induced GPP130 tubules. HeLa cells grown to 50% confluence on 12-mm glass coverslips were placed in 1 ml of normal medium (A), normal medium containing 50 μM H89 (B), hypotonic (210 mOsm) medium (C), or hypotonic medium containing 50 μM H89 (D) for 10 min at 37°C.

Figure 3

H89 blocks both BFA- and nocodazole-induced redistribution of GPP130. HeLa cells grown to 50% confluence on 12-mm glass coverslips were incubated in 2.5 μg/ml BFA in the absence (A) or presence (B) of 50 μM H89 for 20 min at 37°C, or incubated in 10 μg/ml nocodazole in the absence (C) or presence (D) of 50 μM H89 for 60 min at 37°C.

Figure 4

H89 dose response for inhibition of hypotonically (A), BFA (B)-, and nocodazole (C)-induced redistribution. Cells were treated with hypotonic medium (210 mOsm), 2.5 μg/ml BFA, or 10 μg/ml nocodazole in the presence of 0, 25, 50, and 100 μM H89 or 120 μM H8. Hypotonically treated cells were stained with GM130 antibodies and BFA- and nocodazole-treated cells were stained with GPP130 antibodies. The averaged results of two independent experiments are presented.

Figure 5

H89 does not block hypotonically induced ERGIC 53 redistribution to the ER, and H89 alone induces the redistribution of ERGIC 53 to the ER. HeLa cells grown to 50% confluence on 12-mm glass coverslips were placed in 1 ml of normal medium (A), hypotonic (210 mOsm) medium (B), hypotonic medium containing 50 μM H89 (C), or normal medium containing 50 μm H89 (D) at 37°C for 20 min.

Figure 6

H89 treatment induces, and does not block the constitutive recycling of GM130 to the ER (A and B), but does block the nocodazole-induced redistribution of GM130 (C and D). HeLa cells grown to 50% confluence on 12-mm glass coverslips were incubated in the absence (A) or presence (B) of 50 μm H89 for 120 min. In C and D, cells were incubated in the presence of 10 μg/ml nocodazole for 60 min (C), or in the presence of 50 μM H89 and 10 μg/ml nocodazole for 60 min (D).

Figure 7

H89 treatment leads to displacement of Sec13 from peripheral ER exit sites to a soluble cytosolic pool. HeLa cells grown to 50% confluence on 12-mm glass coverslips were incubated in the absence (A) or presence (B) of 50 μm H89 for 10 min, fixed, and stained with antibodies against Sec13. In C and D, HeLa cells stably transfected with HA-tagged Sec13 were grown to 50% confluence and incubated in the absence (C) or presence (D) of 50 μM H89 for 10 min, fixed, and stained with antibodies against the HA epitope. (E) Immunoblot of HASec13-expressing HeLa cells after digitonin extraction under the conditions indicated. H89-treated cells were preincubated for 10 min with 50 μM H89 prior to extraction in 50 μM H89. GTPγS was present at 1 mM where indicated. See MATERIALS AND METHODS for further details.

Figure 8

H89, but not H8, inhibits Sec13 recruitment. HeLa cells grown to 50% confluence on 12-mm glass coverslips were permeabilized in 30 μg/ml digitonin, washed, and incubated on ice as described in MATERIALS AND METHODS. After 20 min, cells were incubated in 50 μl of cytosol (A), buffer (B), cytosol + 50 μm H89 (C), or cytosol + 120 μM H8 (D) for 30 min at 32°C. All incubations included an ATP-regenerating system + 0.5 mM GTPγS. (E) Quantitation of A, C, and D. See MATERIALS AND METHODS for method of quantitation.