A Regulatory Role for cAMP-dependent Protein Kinase in Protein Traffic along the Exocytic Route

doi:10.1074/jbc.271.48.30935

A Regulatory Role for cAMP-dependent Protein Kinase in Protein Traffic along the Exocytic Route*

From the Department of Cell Biology, Faculty of Biology, University of Seville, 41012-Seville, Spain

^∥ To whom correspondence should be addressed:
Dept. of Cell Biology, Faculty of Biology, University of Seville, Avd. Reina Mercedes s/n, 41012-Sevilla, Spain.
Tel.: 34-54557044; Fax: 34-54610261.

↵^‡ The first two authors contributed equally.

Abstract

The influence of protein kinase A activity on transport of newly synthesized vesicular stomatitis virus G glycoprotein along the exocytic pathway was examined. Transport of vesicular stomatitis virus G glycoprotein to the cell surface was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of protein kinase A. This block occurred at the exit of the Golgi complex, whereas transport through the Golgi compartments or from the endoplasmic reticulum to the Golgi was decreased in the presence of H-89. As judged by immunofluorescence endoplasmic reticulum to Golgi transport was accelerated in cells incubated with activators of protein kinase A such as isobutylmethylxanthine (IBMX) or forskolin (FK). Treatment with IBMX and FK also increased transport from the trans-Golgi network to the cell surface. During incubation with IBMX and FK, the organization of the Golgi complex was altered showing intercisternae fusion and miscompartmentalization of resident proteins. These structural changes affected both the kinetics of acquisition of endoglycosidase H resistance and transport activities. These data support a differential regulatory role for protein kinase A in different transport steps along the exocytic pathway. In particular, transport from the trans-Golgi network to the cell surface was dependent on protein kinase A activity. In addition, the results suggest the involvement of this enzyme on the maintenance of the Golgi complex organization.

Footnotes

↵^§ Recipient of a predoctoral fellowship from UNICAJA/Junta de Andalucia.
↵^¶ Supported by a predoctoral fellowship from Agencia Española de Cooperación Internacional.
↵* This work was supported in part by grant PB92-0674 from the Spanish Dirección General de Investigación Cientifica y Técnica and grant 93/0824 from Fondo de Investigaciones Sanitarias. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TGN

trans-Golgi network

ER

endoplasmic reticulum

VSV

vesicular stomatitis virus

VSV-G

VSV G glycoprotein

IBMX

3-isobutyl-1-methylxanthine

FK

forskolin

dibutyryl-cAMP

N⁶,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate

8-Br-cAMP

8-bromoadenosine-3′,5′-cyclic monophosphate

H-89

N-[2-(-p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide

Sp-cBIMPs

5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate Sp isomer

endo H

endoglycosidase H

NRK

normal rat kidney

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline.
↵² M. Alonso, M. Muñiz, J. Hidalgo, and A. Velasco, unpublished observations.
- Received May 10, 1996.
- Revision received July 22, 1996.