Protein kinase A activity is required for the budding of constitutive transport vesicles from the trans-Golgi network

Abstract

We have examined the role played by protein kinase A (PKA) in vesicle-mediated protein transport from the trans-Golgi network (TGN) to the cell surface. In vivo this transport step was inhibited by inhibitors of PKA catalytic subunits (C-PKA) such as the compound known as H89 and a myristoylated form of the inhibitory peptide sequence contained in the thermostable PKA inhibitor. Inhibition by H89 occurred at an early stage during the transfer of vesicular stomatitis virus G glycoprotein from the TGN to the cell surface. Reversal from this inhibition correlated with a transient increase in the number of free coated vesicles in the Golgi area. Vesicle budding fromthe TGN was studied in vitro using vesicular stomatitis virusinfected, permeabilized cells. Addition to this assay of C-PKA stimulated vesicle release while it was suppressed by PKA inhibitory peptide, H89, and antibody against C-PKA. Furthermore, vesicle release was decreased when PKA-depleted cytosol was used and restored by addition of C-PKA. These results indicate a regulatory role for PKA activity in the production of constitutive transport vesicles from the TGN