Effect of Protein Kinase A Activity on the Association of ADP-ribosylation Factor 1 to Golgi Membranes

Maria Esther Martı́N,Josefina Hidalgo,Jose Luis Rosa,Pascal Crottet,Angel Velasco

doi:10.1074/jbc.275.25.19050

Abstract

The small GTP-binding protein ADP-ribosylation factor 1 (ARF1) is an essential component of the molecular machinery that catalyzes the formation of membrane-bound transport intermediates. By using an in vitro assay that reproduces recruitment of cytosolic proteins onto purified, high salt-washed Golgi membranes, we have analyzed the role of cAMP-dependent protein kinase A (PKA) on ARF1 incorporation. Addition to this assay of either pure catalytic subunits of PKA (C-PKA) or cAMP increased ARF1 binding. By contrast, ARF1 association was inhibited following C-PKA inactivation with either PKA inhibitory peptide or RIIalpha as well as after cytosol depletion of C-PKA. C-PKA also stimulated recruitment and activation of a recombinant form of human ARF1 in the absence of additional cytosolic components. The binding step could be dissociated from the activation reaction and found to be independent of guanine nucleotides and saturable. This step was stimulated by C-PKA in an ATP-dependent manner. Dephosphorylated Golgi membranes exhibited a decreased ability to recruit ARF1, and this effect was reverted by addition of C-PKA. Following an increase in the intracellular level of cAMP, ARF proteins redistributed from cytosol to the perinuclear Golgi region of intact cells. Collectively, the results show that PKA exerts a key regulatory role in the recruitment of ARF1 onto Golgi membranes. In contrast, PKA modulators did not affect recruitment of beta-COP onto Golgi membranes containing prebound ARF1.

Highlights

The small GTP-binding protein known as ADP-ribosylation factor 1 (ARF1)1 is required for the recruitment of both COPI and adaptor coat proteins from cytosol and plays an essential regulatory role in membrane trafficking processes along the endocytic and biosynthetic pathways [1,2,3]
While efficient recruitment of cytosolic ARF1 occurred during incubation of Golgi membranes with the other components, no apparent signal was detected when either membranes or cytosol were omitted from the incubation medium (Fig. 2)
ARF1 incorporation was increased 3– 4-fold following preincubation for 30 min at 37 °C of crude cytosol with 10 –20 ␮M okadaic acid, a specific serine/threonine phosphatase inhibitor (Fig. 2). This suggested the involvement of protein kinase activities in ARF1 recruitment from cytosol

Summary

Introduction

The small GTP-binding protein known as ADP-ribosylation factor 1 (ARF1) is required for the recruitment of both COPI and adaptor coat proteins from cytosol and plays an essential regulatory role in membrane trafficking processes along the endocytic and biosynthetic pathways [1,2,3]. We show in this report that cAMP-dependent protein kinase A (PKA) influences the interaction of ARF1 with the Golgi membranes. PKA activation caused ARF redistribution in intact cells These data indicate that PKA exerts a key regulatory role in ARF1 recruitment from cytosol to intracellular membranes. Such effect explains the influence of PKA activity on protein transport processes along the exocytic (34 –39) and endocytic (34, 39 – 42) pathways and is discussed in the context of evagination of transport intermediates from donor membranes. We propose the existence of protein targets in the Golgi membranes that when phosphorylated by PKA act as high affinity sites for ARF1 binding

Methods

Results

Conclusion