Effect of protein kinase A activity on the association of ADP-ribosylation factor 1 to golgi membranes.
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.
Martin
M E
ME
Department of Cell Biology, University of Seville, 41012 Seville, Spain.
Hidalgo
J
J
Rosa
J L
JL
Crottet
P
P
Velasco
A
A
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D020756:Coatomer Protein / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D020756:Coatomer Protein; D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020823:ADP-Ribosylation Factor 1