Regulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) and BIG2 activity via PKA and protein phosphatase 1γ

Abstract

Brefeldin A-inhibited guanine nucleotide-exchange proteins (GEPs) BIG1 and BIG2 activate ADP-ribosylation factor (ARF) GTPases, which are required for vesicular trafficking. Both molecules contain one or more sites for binding protein kinase A, i.e., A kinase-anchoring protein (AKAP) sequences. Elevation of cell cAMP caused PKA-catalyzed phosphorylation and nuclear accumulation of BIG1 but not BIG2. We then asked whether BIG1 phosphorylation altered its GEP activity. Incubation of BIG1 or BIG2 with PKA catalytic subunits and ATP resulted in retardation of their electrophoretic migration, consistent with PKA phosphorylation. Okadaic acid inhibits many protein phosphatases, including protein phosphatase 1 (PP1) and PP2A, that can reverse PKA-catalyzed phosphorylation. Incubation of HepG2 cells with okadaic acid caused concentration-dependent accumulation of presumably phosphorylated BIG1 and BIG2 with decreased mobility, which was increased by subsequent incubation in vitro with specific recombinant phosphatases, PP1γ > PP2A ≫ PP1α. For assays of GEP activity, BIG1 and BIG2 were immunoprecipitated from cells that had been depleted, respectively, of BIG2 and BIG1 by using specific siRNA. GEP activity of each was significantly decreased after incubation with recombinant PKA plus ATP and restored by incubation with PP1γ. In agreement with a role for PP1γ in regulation of BIG, endogenous PP1γ, but not PP1α or β, was immunoprecipitated with BIG1 or BIG2 from microsomal fractions. All observations are consistent with the effects of BIG1 and BIG2 phosphorylation on vesicular trafficking, via alterations in ARF activation and regulatory roles for cAMP, PKA, and PP1γ in ARF activation by BIG1 and BIG2.

Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and effect on ARF1 activity

“…However, active ARF1-GTP bound to GST-GGA3 was decreased 44 Ϯ 5% (n ϭ 3; P Ͻ 0.05) from that in untreated cells (Fig. 7B), consistent with the inhibitory effect of phosphorylation of BIG1 and BIG2 on ARF1 activation that had been demonstrated in vitro (15). ARF1-GTP was decreased even more by 2 other PDE3A siRNAs that decreased PDE3A content by Ͼ90% (Fig.…”

“…Because of demonstrated AKAP characteristics of these GEP molecules with their functions in ARF activation and vesicular trafficking at different intracellular sites, we expected to find a cAMP phosphodiesterase (PDE) component that would terminate the cAMP signal to parallel PP1␥ reversal of PKA phosphorylation (15)(16)(17). We report here the co-IP of PDE3A with BIG1 or BIG2 from HeLa cell cytosol, which contains also isoform PDE3B.…”

“…Differences between mechanisms, and perhaps functions, of BIG1 accumulation in nuclei after cAMP elevation and after serum deprivation remain to be determined. In vitro experiments with BIG1 and BIG2 immunoprecipitated from HepG2 cells that had been selectively depleted, respectively, of BIG2 or BIG1, showed that phosphorylation catalyzed by recombinant PKA catalytic unit decreased GEP activity with recombinant ARF1-GST; activity was restored by incubation with recombinant protein phosphatase PP1␥, which dephosphorylated BIG1 and BIG2 (15).…”

“…PKAcatalyzed phosphorylation of BIG1 and BIG2 in vitro decreased their GEP activities (15). Depletion of PDE3A was expected to result in elevation of cAMP content of domains in which AKAP-tethered PDE3A regulates cAMP concentration.…”

cAMP‐dependent protein kinase A and the dynamics of epithelial cell surface domains: Moving membranes to keep in shape

“…Elevation of cAMP caused PKA-catalyzed phosphorylation of the BIGs and, in an in vitro assay, recombinant PKA altered their GEP activity (71) . The involvement of PKA in Golgi membrane dynamics was recently supported by the notion that the cell-wide downregulation of PKA-RIIα subunits by siRNA results in severe perturbation of Golgi morphology (72) .…”