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Comparative Study
. 2007 Feb 27;104(9):3201-6.
doi: 10.1073/pnas.0611696104. Epub 2007 Feb 21.

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

Affiliations
Comparative Study

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

Fuminobu Kuroda et al. Proc Natl Acad Sci U S A. .

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, PP1gamma > PP2A >> PP1alpha. 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 PP1gamma. In agreement with a role for PP1gamma in regulation of BIG, endogenous PP1gamma, but not PP1alpha or beta, 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 PP1gamma in ARF activation by BIG1 and BIG2.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
In vitro phosphorylation of endogenous BIG1 or BIG2 by PKA. Samples of proteins precipitated from postnuclear supernatants with antibodies against BIG1 (A) or BIG2 (B) were applied directly (NI) to 4% Tris-glycine gel or after incubation at 30°C for 30 min without (C) or with 10 unit of PKA α, β, or γ catalytic subunits and 50 μM ATP in PKA buffer (total volume, 50 μl). Proteins were then separated by SDS/PAGE and reacted with antibodies against BIG1 or BIG2. Distance (millimeter) between the center of each immunoreactive band and the center of 150-kDa protein marker was measured on prints from Fuji Image Gauge software (version 4.0). Data are means ± SD of values from three experiments. ∗, P < 0.01 vs. NI; ∗∗, P < 0.01.
Fig. 2.
Fig. 2.
Effect of phosphatase inhibitors on phosphorylation of BIG1 and BIG2 in cells. Samples (10 μg) of total proteins are from cells that had been incubated for 3 h at 37°C without (C) or with the indicated concentrations of OA (A), CA (B), 100 nM cyclosporin A (CsA) or 500 nM OA (C), and 1 mM sodium orthovanadate (OV) or 500 nM OA (D). Data are reported as in Fig. 1.
Fig. 3.
Fig. 3.
In vitro dephosphorylation of endogenously phosphorylated BIG1 and BIG2. Phosphorylated BIG1 (A) or BIG2 (B), immunoprecipitated from cells that had been incubated with 500 nM OA for 3 h, were incubated at 30°C for 30 min without (NI) or with the indicated amount of PP1α, PP1γ, or PP2A in a total volume of 50 μl before measurement of electrophoretic migration, as in Fig. 1. Data (Right) are means ± SD of values from three experiments, with representative blots from one experiment (Left).
Fig. 4.
Fig. 4.
Effects of OA or H89 on localization of BIG1 and BIG2 in HepG2 cell fractions. Cells were incubated for 3 h at 37°C without (C) or with 500 nM OA or 100 μM H89 before homogenization. Samples (20 μg) of proteins from whole homogenate, cytosol, membrane, and crude nuclear fractions were separated by SDS/PAGE in 4% or 4–12% gel before immunoblotting, as indicated, with antibodies against BIG1 and BIG2 or α-tubulin, Golgi 58K, and histone H1. Means ± SD of densitometric values from three experiments quantified by Fuji Image Gauge version 4.0 and expressed relative to that for C in the same experiment set equal to 100 are shown with blots from a representative experiment. ∗, P < 0.01 vs. untreated cells.
Fig. 5.
Fig. 5.
Effect of OA or H89 on localization of BIG1 and BIG2 in HepG2 cells by confocal immunofluorescence microscopy. Cells were incubated for 75 min at 37°C without (C) or with 500 nM OA or 100 μM H89 before staining of immunoreactive BIG1 (green) or BIG2 (green) and GM130 (red) and visualization by confocal laser-scanning microscopy. Data were similar in two other experiments. (Scale bar, 20 μm.)
Fig. 6.
Fig. 6.
Effect of OA treatment of cells on GEP activity of BIG1 or BIG2. Immunoprecipitated BIG1 (B1) or BIG2 (B2) from cells that had been depleted, respectively, of BIG2 or BIG1 followed by incubation for 3 h without (N) or with 500 nM OA, was assayed for GEP activity with incubation at 37°C for 1 h. Activities were calculated as picomoles of [35S]GTPγS (after correction for binding by beads with nonimmune IgG immunoprecipitate from untreated cells that were incubated without ARF) divided by densitometric value for BIG1 or BIG2 in the immunoprecipitate assayed. Means ± SD of values from three experiments (Left) were calculated after activity of each of the OA-treated samples was expressed as a percentage of its control (N = 100). Representative blots from one experiment (Right). ∗∗, P < 0.05 vs. without OA.
Fig. 7.
Fig. 7.
Effect of BIG1 or BIG2 phosphorylation on GEP activity. Experiments were performed as in Fig. 6, but starting with 15 × 105 cells for each homogenate and 3-fold amounts of all materials, because 95% of each immunoprecipitate from 5 × 105 cells was used for GEP assay. Of the three samples (1, 2, 3) from each preparation of immunoprecipitated BIG1 (B1) or BIG2 (B2), one was incubated in 100 μl of TENDS buffer alone (N) and two with 20 units of PKA Cγ in 100 μl of PKA buffer for 30 min before washing with TENDS (samples 1 and 2) or with PP1 buffer (sample 3), followed by incubation (30 min) in 100 μl of TENDS (samples 1 and 2) or PP1 buffer and 4 units of PP1γ (sample 3), washing with TENDS buffer, and assay of GEP activity (Fig. 6). Means ± SD of values from three experiments (Left) are shown with representative blots from one experiment (Right). ∗, P < 0.01 vs. TENDS buffer alone; ∗∗, P < 0.01 vs. PKA.
Fig. 8.
Fig. 8.
Coimmunoprecipitation of endogenous protein phosphatases from HepG2 cells. HepG2 cell cytosol (A) and microsome (B) fractions were incubated with BIG1 (B1), BIG2 (B2), or normal rabbit IgG (N). Precipitated proteins (IP), 10 μg of cytosol (C), or microsomal fraction (M) were separated by SDS/PAGE before immunoblotting (IB) with indicated antibodies.

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