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. 2006 Feb 21;103(8):2683-8.
doi: 10.1073/pnas.0510571103. Epub 2006 Feb 8.

Effect of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) in HepG2 cell nuclei

Carmen Citterio  1 , Heather D Jones, Gustavo Pacheco-Rodriguez, Aminul Islam, Joel Moss, Martha Vaughan
Affiliations

Effect of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) in HepG2 cell nuclei

Carmen Citterio et al. Proc Natl Acad Sci U S A. .

Abstract

Brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are activators of ADP-ribosylation factor GTPases that are essential for regulating vesicular traffic among intracellular organelles. Biochemical analyses and immunofluorescence microscopy demonstrated BIG1 in nuclei as well as membranes and cytosol of serum-starved HepG2 cells. Within 20 min after addition of 8-Br-cAMP, BIG1 accumulated in nuclei, and this effect was blocked by protein kinase A (PKA) inhibitors H-89 and PKI, suggesting a dependence on PKA-catalyzed phosphorylation. BIG2 localization was not altered by cAMP, nor did BIG2 small interfering RNA influence nuclear accumulation of BIG1 induced by cAMP. Mutant BIG1 (S883A) in which Ala replaced Ser-883, a putative PKA phosphorylation site, did not move to the nucleus with cAMP addition, whereas replacement with Asp (S883D) resulted in nuclear accumulation of BIG1 without or with cAMP exposure, consistent with the mechanistic importance of a negative charge at that site. Mutation (712KPK714) of the nuclear localization signal inhibited BIG1 accumulation in nuclei, and PKA-catalyzed phosphorylation of S883, although necessary, was not sufficient for nuclear accumulation, as shown by the double mutation S883D/nuclear localization signal. A role for microtubules in cAMP-induced translocation of BIG1 is inferred from its inhibition by nocodazole. Thus, two more critical elements of BIG1 molecular structure were identified, as well as the potential function of microtubules in a novel PKA effect on BIG1 translocation.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Effect of 8-Br-cAMP on intracellular distribution of BIG1 and RIα. (A) HepG2 cells were incubated (37°C, 20 min) with 1 mM 8-Br-cAMP before fractionation of homogenates. Samples (5%) of proteins from each fraction corresponding to ≈50 μg from cytosol, 30 μg from membranes, and 20 μg from nuclei were separated by SDS/PAGE before Western blotting with antibodies against BIG1, RIα, or BIG2. Data were similar in three experiments. (B) Cells, incubated as in A without (untreated) or with 8-Br-cAMP were reacted with antibodies against BIG1 (green) and RIα (red) and inspected by confocal laser-scanning microscopy. (Scale bar: 20 μm.) (C) Samples of proteins from cytosol (Cy, 200 μg) and nuclei (Nu, 400 μg) of cells incubated for 20 min without or with 8-Br-cAMP (Nu*), in 500 μl of TKMS buffer (13) were incubated with 4 μg of rabbit IgG (IgG) or anti-BIG1 antibodies overnight at 4°C. Samples (40 μl) of supernatant (S) or of immunoprecipitated proteins (P) eluted from washed beads in 40 μl of gel-loading buffer and 50 μg (2.5%) of total homogenate proteins (Ly) were separated and reacted with antibodies against RIα, BIG2, or BIG1. Data were similar in three experiments.
Fig. 2.
Fig. 2.
Effect of PKA inhibition on 8-Br-cAMP-induced translocation of BIG1. Cells were incubated (20 min, 37°C) without or with 1 mM 8-Br-cAMP and/or 1 mM 8-Br-cGMP, 100 μM H-89, or 10 μM PKI before SDS/PAGE separation of proteins from membrane (30 μg) and nuclear (20 μg) fractions and reaction with antibodies against BIG1 or RIα. Data are means ± SD of values from three experiments, blots from one of which are shown.
Fig. 3.
Fig. 3.
Effect of BIG2 siRNA on BIG1 distribution. (A) HepG2 cells were incubated with BIG2 or nonspecific target (C1) or lamin (C2) siRNA or with vehicle (M) for 72 h before samples (50 μg) of total cell proteins were separated by SDS/PAGE and reacted with antibodies against BIG2, BIG1, or RIα. Below are means ± SD of BIG2 values quantified by densitometry from three replicate experiments. (B) HepG2 cells transfected or not with BIG2 siRNA were incubated without or with 1 mM 8-Br-cAMP (20 min, 37°C) before separation of proteins from cytosol (Cy, 50 μg), membrane (Me, 30 μg), and nuclear (Nu, 20 μg) fractions and reaction with antibodies against BIG1. (C) Untreated or Mock (vehicle) or BIG2 siRNA-transfected HepG2 cells were incubated without or with 8-Br-cAMP (∗) as in B before reaction with BIG2 or BIG1 antibodies for confocal immunofluorescence microscopy. (Scale bar: 8 μm.) Findings were similar in three experiments.
Fig. 4.
Fig. 4.
Effect of nocodazole on BIG1 localization. (A) Cells were incubated (37°C, 60 min) without or with 10 μg/ml nocodazole (Noc) and without or with 1 mM 8-Br-cAMP during the last 20 min before separation of proteins from cytosol (50 μg), membrane (30 μg), and nuclear (20 μg) fractions by SDS/PAGE and reaction with antibodies against BIG1. Data are means ± SD of values from three experiments, blots from one of which are above. (B) Cells treated as in A were reacted with antibodies against α-tubulin (red) and BIG1 (green) and inspected by confocal laser-scanning microscopy. (Scale bar: 8 μm.)
Fig. 5.
Fig. 5.
Overexpression of BIG1 and mutants in HepG2 cells. (A) HepG2 cells, 24 h after transfection with HA-tagged BIG1 wt, S883A, S883D, NLS mutant, or S883D/NLS double mutant were incubated without or with 8-Br-cAMP for 20 min before processing for immunofluorescence with anti-HA antibodies (green). (Scale bar: 8 μm.) (B) Cells were fractionated, and samples of proteins (5% of each fraction) were analyzed by Western blotting with HA antibodies. (C) Proteins (50 μg) from total lysates of HepG2 cells transfected with empty vector (EV) or HA BIG1 wt and mutants were analyzed by Western blotting with BIG1 antibodies to detect both endogenous and overexpressed BIG1.
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