Effect of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) in HepG2 cell nuclei Carmen Citterio*, Heather D. Jones, Gustavo Pacheco-Rodriguez, Aminul Islam, Joel Moss, and Martha Vaughan* Pulmonary–Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Martha Vaughan, December 12, 2005
ADP-ribosylation factor 兩 protein trafficking 兩 A kinase-anchoring protein
M
ammalian ADP-ribosylation factors (ARFs) comprise six 20-kDa GTP-binding proteins with essential regulatory roles in the formation of membrane trafficking vesicles. These vesicles bud from a donor membrane and fuse with a target membrane to deliver cargo molecules (1–3). Conversion of ARF from a cytosolic GDP-bound inactive state to a GTP-bound active form that is tightly membrane-associated is accelerated by guanine nucleotide-exchange factors (GEFs) (4). All known ARF GEFs contain a Sec7 domain of ⬇200 aa that catalyzes ARF activation (5–7). The large-molecular-weight BIG1, BIG2, and GBF1, which localize in Golgi regions, are, with different sensitivities, inhibited by the fungal fatty acid metabolite brefeldin A (8–10). The ⬇200-kDa BIG1 and ⬇190-kDa BIG2 were first purified together from bovine brain cytosol on the basis of their BFA-inhibited GEF activities (11) and appeared to exist as parts of the same macromolecular complex(es) in HepG2 cells (12). When these cells were growing in medium with serum, BIG1 was primarily cytosolic and Golgi-associated. After overnight incubation without serum, a large fraction of endogenous BIG1 was found in the nuclei localized with nucleoporin p62 at the nuclear envelope (probably in transit between nucleus and cytoplasm), as well as in the nuclear matrix and nucleoli (13). The cAMP-dependent protein kinase A (PKA) is a tetrameric protein comprising two catalytic (C) and two regulatory (R) subunits (14). Four isoforms of PKA are designated by their specific R subunits: RI␣ and RI in type I and RII␣ and RII in type II. The holoenzyme is usually tethered by means of www.pnas.org兾cgi兾doi兾10.1073兾pnas.0510571103
interaction of the R dimer with an A kinase-anchoring protein (AKAP) that serves as a scaffold for assembly of the kinase, substrates, and other enzymes, which act in concert to coordinate and localize effects of cAMP in cells (15–17). AKAP domain structures have been reported in diverse proteins, including BIG1 and BIG2 (18). Consistent with an AKAP function for BIG1, it was coimmunoprecipitated with RI␣ and RII␣ as well as with C subunits, from HepG2 cytosol (18). PKA activity has been implicated in several transport pathways (19) and was reported to play a regulatory role in ARF1 recruitment from cytosol to intracellular membranes, perhaps by phosphorylating proteins in the Golgi membrane that serve as binding sites for ARF1 (20). Activation of PKA also altered the subcellular localization of other proteins, increasing their presence in either cytoplasm or nucleus (21, 22). We report here that, in cells incubated with 8-Br-cAMP, BIG1 was redistributed from membrane to nuclear fractions in a process that was blocked by PKA inhibitors. BIG1 accumulation in the nucleus was also specifically blocked by mutation of the putative PKA phosphorylation site or the nuclear localization signal (NLS) in the Sec7 domain of BIG1, consistent with its dependence on both PKA-catalyzed phosphorylation and the predicted NLS. Results Effect 8-Br-cAMP on BIG1 and RI␣ Subcellular Localization. HepG2
cells were incubated overnight (16 h) without serum before experiments. To quantify the distribution of BIG1 and RI␣ in HepG2 cells, we analyzed cell fractions by Western blotting with densitometry. We had found that, during incubation of cells with 8-Br-cAMP, amounts of BIG1 decreased in the membrane fraction to ⬇40% of the zero time level after 20–30 min, at the same time increasing to approximately twice the initial level in nuclei (data not shown). Effects of cAMP at concentrations of 1–1,000 M were compared in 20-min incubations. Statistically significant effects of 1 mM cAMP confirmed those seen in time-course experiments (data not shown). After exposure to cAMP, amounts of BIG1 in the cytosol were unchanged, whereas BIG1 was significantly decreased in membrane fractions and concomitantly increased in nuclear fractions (Fig. 1A). RI␣ was present in all subcellular fractions with no statistically significant change after cAMP treatment (Fig. 1 A). BIG2 was not detected in the nuclear fraction, and cAMP did not alter its distribution (Fig. 1 A). On confocal immunofluorescence microscopy, endogenous BIG1 and RI␣ were seen throughout the cells with partial Conflict of interest statement: No conflicts declared. Abbreviations: AKAP, A kinase-anchoring protein; BIG1, brefeldin A-inhibited guanine nucleotide-exchange protein 1; siRNA, small interfering RNA; HA, hemagglutinin; NLS, nuclear localization signal; ARF, ADP-ribosylation factor; PKA, protein kinase A; wt, wild type. *To whom correspondence may be addressed at: Building 10, Room 5N-307, National Institutes of Health, Bethesda, MD 20892-1434. E-mail:
[email protected] or
[email protected].
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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兾 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.
Fig. 1. Effect of 8-Br-cAMP on intracellular distribution of BIG1 and RI␣. (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 g from cytosol, 30 g from membranes, and 20 g from nuclei were separated by SDS兾PAGE before Western blotting with antibodies against BIG1, RI␣, 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 RI␣ (red) and inspected by confocal laser-scanning microscopy. (Scale bar: 20 m.) (C) Samples of proteins from cytosol (Cy, 200 g) and nuclei (Nu, 400 g) of cells incubated for 20 min without or with 8-Br-cAMP (Nu*), in 500 l of TKMS buffer (13) were incubated with 4 g of rabbit IgG (IgG) or anti-BIG1 antibodies overnight at 4°C. Samples (40 l) of supernatant (S) or of immunoprecipitated proteins (P) eluted from washed beads in 40 l of gel-loading buffer and 50 g (2.5%) of total homogenate proteins (Ly) were separated and reacted with antibodies against RI␣, BIG2, or BIG1. Data were similar in three experiments.
colocalization most evident in nuclei (Fig. 1B). After incubation of cells with cAMP for 20 min, BIG1 was clearly more concentrated in the nuclei, consistent with the increase quantified in Fig. 1 A. RI␣ distribution seemed somewhat different, although the two proteins still appeared partially colocalized in nuclei (Fig. 1B). RI␣ immunoprecipitated with BIG1 from the nuclear fraction both before and after cAMP treatment (Fig. 1C). The amount of anti-BIG1 antibodies that completely precipitated BIG1 from nuclei of untreated cells failed to immunoprecipitate 100% of the larger amount of BIG1 in cAMP-treated cells and also failed to immunoprecipitate all of the RI␣. Antibodies against BIG1 immunoprecipitated BIG2 from cytosolic but not nuclear fractions (Fig. 1C), consistent with failure to detect BIG2 in nuclei microscopically or on Western blotting. 2684 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0510571103
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兾or 1 mM 8-Br-cGMP, 100 M H-89, or 10 M PKI before SDS兾PAGE separation of proteins from membrane (30 g) and nuclear (20 g) fractions and reaction with antibodies against BIG1 or RI␣. Data are means ⫾ SD of values from three experiments, blots from one of which are shown.
Effect of PKA Inhibition on 8-Br-cAMP-Induced BIG1 Translocation. To assess PKA-catalyzed phosphorylation involvement in BIG1 redistribution, effects of PKA inhibitors H-89 and PKI on BIG1 distribution in cells incubated with cAMP were investigated. BIG1 distribution was not altered by H-89 or PKI alone, but each inhibitor significantly decreased the effects of cAMP on BIG1 content of membranes and nuclei (Fig. 2). 8-Br-cGMP did not mimic the effects of cAMP on BIG1 content of membranes and nuclei, consistent with a role for PKA in the trafficking of this protein. RI␣ distribution was not altered by PKA inhibitors or by 8-Br-cGMP treatment (Fig. 2), which likewise had no effects on amounts of BIG2 and RI␣ in cytosol (data not shown). BIG1 Distribution in Cells Incubated with BIG2 Small Interfering RNA (siRNA). Because BIG1 and BIG2 had appeared to exist in the
same macromolecular complex(es) in HepG2 cytosol (12), we investigated the effect on BIG1 distribution of ‘‘knocking down’’ BIG2 with siRNA. Incubation of HepG2 cells for 72 h with BIG2 siRNA markedly decreased BIG2 protein levels (Fig. 3A) and Citterio et al.
resulted in the virtual disappearance of BIG2 staining in cells (Fig. 3C). There were, however, no changes in amounts or distribution of BIG1 and RI␣ proteins (Fig. 3A). Similarly, effects of cAMP on BIG1 distribution were not altered in cells transfected with BIG2 siRNA (Fig. 3B), nor was the appearance of BIG1 in nuclei of cAMP-treated cells (Fig. 3C). Effect of Nocodazole on BIG1 Distribution. Transport by means of
microtubules has been implicated in the delivery of proteins through the cytoplasm to nuclear pores (23). We investigated the effect of microtubule disruption by nocodazole on the nuclear accumulation of BIG1. After incubation of cells for 60 min with nocodazole, BIG1 in cytosol was significantly increased, and that in the membrane fraction decreased, whereas it had disappeared totally from nuclei (Fig. 4A). Addition of cAMP during the last 20 min of incubation with nocodazole had no effect on BIG1 Citterio et al.
distribution, which was thus higher in cytosol than it was in cells exposed to cAMP in the absence of nocodazole and much lower (i.e., not detectable) in nuclei (Fig. 4A). Confocal immunofluorescence microscopy revealed BIG1 partially colocalized with ␣-tubulin in cytoplasm especially in the perinuclear region (Fig. 4B). Colocalization was less in cells incubated with cAMP, where BIG1 was accumulated in nuclei with the remaining cytoplasmic BIG1 still partially associated with ␣-tubulin, but in a different pattern (Fig. 4B). In cells treated with nocodazole for 1 h, the microtubule network was disrupted, no BIG1 was seen in nuclei, and effects of cAMP on BIG1 localization were apparently completely abolished (Fig. 4B). Nuclear Accumulation of BIG1 by Means of Phosphorylation by PKA.
To assess the role in nuclear accumulation of a potential PKA phosphorylation site in the BIG1 Sec-7 domain, Ser-883 was replaced by alanine or aspartate to generate, respectively, hemagglutinin (HA)-tagged BIG1(S883A) or BIG1(S883D). Localization of overexpressed HA-tagged wild-type (wt) BIG1 resembled that of endogenous BIG1 (Figs. 1B and 4B), but it was perhaps more concentrated in the perinuclear region (Fig. 5A). Punctate collections of HA–BIG1(S883A) were more widely scattered in the cells than wt, whereas HA–BIG1(S883D), which PNAS 兩 February 21, 2006 兩 vol. 103 兩 no. 8 兩 2685
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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 g) of total cell proteins were separated by SDS兾PAGE and reacted with antibodies against BIG2, BIG1, or RI␣. 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 g), membrane (Me, 30 g), and nuclear (Nu, 20 g) 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 m.) Findings were similar in three experiments.
Fig. 4. Effect of nocodazole on BIG1 localization. (A) Cells were incubated (37°C, 60 min) without or with 10 g兾ml nocodazole (Noc) and without or with 1 mM 8-Br-cAMP during the last 20 min before separation of proteins from cytosol (50 g), membrane (30 g), and nuclear (20 g) fractions by SDS兾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 ␣-tubulin (red) and BIG1 (green) and inspected by confocal laser-scanning microscopy. (Scale bar: 8 m.)
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兾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 m.) (B) Cells were fractionated, and samples of proteins (5% of each fraction) were analyzed by Western blotting with HA antibodies. (C) Proteins (50 g) 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.
may resemble wt BIG1 phosphorylated by PKA, was concentrated in nuclei (Fig. 5A). Treatment of cells with cAMP induced nuclear accumulation of wt HA–BIG1 but did not alter the distribution of HA–BIG1(S883A), which remained in the cytosol, or the distribution of HA–BIG1(S883D), which had been concentrated in nuclei in the absence of cAMP (Fig. 5A). No HA–BIG1(S883D) was seen in nuclei of cells after incubation for 1 h with nocodazole (data not shown), consistent with the requirement for microtubule function in the nuclear accumulation of BIG1. BIG1(NLS), in which the NLS was mutated, was not seen in nuclei of cells exposed to cAMP, nor was the double mutant BIG1(S883D兾NLS), as expected if the NLS is required for nuclear localization (Fig. 5A). Distribution of HA–BIG1 wt and mutants was also assessed by Western blotting with anti-HA antibody. wt BIG1 and all of the mutants were present in the cytosol with no apparent change in cells exposed to cAMP (Fig. 5B). After incubation of cells with cAMP, the amount of wt HA–BIG1 in nuclei was clearly increased and that in membranes was perhaps decreased, but cAMP had no effects on amounts of any of the mutant proteins recovered in membrane or nuclear fractions (Fig. 5B). Amounts of BIG1(S883D) in nuclei were similar before and after cAMP treatment (Fig. 5B). Western blotting with BIG1 antibodies of proteins in lysates of HepG2 cells transfected with empty vector or the HA–BIG constructs revealed the overexpressed proteins at similar levels, all greater than the amount of endogenous BIG1 in cells transfected with empty vector (Fig. 5C). Amounts of endogenous BIG1 in cells overexpressing the several constructs were ⬇30% of those in the control (empty vector) cells and ⬍15% of those of the overexpressed proteins (Fig. 5C). Discussion The intracellular trafficking of BIG1 and BIG2 remains incompletely understood, and whether a multiprotein complex is involved in the intracellular actions of these proteins is not clear, although the proteins were initially purified together from bovine brain cytosol in an ⬇670-kDa complex (11). Subse2686 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0510571103
quently, yeast two-hybrid screens and coimmunoprecipitation revealed the association of BIG1 and BIG2 with other proteins, such as PKA regulatory and catalytic subunits (18). As reported here, BIG1 was present in the cytosol, membrane, and nuclear fractions of HepG2 cells and rapidly accumulated in the nuclei when cells were incubated with cAMP; i.e., the intracellular redistribution of BIG1 appeared to reflect changes in cell cAMP concentration and subsequent PKA activation. In fact, when PKA activity was inhibited with relatively specific kinase inhibitors H-89 and PKI, nuclear accumulation of BIG1 was prevented, consistent with a requirement for PKA-catalyzed phosphorylation. BIG2, however, was not detected in nuclei, whether or not BIG1 was present. When cellular cAMP levels are low, PKA exists as holoenzyme, which is too large to enter the nucleus by diffusion (15). Upon binding cAMP, the R dimer dissociates from two active C subunits that then diffuse into the nucleus, where they can phosphorylate cAMP response element-binding proteins, leading to cAMP effects on transcription (15). Structures and functions of the R and C subunits are rather well understood (14), and an additional level of regulation is established by means of interaction of the R dimer with an A kinase-anchoring protein (AKAP) that tethers PKA and other proteins with which it functions as an integrated molecular machine (16). A variety of such multimolecular complexes comprise different assortments of protein components assembled on specialized AKAP scaffolds at specific subcellular localizations (17). Most AKAPs had initially been thought to interact exclusively with RII subunits, but PKA can also be tethered via RI␣ and RI (17). Three short sequences in the N-terminal region of the BIG2 molecule were identified as AKAP domains that bind different PKA R subunits (18). In a yeast two-hybrid screen and then with coimmunoprecipitation of in vitro translated, epitopetagged proteins, Li et al. (18) demonstrated the interaction of RI␣ with BIG2. They also reported that antibodies against RI␣ precipitated BIG1 and BIG2 from HepG2 cytosol; RI␣ was precipitated by antibodies against BIG2 or BIG1 (18). Here, we have shown coimmunoprecipitation of BIG1 and RI␣ from Citterio et al.
Citterio et al.
phorylations catalyzed by protein kinase CK2 and the cyclindependent kinase cdc2. Phosphorylation of the CK2 site accelerated NLS-dependent nuclear import, whereas phosphorylation of the cdc2 site adjacent to the NLS inhibited transport and markedly reduced maximal nuclear accumulation (28). A general role for this ‘‘CcN motif’’ in regulating nuclear protein transport was suggested by the similar arrangements of CK2 and cdc2 kinase sites with NLS in several other proteins. To begin to understand how BIG1 is delivered to a nuclear pore for translocation to its intranuclear site(s) and function(s) that remain to be defined, we assessed the involvement of microtubules, which function widely in both endocytotic and exocytotic trafficking pathways. Many animal viruses rely on microtubule-based transport for delivery to the nuclear envelope, where the viral genome is released through the nuclear pores (29, 30). Salman et al. (23) demonstrated integration of the microtubule delivery and nucleocytoplasmic transport systems in an animal cell model, showing that the same NLS responsible for translocation through nuclear pores also invoked active, dyneinmediated transport along microtubules. In intact cells, the microtubule network radiates from the centrosome near the nucleus with plus ends pointing toward the cell periphery. In this orientation, dynein could move NLS-bearing molecules to the nuclear envelope for nuclear import. The absence of nuclear accumulation of BIG1 in cells after depolymerization of microtubules by nocodazole is consistent with their involvement in the delivery of BIG1 for nuclear import. BIG1 was present, along with BIG2 and exocyst proteins, in microtubules purified from HepG2 cells by taxol polymerization (31). It seems most probable that the interactions of BIG1 and BIG2 with microtubules are independent and quite different, both structurally and functionally. Materials and Methods HepG2 human liver carcinoma cells were purchased from American Type Culture Collection; penicillin, streptomycin, 8-bromocAMP, 8-bromo-cGMP, and nocodazole were purchased from Sigma; H-89 (catalog no. EI-196) and PKI (catalog no. P-203) were purchased from Biomol; and goat and horse sera were purchased from Vector Laboratories. Antibodies. Preparation and purification of antibodies against
BIG1 and BIG2 were reported in refs. 12 and 31. Mouse antibody against ␣-tubulin was purchased from Sigma, chicken polyclonal anti-RI␣ antibody was purchased from Biomol, and polyclonal antibody against HA was purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Amersham Pharmacia, and anti-chicken IgY was purchased from Promega. Cell Culture. HepG2 cells were grown and incubated, unless
otherwise indicated, on collagen I-coated 10-cm dishes (Becton Dickinson) at 37°C, 5% CO2兾95% air in DMEM (GIBCO) with 10% FBS (GIBCO), penicillin (100 units兾ml), and streptomycin (100 g兾ml). Before experiments, cells were incubated overnight (16 h) in the same medium without FBS. Cell Fractionation, Western Blotting, and Immunoprecipitation. Pro-
teins from HepG2 cell fractions prepared as described in ref. 13 were separated by SDS兾PAGE in 10-well 4–12% gels (Invitrogen) and transferred to nitrocellulose membranes, which were divided for reaction with anti-BIG1 (0.5 g兾ml), anti-RI␣ (5 g兾ml), or anti-BIG2 (1:200). Secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG, were detected with SuperSignal chemiluminescent substrate (Pierce). Densitometry was performed by using a ChemiImager 5500 (Alpha Innotech, San Leandro, CA). Immunoprecipitation is described in the Fig. 1 legend. PNAS 兩 February 21, 2006 兩 vol. 103 兩 no. 8 兩 2687
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HepG2 cell nuclei. This interaction was not dependent on BIG2, which was not detected in the nuclei. The BIG1–RI␣ interaction is consistent with the reported presence in BIG1 of an AKAP sequence identical to one in BIG2 that interacted with both RI and RII subunits in yeast two-hybrid experiments (18). Proteins larger than 45 kDa require a nuclear localization sequence (NLS) for entry into the nucleus (15). Nuclear importation of proteins is a two-step process involving the dimeric importin-␣兾, in which the ␣-subunit directly binds the NLS motif and serves as an adaptor for importin-. NLS–importin-␣ complexes interact with nuclear pore complexes by means of importin- and are translocated into the nucleus in an energydependent process (19, 24). BIG1 was described in HepG2 cells colocalized, in part, with nucleoporin p62 at the nuclear envelope, perhaps in transit between nuclear and cytoplasmic compartments (13). The NLS in a protein destined for nuclear localization contains a unipartite, or a bipartite, basic amino acid cluster, such as KKKRK in SV40 large tumor antigen (25) or RKR-Xn-RKRKR in T cell protein tyrosine phosphatase (26), which is recognized by an importin-␣兾 heterodimer. Protein phosphorylation in the vicinity of the NLS is reported to play a major role in modulating NLS-dependent nuclear import and can facilitate NLS recognition by the NLS-binding importin-␣ subunit (23, 27, 28). Protein kinases, including PKA, regulate the subcellular localization of a number of proteins. Phosphorylation of S312 in the dorsal protein of Drosophila by PKA increased its affinity for importin-␣ and was accompanied by enhanced nuclear accumulation (21). The contribution of a negative charge to that effect was suggested by the observation that replacement of Ser-312 with Glu lowered the affinity slightly less than did the Ala substitution (21). BIG1 is a protein of 1,849 aa with a predicted NLS sequence 711KKPKR715, which belongs to the same class of monopartite NLS modules present in SV40 T antigen. The BIG1 sequence 880KKIS883 was identified as a potential PKAphosphorylation site, C-terminal to the NLS sequence. Mutagenesis of this site in BIG1 showed that the PKA-catalyzed phosphorylation of Ser-883 was necessary for nuclear accumulation of BIG1 in response to 8-Br-cAMP. The mutant in which Ala replaced Ser-883, BIG1(S883A), was not present in nuclei after cAMP stimulation, whereas the S883D mutant, in which Asp may resemble a phosphorylated Ser, was present in nuclei whether or not cells were treated with cAMP. The effect of Ser-883 replacement by Asp is consistent with a mechanistic importance for the negative charge at that site, as suggested earlier (21). Mutation of the NLS in BIG1 resulted in the absence of its nuclear localization with or without cAMP treatment. The double-mutant S883D兾NLS, not surprisingly, also failed to accumulate in nuclei after incubation with cAMP. PKAcatalyzed phosphorylation of Ser-883 was necessary for nuclear translocation, but not sufficient, because the presence of a functional NLS was required. The phosphorylated S883 presumably represents a signal in addition to, or recognized in concert with, the NLS, which is specifically recognized by the nuclear transport apparatus. We note that BIG2, which has not yet been found in nuclei, contains a NLS corresponding to that in BIG1. BIG2 appears to lack a PKA substrate site (10), but perhaps phosphorylation by another kinase(s) will result in its nuclear accumulation. The NLS is, clearly, not a sole determinant of nuclear localization. Phosphorylation sites together with the NLS constitute regulatory modules for nuclear localization, which have been called phosphorylation-regulated NLSs (or prNLSs) (28). Although numerous prNLSs have been identified, the mechanism that underlies prNLS-dependent regulation of nuclear transport is still unclear. One example of a prNLS is the CcN motif of the viral T antigen, transport of which is regulated by dual phos-
Experiments with siRNA. The siRNA for BIG2 was designed
with the S883D primers. All new clones were fully sequenced for verification.
according to the manufacturer’s instructions and synthesized by Dharmacon Research (Lafayette, CO). HepG2 cells were transfected with 100 nM BIG2 siRNA oligonucleotides or control scrambled siRNA or lamin siRNA as a negative control by using DharmaFECT 4 Transfection Reagent (Dharmacon) according to the manufacturer’s directions. Experiments were performed 72 h after transfection with removal of serum for the last 16 h.
Germany) T-28 protocol was used for transfection of 2 ⫻ 106 HepG2 cells with 3 g of HA–BIG1 in 100 l of Nucleofector solution V with incubation in growth medium for 8 h and in medium without serum for 16 h before experiments.
Cloning of pCMV-HA BIG1. A 4,478-bp fragment of BIG1 cDNA,
Confocal Immunofluorescence Microscopy. HepG2 cells (4 ⫻ 104
excised by using an EcoRI site at 1,121 bp and a BglII site in the 3⬘ UTR, was ligated to the mammalian expression vector pCMV-HA (CLONTECH) by means of EcoRI and BglII sites. A 1,464-bp fragment of the 5⬘ terminus of BIG1 was generated by PCR by using a 5⬘ primer that introduced a SfiI restriction site in-frame with the start codon for BIG1. Digestion of the PCR product and the pCMV-HA-partial BIG1 clone with SfiI and EcoRI yielded fragments that were ligated by means of the SfII and EcoRI sites to generate a complete pCMV-HA–BIG1 clone. This clone was fully sequenced to verify correct frame and sequence, matching the curated BIG1 sequence in the National Center for Biotechnology Information database (accession no. NM㛭006421). Generation of pCMV-HA BIG1 Mutations in PKA-Phosphorylation Site and NLS. BIG1 sequence was analyzed by using the Protein
Analysis Toolkit (32). Amino acids 880–883 (KKIS) were identified as a putative PKA phosphorylation site, and amino acids 711–715 (KKPKR) were identified as two overlapping potential NLS (KKPK and KPKR). The Stratagene QuikChange II XL Site-Directed Mutagenesis kit and appropriate primers were used to generate the following mutations of pCMV-HA BIG1 in separate reactions: serine to alanine (S883A); serine to aspartate (S883D); and 712KPK714 to alanine–alanine–alanine (NLS). The double-mutant S883D兾NLS was generated by performing a second round of site-directed mutagenesis on the NLS mutant 1. Bonifacino, J. S. & Lippincott-Schwartz, J. (2003) Nat. Rev. Mol. Cell Biol. 4, 409–414. 2. Moss, J. & Vaughan, M. (1998) J. Biol. Chem. 273, 21431–21434. 3. Chavrier, P. & Goud, B. (1999) Curr. Opin. Cell Biol. 11, 466–475. 4. Jackson, C. L. & Casanova, J. E. (2000) Trends Cell Biol. 10, 60–67. 5. Franzusoff, A. & Schekman, R. (1989) EMBO J. 8, 2695–2702. 6. Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C. L. & Chabre, M. (1996) Nature 384, 481–484. 7. Donaldson, J. G. & Jackson, C. L. (2000) Curr. Opin. Cell Biol. 12, 475–482. 8. Morinaga, N., Moss, J. & Vaughan, M. (1997) Proc. Natl. Acad. Sci. USA 94, 12926–12931. 9. Claude, A., Zhao, B.-P., Kuziemsky, C. E., Dahan, S., Berger, S. J., Yan, J.-P., Armold, A. D., Sullivan, E. M. & Melancon, P. (1999) J. Cell Biol. 146, 71–84. 10. Togawa, A., Morinaga, N., Ogasawara, J., Moss, J. & Vaughan, M. (1997) J. Biol. Chem. 274, 12308–12315. 11. Morinaga, N., Tsai, S.-C., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 12856–12860. 12. Yamaji, R., Adamik, R., Takeda, K., Togawa, A., Pacheco-Rodriguez, G., Ferrans, V. J., Moss, J. & Vaughan, M. (1999) Proc. Natl. Acad. Sci. USA 97, 2567–2572. 13. Padilla, P. I., Pacheco-Rodriguez, G., Moss, J. & Vaughan, M. (2004) Proc. Natl. Acad. Sci. USA 101, 2752–2757. 14. Taylor, S. S., Buechler, J. A. & Yonemoto, W. (1990) Annu. Rev. Biochem. 59, 971–1005. 15. Wen, W., Meinkoth, J. L., Tsien, R. Y. & Taylor, S. S. (1995) Cell 82, 463–473. 16. Dell’Acqua, M. L. & Scott, J. D. (1997) J. Biol. Chem. 272, 12881–12884. 17. Wong, W. & Scott, J. D. (2004) Nat. Rev. Mol. Cell Biol. 5, 959–970.
2688 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0510571103
Transfection. The Nucleofector (Amaxa Biosystem, Cologne,
cells per well) grown on two-well, collagen-coated culture slides (Becton Dickinson) for 24 h in growth medium were incubated overnight (16 h) in the same medium without FBS, treated as described in the figure legends, washed three times with PBSCM (PBS containing 1 mM CaCl2 and 1 mM MgCl2), and fixed for 20 min at room temperature in 4% paraformaldehyde (Electron Microscopy Services, Washington, PA) in PBSCM. After washing three times with PBSCM, cells were permeabilized with 0.05% Triton X-100 in PBSCM for 3 min, washed three times with PBSCM, incubated for 1 h in blocking buffer (PBSCM containing 6% BSA, 5% goat serum, and 5% horse serum) at room temperature, then overnight (4°C) in blocking buffer with primary antibody (1:100 anti-BIG1, 1:50 anti-RI␣, 1:100 antiBIG2, 1:500 anti-␣-tubulin, or 1:100 anti-HA), and washed three times with PBSCM before incubation for 2 h at room temperature with secondary antibodies, Alexa Fluor 488 goat antirabbit IgG or Alexa Fluor 568 goat anti-mouse IgG (Molecular Probes) in PBSCM. Cells were washed three times with PBSCM, mounting medium with 4⬘,6-diamidino-2-phenylindole dihydrochloride (Vectashield; Vector Laboratories) was added, and coverslips were sealed with nail polish (Electron Microscopy Sciences). Images were collected by using a Leica SP laser scanning confocal microscope. We thank Dr. Philip I. Padilla for helpful technical assistance and Michael Spencer for his help with the artwork. This work was supported by the Intramural Research Program of the National Institutes of Health, National Heart, Lung, and Blood Institute. 18. Li, H., Adamik, R., Pacheco-Rodriguez, G., Moss, J. & Vaughan, M. (2003) Proc. Natl. Acad. Sci. USA 100, 1627–1632. 19. Jans, D. A. & Hu ¨bner, S. (1996) Physiol. Rev. 76, 651–685. 20. Martin, M. E., Hidalgo, J., Rosa, J. L., Crottet, P. & Velasco, A. (2000) J. Biol. Chem. 275, 19050–19059. 21. Briggs, L. J., Stein, D., Goltz, J., Corrigan, V. C., Efthvmiadis, A., Heubner, S. & Jans, D. A. (1998) J. Biol. Chem. 273, 22745–22752. 22. Go ¨rner, W., Durschlag, E., Martinez-Pastor, M. T., Estruch, F., Ammerer, G., Hamilton, B., Ruis, H. & Schueller, C. (1998) Genes Dev. 12, 589–597. 23. Salman, H., Abu-Arish, A., Oliel, S., Loyter, A., Klafter, J., Granek, R. & Elbaum, M. (2005) Biophys. J. 89, 2134–2145. 24. Nigg, E. A. (1997) Nature 386, 779–787. 25. Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. (1984) Cell 39, 499–509. 26. Tiganis, T., Flint, A. J., Adam, S. A. & Tonks, N. K. (1997) J. Biol. Chem. 272, 21548–21557. 27. Hennekes, H., Peter, M., Weber, K. & Nigg, E. A. (1993) J. Cell Biol. 120, 1293–1304. 28. Hu ¨bner, S., Xiao, C.-Y. & Jans, D. A. (1997) J. Biol. Chem. 272, 17191–17195. 29. Suikkanen, S., Aaltonen, T., Nevalainen, M., Va ¨lilehto, O., Lindholm, L., Vuento, M. & Vihinen-Ranta, M. (2003) J. Virol. 77, 10270–10279. 30. Kelkar, S. A., Pfister, K. K., Crystal, R. G. & Leopold, P. L. (2004) J. Virol. 78, 10122–10132. 31. Xu, K.-F., Shen, X., Li, H., Pacheco-Rodriguez, G., Moss, J. & Vaughan, M. (2005) Proc. Natl. Acad. Sci. USA 102, 2784–2789. 32. Gracy, J. & Chiche, L. (2005) Nucleic Acids Res. 33, W65–W71.
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