Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2)

Yeast Two-Hybrid System.

Yeast strains EGY48 [MATα, his3, trp1, ura3, LexAop (x6)-LEU2] and YM4271 (MATa, ura3–52, his3–200, lys2–801, ade2–101, ade5, trp1–901, leu2–3, 112, tyr1–501, gal4-Δ512, gal80-Δ538, ade5∷hisG), MATCHMAKER vectors, and the lacZ reporter plasmid for the LexA system were purchased from CLONTECH and used according to the manufacturer's instructions in published procedures (16).

Antibodies.

Mouse monoclonal antibodies against RIα, RIIα, and C subunits were purchased from BD Signal Transduction (Lexington, KY), chicken polyclonal anti-RIα antibody from Biomol (Plymouth Meeting, PA), horseradish peroxidase-conjugated anti-rabbit IgG, anti-mouse IgG, and anti-chicken IgY from Promega, normal mouse and rabbit IgG from Vector Laboratories, monoclonal LexA and c-myc antibodies from CLONTECH, polyclonal anti-c-myc and anti-hemagglutinin (HA) antibodies from Santa Cruz Biotechnology, and anti-FLAG antibodies from Sigma. Polyclonal antibodies against BIG1 and BIG2 are described in ref. 6.

Cloning of BIG2 Fragments.

Three fragments of BIG2 (see Fig. 1A) were amplified from plasmid pBluescript-BIG2full (provided by Ryoichi Yamaji, see ref. 6) with the following primers (restriction enzyme sites italicized). Forward primer 1 has an EcoRI site (5′-GCGGAATTCATGCAGGAGAGCCAG), reverse primers 2 and 3 have XhoI sites (5′-CGCCTCGAGCTATTGTTGCTTGATG and 5′-CGCCTCGAGCTATGTTTCTTTCATTG), and forward primer 4 has an EcoRI site (5′-GCGGAATTCCAAAAAGAAATCATTGAAC). Primers 1 and 2 were used to prepare constructs encoding BIG2 amino acids 1–643, primers 1 and 3 for 1–832, and primers 4 and 3 for 644–832 (Sec 7 domain). Initiation and termination codons are underlined. PCR products were excised with EcoRI plus XhoI and ligated into pGilda. LexA-BIG2 constructs were sequenced and transformed into the yeast EGY48 to determine that fusion proteins were translocated into the nucleus and had no intrinsic transactivation capability. Stable expression of LexA fusion proteins was verified by Western blotting.

cDNA Library Screening for BIG2 Interactions.

Library plasmids were extracted, purified (Qiagen, Chatsworth, CA; mega kit), and transformed into yeast EGY48, which had been transformed with the bait plasmids and the lacZ reporter plasmid (p8op-lacZ). Yeast were grown on SD/-His/-Trp/-Ura glucose plates and selected on SD/-His/-Trp/-Ura/-Leu galactose and raffinose plates. Approximately 5 × 10⁷ transformants were screened for interactions evidenced by growth in leucine-free medium and β-galactosidase activity that were dependent on galactose. Specificity of the interaction was confirmed by using as bait cDNAs encoding human lamin C and murine p53. Plasmids were isolated from the positive colonies, inserts amplified by PCR, and PCR products characterized by digestion with AluI, HaeIII, EcoRI, and XhoI to sort 51 positive colonies from p2A-13-1 into seven unique clones; 48 positive colonies from p3-9-2 represented five clones. Positive AD/library plasmids were rescued in KC8 Escherichia coli.

DNA Sequencing and Analysis.

Purified positive AD/library plasmids were initially sequenced by automated sequencing (373 DNA sequenator, Applied Biosystems) with pB42AD sequencing primer 5′-CAGCCTCTTGCTGAGTGGAGATGCC to obtain the 5′ regions of the inserts. Multiple primers located inside of the inserts were used to complete insert sequences. Sequence data were analyzed using GENEWORKS 2.5.1 and compared with nucleotide sequence databases (all GenBank, RefSeq Nucleotides, EMBL, DDBJ, and PDB) by using BLAST (www.ncbi.nlm.nih.gov/BLAST/). p2-1-1 encoded full-length human PKA RIα (NM_002734) and p3-24-2 amino acids 1–59.

Protein Synthesis in Reticulocyte Lysate.

Positive library inserts were excised from pB42AD with EcoRI plus XhoI and inserted into plasmid pGADT7 (CLONTECH) to add an N-terminal HA tag. Bait inserts were excised with EcoRI plus SalI and inserted into the plasmid pGBKT7 (CLONTECH) to add an N-terminal c-myc tag. T7-coupled reticulocyte lysate system (TNT, Promega) was used to synthesize [³⁵S]methionine-labeled bait and library proteins according to the manufacturer's instructions. Samples (5 μl) of in vitro translated bait and prey proteins, combined as indicated, were incubated at 30°C for 1 h before the addition of 470 μl of immunoprecipitation (IP) buffer (20 mM Tris⋅HCl, pH 7.5/150 mM NaCl/1 mM DTT/0.5 mM phenylmethylsulfonyl fluoride/0.1% Tween 20/aprotinin, 5 μg/ml) containing 10 μl (1 μg) of anti-c-myc mAb, anti-HA IgG, or anti-FLAG IgG and 10 μl of protein G-agarose beads. After rocking for 2 h at 4°C, beads were washed three times with 500 μl of TBST buffer (20 mM Tris⋅HCl, pH 7.5/150 mM NaCl/0.1% Tween 20). Proteins eluted in 10 μl of sample buffer were separated by SDS/PAGE (8–16% or 10–20% gel). Dried gels were exposed to x-ray film.

Cloning of PKA Regulatory Subunits.

cDNAs for R subunits were amplified by PCR from a human heart cDNA library (CLONTECH) using for RIα (NM_002734), forward primer 5′-GCGGGATCCATGGAGTCTGGCAGTAC with a BamHI site and reverse primer 5′-CGCCTCGAGTCAGACAGACAGTGACAC with a XhoI site; and for RIIβ (M31158), forward primer 5′-GCGGAATTCATGAGCATCGAGATCCCG with an EcoRI site and reverse primer 5′-CGCCCATGGTCATGCAGTGGGTTCAAC with a NcoI site. RIβ (M65066) and RIIα (X14968) were amplified from a human testis cDNA library (CLONTECH) with forward primers (5′-GCGGAATTCATGGCCTCCCCGCC and 5′-GCGGGATCCCCCAACCCGTCTATC) and reverse primers with XhoI sites (5′-CGCCTCGAGTCAGACGGTGAGGGAG and 5′-CGCCTCGAGCACAGCAATGGCAGCAG), respectively. PCR products were excised with appropriate enzymes and inserted into vector pGilda to be transformed into yeast YM4271. BIG2 (1–832), excised from pGilda with EcoRI and XhoI, was ligated to pB42AD to produce pB3-9-2 and transformed into EGY48[p8op-lacZ] for mating experiments (see Fig. 4A).

For the experiment in Fig. 4B, PCR with pGilda-RIα, -RIβ, -RIIα, and -RIIβ templates was used to construct pGADT7 plasmids with the corresponding inserts. Forward primers with NdeI sites (5′-GCGCATATGGAGTCTGGCAGTACCGCC, 5′-GCGCATATGGCCTCCCCGCCCGCCTGC, and 5′-GCGCATATGAGCCACATCCAGATCCCG) and reverse primers with XhoI sites (5′-CGCCTCGAGTCAGACAGACAGTGACAC, 5′-CGCCTCGAGTCAGACGGTGAGGGAG, and 5′-CGCCTCGAGCTACTGCCCGAGGTTGC) were used for RIα, RIβ, and RIIα, respectively. For RIIβ, forward primer 5′-GCGGAATTCATGAGCATCGAGATCCCG with an EcoRI site and reverse primer 5′-CGCATCGATTCATGCAGTGGGTTCAAC with a ClaI site were used.

Immunoprecipitation and Western Blot.

To prepare cytosol, confluent HepG2 cells, grown on dishes coated with type I collagen in DMEM (Invitrogen) containing 25 mM Hepes, with 1 mM sodium pyruvate, 10% FBS, penicillin G (100 units/ml), and streptomycin (100 μg/ml) at 37°C in an atmosphere of 5% CO₂/95% air, were harvested by scraping in cold PBS. Cells from ≈40 dishes were homogenized in 5 ml of TENDS buffer (20 mM Tris·HCl, pH 8.0/1 mM EDTA/1 mM NaN₃/2 mM DTT/0.25 M sucrose), 8 ml per dish, with 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 0.5 mM benzamidine hydrochloride, and protease inhibitors (soybean and lima bean trypsin inhibitors, leupeptin, and aprotinin, each 1 μg/ml). The homogenate (30 strokes, glass Dounce tissue grinder) was centrifuged (105,000 × g for 1.5 h) to yield cytosol. Samples of cytosol (1.2 mg of protein) were incubated (rocking, 4°C, 4 h) with 5 μg of affinity-purified anti-BIG1, 6 μg of anti-BIG2 IgG, or 5 μg of monoclonal antibodies against RIα, RIIα, or C subunit in TENDS buffer with 2 μM MgCl₂, 0.1% ovalbumin, and 0.5 mM AEBSF (total volume 0.5 ml). Controls were normal rabbit or mouse IgG or no antibody. After adding 60 μl of a 50% slurry of protein G-Sepharose CL-4B (Amersham Pharmacia) in PBS and incubation at 4°C overnight, beads were washed three times with 1 ml of ice-cold PBS containing 0.5 mM AEBSF. Proteins bound to beads were eluted in 80 μl of loading buffer at 65°C for 10 min, separated by SDS/PAGE in 4–12% gradient or 8% gels, and transferred to nitrocellulose membrane for incubation with rabbit anti-BIG1 or anti-BIG2 IgG, chicken anti-RIα IgY, or mAb against RIIα or C subunit, followed by appropriate horseradish peroxidase-conjugated second antibodies and detection using Super Signal Chemiluminescent substrate (Pierce).

After determination of the amount of anti-BIG2 IgG or RIα mAb needed for complete IP, samples (50 μg of protein) of HepG2 cytosol were incubated (4°C, 4 h, rocking) with 2 μg of anti-BIG2 IgG or 1.5 μg of anti-RIα mAb in TENDS buffer plus 2 μM MgCl₂, 0.5 mM AEBSF, and 0.1% ovalbumin (total volume 250 μl), followed by addition of 40 μl of protein G-Sepharose CL-4B (50% slurry in PBS) and incubation overnight. After centrifugation, the supernatant was subjected to a second IP in the same medium with 2 μg of anti-BIG2 IgG or 1.5 μg of anti-RIα mAb (total volume 0.5 ml), followed by incubation overnight at 4°C with 40 μl of protein G-Sepharose CL-4B (50% slurry in PBS). After washing beads three times with 1 ml of ice-cold PBS containing 0.5 mM AEBSF, bound proteins were eluted with 30 μl of SDS sample buffer and separated by SDS/PAGE (8% gel). Immunoreactive proteins were quantified by using a ChemiImager 5500 (Alpha Innotech, San Leandro, CA).

Interaction of BIG2 with RIα in Vitro and in Vivo.

In a yeast two-hybrid screen of a human heart cDNA library (≈5 × 10⁷ transformants), BIG2 constructs (Fig. 1A) encoding amino acids 1–832 (3-9-2) and 1–643 (2A-13-1) each yielded multiple positive clones, two of which were p2-1-1, encoding full-length PKA RIα, and p3-24-2, encoding the first 59 amino acids of RIα (Fig. 1B). The Sec 7 domain (644–832), which is responsible for ARF GEP activity, yielded no RIα clones. Specificity of the interactions was confirmed by mating experiments (Fig. 1C).

Interaction of BIG2 and RIα was also demonstrated by coimmunoprecipitation of [³⁵S]methionine-labeled epitope-tagged proteins synthesized in a reticulocyte lysate system (Fig. 2). myc-BIG2 (1–832) and HA-RIα (1–381) or HA-RIα (1–59) were individually synthesized and mixed as indicated. Proteins immunoprecipitated with anti-myc, anti-HA, or anti-FLAG (control) antibodies and separated by SDS/PAGE demonstrated an interaction of the N-terminal fragment of recombinant RIα (1–59) and BIG2 (1–832).

To investigate the association of endogenous BIG2 and RIα in human cells, proteins precipitated from HepG2 cell cytosol with anti-BIG2, anti-BIG1, or anti-RIα antibodies were separated by SDS/PAGE followed by immunoblotting. Antibodies against RIα precipitated BIG1 as well as BIG2, and RIα was precipitated by both anti-BIG1 and anti-BIG2 antibodies (Fig. 3A). Coimmunoprecipitation of significant fractions of BIG1 and BIG2 from HepG2 cells had already been reported (6), but whether or not they interact directly (or whether RIα interacts directly with BIG1) remains to be determined.

To estimate the fractions of total BIG2 and RIα that were associated, HepG2 cytosolic proteins were immunoprecipitated with anti-BIG2 or anti-RIα antibodies followed by a second immunoprecipitation with the indicated antibodies (Fig. 3B) and Western blot analysis of precipitated proteins. Immunoreactive BIG2 was precipitated by anti-RIα, RIα was precipitated by anti-BIG2, and neither BIG2 nor RIα was detected on second immunoprecipitation with the same antibody (Fig. 3B). In three experiments with densitometric quantification, ≈40% RIα was precipitated along with 100% of BIG2, and antibodies against RIα coprecipitated 30–70% of BIG2.

The association of endogenous BIG2 and C subunit of PKA was demonstrated by immunoprecipitation from HepG2 cell cytosol. Antibody against C subunit precipitated both BIG2 and BIG1 (Fig. 3C).

BIG2 Interaction with RIβ, RIIα, and RIIβ.

In additional immunoprecipitation experiments (Fig. 3C), monoclonal antibodies against RIIα subunit, like those against RIα, precipitated BIG1 and BIG2 from HepG2 cell cytosol. To evaluate potential interactions of BIG2 with different R subunits, yeast mating experiments were carried out with transformants expressing BIG2 (1–832) and those expressing RIα, RIβ, RIIα, or RIIβ (Fig. 4A). All diploid colonies were positive for β-galactosidase activity, and controls were negative. Interactions of BIG2 with each of the R subunits was also observed in experiments like that in Fig. 2, in which [³⁵S]methionine-labeled epitope-tagged R subunits and BIG2 (1–832) synthesized in reticulocyte lysates were mixed and immunoprecipitated (Fig. 4B).

Multiple PKA R Subunit-Binding Sites in BIG2.

To map the R subunit-binding site(s) within the first 643 residues of BIG2, 28 different fragments were expressed in plasmid pB42AD, and the four R subunits were expressed in the BD fusion plasmid pGilda. They were individually transformed, respectively, into yeast YM4271 and EGY48[p8op-lacZ], and interactions were evaluated in mating experiments (Fig. 5). Three R-binding regions were identified in BIG2. Domain A (amino acids 27–48) interacted with RIα and RIβ, domain B (amino acids 284–301) interacted with RIIα and RIIβ, and domain C (amino acids 517–538) interacted with RIα, RIIα, and RIIβ. An AKAP consensus sequence for RII binding contains conserved hydrophobic residues and forms an amphipathic helix (12, 13). Alignment of BIG2 domains A, B, and C with R-binding domains of known AKAPs shows conserved hydrophobic amino acids and helical structures (Fig. 6).

Translocation of BIG2 to Membranes in HepG2 Cells.

BIG2 and BIG1 had been initially localized by confocal immunofluorescence microscopy in Golgi structures and in a punctate distribution throughout the cytosol in resting conditions (6). Incubation of cells with 8-Br-cAMP or forskolin resulted in movement of BIG1 and BIG2 from cytosolic to membrane fractions, whereas clathrin was unchanged; no effect of 8-Br-cGMP was seen (Fig. 7).