AKAP-Lbc Anchors Protein Kinase A and Nucleates Gα12-selective Rho-mediated Stress Fiber Formation
2001; Elsevier BV; Volume: 276; Issue: 47 Linguagem: Inglês
10.1074/jbc.m106629200
ISSN1083-351X
AutoresDario Diviani, Jacquelyn A. Soderling, John D. Scott,
Tópico(s)Ubiquitin and proteasome pathways
ResumoGuanine nucleotide exchange factors of the Dbl family relay signals from membrane receptors to Rho family GTPases. We now demonstrate that a longer transcript of the Lbc gene encodes a chimeric molecule, which we have called AKAP-Lbc, that functions as an A-kinase-anchoring protein (AKAP) and a Rho-selective guanine nucleotide exchange factor. Expression of AKAP-Lbc in fibroblasts favors the formation of stress fibers in a Rho-dependent manner. Application of lysophosphatidic acid or selective expression of Gα12 enhances cellular AKAP-Lbc activation. Furthermore, biochemical studies indicate that AKAP-Lbc functions as an adaptor protein to selectively couple Gα12 to Rho. Thus, AKAP-Lbc anchors PKA and nucleates the assembly of a Rho-mediated signaling pathway. Guanine nucleotide exchange factors of the Dbl family relay signals from membrane receptors to Rho family GTPases. We now demonstrate that a longer transcript of the Lbc gene encodes a chimeric molecule, which we have called AKAP-Lbc, that functions as an A-kinase-anchoring protein (AKAP) and a Rho-selective guanine nucleotide exchange factor. Expression of AKAP-Lbc in fibroblasts favors the formation of stress fibers in a Rho-dependent manner. Application of lysophosphatidic acid or selective expression of Gα12 enhances cellular AKAP-Lbc activation. Furthermore, biochemical studies indicate that AKAP-Lbc functions as an adaptor protein to selectively couple Gα12 to Rho. Thus, AKAP-Lbc anchors PKA and nucleates the assembly of a Rho-mediated signaling pathway. The transmission of information from the plasma membrane to the actin cytoskeleton is essential to control a variety of dynamic cellular processes including cell shape, motility, and adherence (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar). Critical mediators of these events are the Rho family small molecular weight GTPases, Rho, Rac, and Cdc42, which regulate distinct actin remodeling events. Rho is primarily responsible for the assembly of actin stress fibers and focal adhesions, and Rac controls the formation of lamellipodia, while Cdc42 induces filopodial formation (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar, 2Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1679) Google Scholar). In addition to these effects, Rho has also been implicated in a variety of other critical cellular functions including gene transcription (3Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1197) Google Scholar, 4Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar) and progression through the cell cycle (5Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1058) Google Scholar). Lysophosphatidic acid (LPA) and thrombin are the extracellular ligands that induce Rho signaling events (2Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1679) Google Scholar). Binding of either ligand to distinct classes of cell surface receptors triggers a series of events that mobilize the pertussis toxin-insensitive heterotrimeric G protein subunits Gα12 and Gα13 (6Strathmann M.P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5582-5586Crossref PubMed Scopus (204) Google Scholar, 7Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). The intracellular targets for either Gα subunit are a growing family of guanine nucleotide exchange factors (GEFs) (8Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (888) Google Scholar). These exchange factors dock with activated Gα12 and Gα13 and facilitate GTP loading of Rho. Individual cells express several Rho-GEFs, which activate distinct Rho signaling pathways (9Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar). For example, p115 Rho-GEF interacts with Gα13 through a regulator of G protein signaling (RGS) domain located in the amino-terminal region of the exchange factor (10Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (740) Google Scholar, 11Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar). Likewise, a related module, the Lsc homology domain, governs docking of Gα13 to exchange factors such as PDZ Rho-GEF, KIAA0380, or GTRAP48 (12Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 13Togashi H. Nagata K. Takagishi M. Saitoh N. Inagaki M. J. Biol. Chem. 2000; 275: 29570-29578Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 14Jackson M. Song W. Liu M.Y. Jin L. Dykes-Hoberg M. Lin C.I. Bowers W.J. Federoff H.J. Sternweis P.C. Rothstein J.D. Nature. 2001; 410: 89-93Crossref PubMed Scopus (204) Google Scholar). A universal hallmark of exchange factors that activate GTPases of the Rho family is a conserved region of ∼250 residues that contains a Dbl homology (DH) domain followed by a pleckstrin homology domain (9Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar). The DH domain contains the nucleotide exchange activity, whereas the pleckstrin homology domain is thought to be involved in the subcellular localization of GEFs (2Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1679) Google Scholar). GTPase selectivity is governed by determinants located within the DH domain that discriminate Rho-specific from Rac or Cdc42-specific exchange factors. Several families of Rho-specific exchange factors have been recognized. Members of the Lbc family were originally identified in a screen for transforming genes from human myeloid leukemias (15Toksoz D. Williams D.A. Oncogene. 1994; 9: 621-628PubMed Google Scholar). Onco-Lbc is a 424-residue oncogenic protein with unregulated exchange factor activity that transforms NIH-3T3 cells in a Rho-dependent manner (16Zheng Y. Olson M.F. Hall A. Cerione R.A. Toksoz D. J. Biol. Chem. 1995; 270: 9031-9034Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Subsequently, a proto-oncogenic form has been isolated with a COOH-terminal region that attenuates its transforming potential (17Sterpetti P. Hack A.A. Bashar M.P. Park B. Cheng S.D. Knoll J.H. Urano T. Feig L.A. Toksoz D. Mol. Cell. Biol. 1999; 19: 1334-1345Crossref PubMed Scopus (69) Google Scholar). More recently, a splice variant called Brx has been identified that is specifically expressed in testis and estrogen-sensitive tissues (18Rubino D. Driggers P. Arbit D. Kemp L. Miller B. Coso O. Pagliai K. Gray K. Gutkind S. Segars J. Oncogene. 1998; 16: 2513-2526Crossref PubMed Scopus (71) Google Scholar). Interestingly, Lbc does not possess RGS-like domains, suggesting that different mechanisms might be involved in its activation in response to extracellular signals. In this study, we demonstrate that a novel Lbc splice variant, AKAP-Lbc, is also an A-kinase-anchoring protein (AKAP). AKAPs are a group of functionally related proteins that coordinate cAMP-responsive events at defined subcellular compartments by directing PKA toward preferred substrates (19Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 20Scott J.D. Pawson T. Sci. Am. 2000; 282: 72-79Crossref PubMed Scopus (65) Google Scholar, 21Diviani D. Scott J.D. J. Cell Sci. 2001; 114: 1431-1437Crossref PubMed Google Scholar). Many AKAPs contain distinct binding sites for PKA and other signaling enzymes such as phosphatases (19Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar), phosphodiesterases (22Dodge K.L. Khouangsathiene S. Kapiloff M.S. Mouton R. Hill E.V. Houslay M.D. Langeberg L.K. Scott J.D. EMBO J. 2001; 20: 1921-1930Crossref PubMed Scopus (401) Google Scholar), and other protein kinases (23Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (482) Google Scholar, 24Nauert J.B. Klauck T.M. Langeberg L.K. Scott J.D. Curr. Biol. 1997; 7: 52-62Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 25Takahashi M. Shibata H. Shimakawa M. Miyamoto M. Mukai H. Ono Y. J. Biol. Chem. 1999; 274: 17267-17274Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 26Takahashi M. Mukai H. Oishi K. Isagawa T. Ono Y. J. Biol. Chem. 2000; 275: 34592-34596Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Through modular interactions, multienzyme complexes are assembled at specific sites in the cell to process and integrate various signals. For a decade, a PKA binding fragment called Ht31 has served as the prototype for a structural elucidation of how the PKA holoenzyme interacts with AKAPs (27Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google Scholar, 28Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Crossref PubMed Scopus (181) Google Scholar). We now show that the original Ht31 fragment is part of a larger molecule with Rho-specific guanine nucleotide exchange activity. Cell-based experiments demonstrate that AKAP-Lbc nucleates a Gα12-mediated Rho activation pathway that responds to LPA. A 3045-base pair cDNA clone encoding for an RII-binding protein fragment designated as Ht31 was originally isolated from a human thyroid expression library (29Carr D.W. Hausken Z.E. Fraser I.D.C. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar). The sequence upstream of the Ht31 clone was isolated by rapid amplification of cDNA ends using human heart marathon-adapted cDNA (CLONTECH). To isolate a full-length AKAP-Lbc cDNA, human heart poly(A)+ mRNA was reverse-transcribed using a reverse primer corresponding to nucleotides 4302–4330 of the proto-Lbc cDNA. The resulting cDNA was PCR-amplified using specific primers and subcloned into pCDNA3 or pEGFP. Three independent PCR products encompassing the entire AKAP-Lbc cDNA were sequenced in both directions. DNA probes were radiolabeled with [α-32P]dCTP to a specific activity of 109cpm/μg of DNA. Multiple Tissue Northern blots (CLONTECH) were hybridized using a probe corresponding to nucleotides 1–500 of the AKAP-Lbc cDNA according to the manufacturer's protocol. The A1251P/I1260P and Y2153F mutants of AKAP-Lbc were generated using the QuikChange mutagenesis method (Stratagene). The RhoA, Rac1, and Cdc42 expression constructs were provided by Andrew Thorburn. The G14V and T19N mutations in the NH2 terminus of RhoA were made using the QuikChange mutagenesis method (Stratagene). The FLAG-tagged RhoA, RhoB, and RhoC constructs were generously provided by Melvin Simon. For the construction of the GST fusion protein of the Rho-binding domain (RBD) of rhotekin, the RBD was PCR-amplified from mouse brain cDNA (CLONTECH) and subcloned into pGEX-4T. The expression constructs for the constitutively active mutants of Gα12, Gα13, Gαq, Gα11, Gαi2, and Gαs were obtained form the Guthrie cDNA Resource Center. The dominant negative mutants of Gα12 and Gα13 were generated using the QuikChange mutagenesis method (Stratagene). Antibodies to AKAP-Lbc were generated in rabbits using a recombinant His6-tagged AKAP-Lbc protein fragment (residues 789–1186) as the immunogen. Affinity purification of this antiserum was accomplished using the immunogen immobilized on Affi-Gel 15 resin (Bio-Rad) and following the manufacturer's instructions. AKAP-Lbc serum was used at a 1:200 dilution for immunoprecipitations and at a 1:5000 dilution for immunoblots, whereas affinity-purified AKAP-Lbc antibodies were used at a concentration of 10 μg/ml for immunoprecipitations and 1 μg/ml for immunoblots. The following affinity-purified primary antibodies were used for immunoblotting: rabbit polyclonal antibody to RhoA (200 μg/ml, 1:250 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse monoclonal antibodies to RhoA, Rac1, or Cdc42 (200 μg/ml, 1:250 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to PKA catalytic subunit (clone 5B, 1:1000 dilution; Transduction Laboratories); mouse monoclonal to PKA type II regulatory subunit (1:250 dilution; Transduction Laboratories); mouse monoclonal antibody to the GST tag (1:500 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to the FLAG tag (1:2000 dilution; Sigma); rabbit polyclonal antibody to the GFP tag (1:100 dilution; Invitrogen); rabbit polyclonal antibody to Gα12 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to Gα13 (1:250 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to Gαq (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to Gα11 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to Gαi2 (1:500 dilution; Santa Cruz Biotechnology); and rabbit polyclonal antibody to Gαs (1:500 dilution; Santa Cruz Biotechnology). RhoA, Rac1, and Cdc42 and the Rho-binding domain of rhotekin were expressed as NH2-terminal GST fusion proteins in bacteria (BL21DE3) (30Self A.J. Hall A. Methods Enzymol. 1995; 256: 67-76Crossref PubMed Scopus (115) Google Scholar). Recombinant proteins expressed in bacteria were purified using glutathione-Sepharose (Amersham Pharmacia Biotech). Bacterial extracts containing GST fusion proteins were prepared by centrifugation of bacterial cultures; lysis of pelleted bacteria in 20 mmTris, 50 mm NaCl, 5 mm MgCl2, 0.5% (w/v) Triton X-100, 1 mm benzamidine, 2 μg/ml leupeptin, 2 μg/ml pepstatin; sonication; and centrifugation at 38,000 ×g for 30 min at 4 °C. The supernatant was incubated with glutathione-Sepharose beads (Amersham Pharmacia Biotech) overnight. The resin was washed with 10 bed volumes of lysis buffer and stored at 4 °C. GST fusion proteins were eluted from the resin with 5 mm reduced glutathione for 15 min at room temperature and dialyzed. HEK293 cells were transfected at 50–80% confluence using the LipofectAMINE Plus Reagent kit (Life Technologies, Inc.) with 0.1–6 μg of the various cDNA expression vectors. Cells were incubated with the DNA for 5 h in serum-free Dulbecco's modified Eagle's medium at 37 °C under 5% CO2 and further incubated for 24–72 h in normal growth medium (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1% penicillin, 1% streptomycin) before harvesting. For immunoprecipitation of AKAP-Lbc complexes from heterologous expression systems, cells grown in 100-mm dishes were transfected at 70–80% confluence using 6 μg of AKAP-Lbc cDNA. Cells were harvested and lysed 24 h after transfection in 500 μl of IP buffer (10 mm phosphate, 150 mm NaCl, 5 mmEDTA, 5 mm EGTA, 1 mm benzamidine, 10 μg/ml pepstatin, 10 μg/ml leupeptin, and 1 mm4-(2-aminoethyl)-benzenesulfonyl fluoride) containing 1% Triton X-100 and 0.2% sodium deoxycholate and then incubated for 4 h at 4 °C. Lysates were spun at 100,000 × g for 30 min and dialyzed twice against lysis buffer without deoxycholate. Supernatants were incubated with 5 μg of antibody or control nonimmune IgG and 40 μl of protein A- or G-agarose beads. Following overnight incubation at 4 °C, the immunocomplexes were pelleted by centrifugation (3000 × g, 1 min); washed four times with lysis buffer plus 650 mm NaCl, twice with lysis buffer, and twice with PBS; and eluted with 2× SDS-PAGE sample buffer. Bound proteins were analyzed by immunoblotting. For immunoprecipitation of AKAP-Lbc from human tissues or HeLa cells, extracts were processed as described above. To detect PKA catalytic activity in AKAP-Lbc immunoprecipitates, immunocomplexes were incubated with 1 mm cAMP for 10 min. PKA catalytic activity in eluates was assayed using Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) as substrate as described (31Corbin J.D. Reimann E.M. Methods Enzymol. 1974; 38: 287-294Crossref PubMed Scopus (391) Google Scholar). PKA activity was defined as the activity inhibited by the PKI-(5–24) inhibitor peptide. For immunoblots, the nitrocellulose filters were blocked overnight with Blotto plus 0.1% bovine serum albumin in TBS (100 mm Tris, pH 7.4, 140 mm NaCl, and 5% nonfat dry milk) at room temperature, washed three times with TTBS (0.05% Tween 20 in TBS), and then incubated with the specific primary antibody diluted in TTBS for 2 h at room temperature. After three washes with TTBS, filters were probed with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech) in TTBS for 1 h, washed with TBS, and developed using the enhanced chemiluminescence method according to the manufacturer's protocol (Amersham Pharmacia Biotech). For [32P]RII overlays, the filters were blocked 1 h with Blotto plus 1% bovine serum albumin in TBS at room temperature and then incubated 4–16 h with 100,000 cpm/ml PKA-phosphorylated [32P]RII in Blotto plus 0.1% bovine serum albumin. After extensive washes in TTBS, the blots were visualized by autoradiography. HEK293 cells grown in 100-mm dishes were transfected with 6 μg of the AKAP-Lbc/pEGFP or AKAP-Lbc Y2153F/pEGFP constructs. 24 h after transfection, cells were lysed in IP buffer containing 5 mm MgCl2, 1% Triton X-100, and 0.2% sodium deoxycholate. Lysates were then centrifuged at 100,000 × g for 30 min at 4 °C and incubated overnight with a polyclonal anti-AKAP-Lbc antibody and Protein A-Sepharose at 4 °C. Beads were then washed four times in IP buffer with 650 mmNaCl and twice in IP buffer. Beads were subsequently incubated with 0.5 μg of GST-RhoA, GST-Rac1, or GST-Cdc42 previously loaded with GDP or GTPγS or nucleotide-depleted for 4 h at 4 °C. After five washes in IP buffer with 1% (w/v) Triton X-100, proteins were eluted with Laemmli buffer and separated by SDS-PAGE. The exchange assays were performed as previously described (32Zheng Y. Hart M.J. Cerione R.A. Methods Enzymol. 1995; 256: 77-84Crossref PubMed Scopus (70) Google Scholar). For GTPγS binding, 2 μg of the recombinant GTPases were initially incubated for 5 min in 60 μl of loading buffer (20 mm Tris-HCl, pH 8.0, 100 mmNaCl, 2 mm EDTA, 0.2 mm dithiothreitol, 100 μm AMP-PNP, and 10 μm GDP) at room temperature. MgCl2 was then added to a final concentration of 5 mm, and the incubation continued for an additional 15 min. Finally, aliquots (20 μl) of GDP-loaded GTPases were mixed with 100 μg of lysates from cells overexpressing either AKAP-Lbc or AKAP-Lbc Y2153F diluted in reaction buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 10 mm MgCl2, 100 μm AMP-PNP, 0.5 mg/ml bovine serum albumin, and 5 μm [35S]GTPγS (11,000 cpm/pmol)) to initiate the exchange reaction (final volume 100 μl) at room temperature. Aliquots (15 μl) of samples were taken at various time points from the reaction mixture and added to 10 ml of ice-cold PBS. Bound and free nucleotides were separated by filtration through BA85 nitrocellulose filters. For the GDP dissociation assay, 10 μm radiolabeled [3H]GDP was used in the loading buffer instead of GDP, and 1 mm GTP was used in the reaction buffer instead of [35S]GTPγS. HEK293 cells grown in 100-mm dishes were transfected with 6 μg of the AKAP-Lbc/pEGFP or AKAP-Lbc Y2153F/pEGFP constructs. 24 h after transfection, cells were lysed in RBD lysis buffer (50 mm Tris, pH 7.2, 1% (w/v) Triton X-100, 0.5% sodium deoxycholate, 0.1% (w/v) SDS, 500 mmNaCl, 10 mm MgCl2, 1 mmbenzamidine, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride). Lysates were subjected to centrifugation at 38,000 × g for 15 min at 4 °C and incubated with 30 μg of RDB beads for 45 min at 4 °C. Beads were then washed three times with RBD buffer without deoxycholate, resuspended in 2× Laemmli buffer, and analyzed by SDS-PAGE. Cells grown on coverslips were transfected at 40% confluence using the LipofectAMINE Plus Reagent kit (Life Technologies, Inc.), washed twice with PBS, and then fixed for 10 min in PBS plus 3.7% formaldehyde and permeabilized for 5 min with 0.2% (w/v) Triton X-100 in PBS. Cells were blocked for 30 min in PBS plus 1% bovine serum albumin and then incubated for 1 h either with a 1:1000 dilution of polyclonal anti-AKAP-Lbc followed by 1 h in fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch) and Texas-red phalloidin (Molecular Probes, Inc., Eugene, OR) or Texas Red phalloidin alone. The cells were mounted using Prolong (Molecular Probes). Immunofluorescent staining or intrinsic GFP fluorescence was visualized on a laser-scanning confocal microscope (Bio-Rad). The Ht31 cDNA represents a partial clone encoding a 1015-amino acid PKA binding fragment that has proved to be a valuable tool for defining the molecular mechanism of PKA anchoring (29Carr D.W. Hausken Z.E. Fraser I.D.C. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar). The identity and function of the full-length anchoring protein are unknown. A data base search of GenBankTMrevealed that residues 631–1015 of Ht31 were identical to the amino terminus of Brx (18Rubino D. Driggers P. Arbit D. Kemp L. Miller B. Coso O. Pagliai K. Gray K. Gutkind S. Segars J. Oncogene. 1998; 16: 2513-2526Crossref PubMed Scopus (71) Google Scholar), a recently identified splice variant of the guanine nucleotide exchange factor Lbc (Fig.1 A). Lbc variants include Brx, the oncogene, onco-Lbc, and a proto-oncogene called proto-Lbc (15Toksoz D. Williams D.A. Oncogene. 1994; 9: 621-628PubMed Google Scholar, 17Sterpetti P. Hack A.A. Bashar M.P. Park B. Cheng S.D. Knoll J.H. Urano T. Feig L.A. Toksoz D. Mol. Cell. Biol. 1999; 19: 1334-1345Crossref PubMed Scopus (69) Google Scholar,18Rubino D. Driggers P. Arbit D. Kemp L. Miller B. Coso O. Pagliai K. Gray K. Gutkind S. Segars J. Oncogene. 1998; 16: 2513-2526Crossref PubMed Scopus (71) Google Scholar). Our data now suggest that a fourth and larger transcript exists that includes the Ht31 sequence (Fig. 1 A). This was confirmed by the PCR amplification of a 6693-base pair fragment encompassing the 5′ coding region of Ht31 and the 3′-untranslated sequence of proto-Lbc (Fig. 1 A). A further 2800 base pairs of message was obtained by 5′ rapid amplification of cDNA ends, and a full-length cDNA of 10.1 kb was amplified by reverse transcriptase-PCR (Fig. 1 A). Three independent PCR products were sequenced in both directions, revealing a coding sequence of 8451 nucleotides, encoding a protein of 2817 amino acids with a predicted molecular mass of 312 kDa (Fig. 1 C). On the basis of the homology with Lbc, we have named this anchoring protein AKAP-Lbc. The sequence has been deposited in GenBankTM (accession number AF406992). The AKAP-Lbc gene includes 37 exons and spans a region of 250 kb on chromosome 15 q24–25. Exons 1–9 encode unique sequences including the PKA-anchoring region, exons 10–20 encode sequences that are shared by AKAP-Lbc and Brx, and exons 21–37 encode a common region present all Lbc splice variants (Fig. 1 B). Several protein interaction modules are present including ankyrin repeats, the RII-binding domain, and a cysteine-rich motif homologous to the C1 region of PKC (Fig.1 C). The carboxyl-terminal region contains a Dbl homology and a pleckstrin homology domain that are characteristic of Rho family GEFs (9Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar) (Fig. 1 C). Earlier Northern blot analyses of Lbc mRNA tissue distribution revealed a variety of Lbc transcripts ranging from 6 to 10 kb in most human tissues (15Toksoz D. Williams D.A. Oncogene. 1994; 9: 621-628PubMed Google Scholar, 17Sterpetti P. Hack A.A. Bashar M.P. Park B. Cheng S.D. Knoll J.H. Urano T. Feig L.A. Toksoz D. Mol. Cell. Biol. 1999; 19: 1334-1345Crossref PubMed Scopus (69) Google Scholar, 18Rubino D. Driggers P. Arbit D. Kemp L. Miller B. Coso O. Pagliai K. Gray K. Gutkind S. Segars J. Oncogene. 1998; 16: 2513-2526Crossref PubMed Scopus (71) Google Scholar). A consistent 5-kb band corresponding to Brx was specifically detected in the testis (18Rubino D. Driggers P. Arbit D. Kemp L. Miller B. Coso O. Pagliai K. Gray K. Gutkind S. Segars J. Oncogene. 1998; 16: 2513-2526Crossref PubMed Scopus (71) Google Scholar), whereas a single 10-kb band was detected in the heart. Here, using a probe specific to the 5′ region of the AKAP-Lbc coding sequence, a single mRNA transcript of 10 kb was predominantly detected in the heart, although lower levels of this message were evident in the lung, placenta, kidney, pancreas, skeletal muscle, and liver (Fig.2 A, left panel). AKAP-Lbc message was also detected in HeLa S3 cells (Fig. 2 A, right panel). The tissue distribution of AKAP-Lbc protein was determined by immunoblot using antibodies raised against a recombinant fragment encompassing residues 769–1168 of the anchoring protein. A single protein species of ∼320 kDa was detected in tissue extracts of human heart but not in the brain or liver (Fig. 2 B). No bands were detected when the antibody was added in the presence of a 1 μm concentration of the recombinant AKAP-Lbc fragment encompassing residues 769–1168 or when the membranes were incubated with preimmune serum (results not shown). Due to the limited availability of healthy human heart tissues, subcellular fractionation (Fig. 2 C) and immunocytochemical analyses (Fig. 2 D) were performed on HeLa S3 cells, which endogenously expressed AKAP-Lbc. HeLa cell lysates were solubilized with increasing concentrations of Triton X-100 or sodium deoxycholate and fractionated by high speed centrifugation. AKAP-Lbc was only detected in deoxycholate fractions (Fig. 2 C,lanes 4 and 5). Immunocytochemical analysis demonstrated that AKAP-Lbc staining (green) was uniformly distributed throughout the cells but was excluded from the nucleus (blue) (Fig. 2 D). No signal was detected when the antibody was added in the presence of 1 μm of the recombinant AKAP-Lbc fragment encompassing residues 769–1168 or when the cells were incubated with preimmune serum (results not shown). These results suggest that AKAP-Lbc is predominantly expressed in the heart and is associated with Triton-insoluble structures within the cytoplasm. AKAPs are defined as proteins that tether the PKA holoenzyme inside cells (21Diviani D. Scott J.D. J. Cell Sci. 2001; 114: 1431-1437Crossref PubMed Google Scholar). Endogenous AKAP-Lbc was immunoprecipitated from HeLa cells (Fig.3 A, top panel). Both RII (Fig. 3 A, middle panel) and the catalytic subunit of PKA (C subunit) (Fig.3 A, bottom panel) were co-immunoprecipitated as identified by immunoblot. PKA activity was enriched 3.2 ± 0.5-fold (n = 3) upon immunoprecipitation of the anchoring protein when Kemptide was used as a substrate (Fig. 3 B). Control experiments using preimmune serum did not co-purify PKA subunits or enrich for cAMP-dependent kinase activity (Fig. 3, A and B). These experiments demonstrate that endogenous AKAP-Lbc and the PKA holoenzyme form a complex inside HeLa cells. Disruption of the secondary structure within the RII-binding domain of AKAP-Lbc abolished PKA anchoring, as shown by the fact that the AKAP-Lbc PP mutant, in which alanine 1251 and isoleucine 1260 within the PKA anchoring motif were mutated to proline, did not bind RII in the overlay assay (Fig. 3 C, top panel,lane 3). In contrast, wild type AKAP-Lbc retained the PKA anchoring function (Fig. 3 C, top panel, lane 2). Control experiments demonstrated that equal levels of both AKAP-Lbc forms were immunoprecipitated from HEK293 cells (Fig. 3 C,bottom panel). Furthermore, immunoprecipitation of recombinant AKAP-Lbc enriched kinase activity 15 ± 3.2-fold (n = 3) as compared with preimmune control, whereas immunoprecipitation of AKAP-Lbc PP did not (Fig. 3 D). These data indicate that endogenous and recombinant AKAP-Lbc function as PKA-anchoring proteins inside cells. Previous studies have demonstrated that the guanine nucleotide exchange factor Lbc interacts with RhoA in a nucleotide-dependent manner (15Toksoz D. Williams D.A. Oncogene. 1994; 9: 621-628PubMed Google Scholar, 17Sterpetti P. Hack A.A. Bashar M.P. Park B. Cheng S.D. Knoll J.H. Urano T. Feig L.A. Toksoz D. Mol. Cell. Biol. 1999; 19: 1334-1345Crossref PubMed Scopus (69) Google Scholar, 18Rubino D. Driggers P. Arbit D. Kemp L. Miller B. Coso O. Pagliai K. Gray K. Gutkind S. Segars J. Oncogene. 1998; 16: 2513-2526Crossref PubMed Scopus (71) Google Scholar). Therefore, we initiated experiments to establish whether AKAP-Lbc binds GTPases of the Rho family. Recombinant AKAP-Lbc was immunoprecipitated from HEK293 cells and incubated with GST fusion proteins of RhoA, Rac1, or Cdc42 (Fig.4 A, lanes 1–3). AKAP-Lbc only bound to RhoA in its GDP-bound or nucleotide-free conformations (Fig. 4 A, lanes 4 and 5); preloading RhoA with GTPγS abolished the interaction (Fig. 4 A, lane 6). Similar results were obtained with the RhoB and RhoC isoforms (data not shown). AKAP-Lbc interaction with Rac1 and Cdc42 was not detected (Fig.4 A, lanes 7–9 and 10–12), suggesting that the anchoring protein specifically associates with Rho. A more rigorous test of this hypothesis was to determine whether AKAP-Lbc retained its specificity for Rho inside cells. Therefore, endogenous AKAP-Lbc was immunoprecipitated from HeLa lysates. Endogenous RhoA was detected by immunoblot in AKAP-Lbc immunoprecipitates (Fig. 4 B, top panel, lane 3) but not in immunocomplexes isolated with preimmune serum (Fig. 4 B,top panel, lane 2). Control experiments confir
Referência(s)