FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1973172146> ?p ?o ?g. }

• W1973172146 endingPage "15412" @default.
• W1973172146 startingPage "15405" @default.
• W1973172146 abstract "AKAP-Lbc is a novel member of the A-kinase anchoring protein (AKAPs) family, which functions as a cAMP-dependent protein kinase (PKA)-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We recently demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the α-subunit of the heterotrimeric G protein G12, whereas phosphorylation of AKAP-Lbc by the anchored PKA induces the recruitment of 14-3-3, which inhibits its GEF function. In the present report, using co-immunoprecipitation approaches, we demonstrated that AKAP-Lbc can form homo-oligomers inside cells. Mutagenesis studies revealed that oligomerization is mediated by two adjacent leucine zipper motifs located in the C-terminal region of the anchoring protein. Most interestingly, disruption of oligomerization resulted in a drastic increase in the ability of AKAP-Lbc to stimulate the formation of Rho-GTP in cells under basal conditions, suggesting that oligomerization maintains AKAP-Lbc in a basal-inactive state. Based on these results and on our previous findings showing that AKAP-Lbc is inactivated through the association with 14-3-3, we investigated the hypothesis that AKAP-Lbc oligomerization might be required for the regulatory action of 14-3-3. Most interestingly, we found that mutants of AKAP-Lbc impaired in their ability to undergo oligomerization were completely resistant to the inhibitory effect of PKA and 14-3-3. This suggests that 14-3-3 can negatively regulate the Rho-GEF activity of AKAP-Lbc only when the anchoring protein is in an oligomeric state. Altogether, these findings provide a novel mechanistic explanation of how oligomerization can regulate the activity of exchange factors of the Dbl family. AKAP-Lbc is a novel member of the A-kinase anchoring protein (AKAPs) family, which functions as a cAMP-dependent protein kinase (PKA)-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We recently demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the α-subunit of the heterotrimeric G protein G12, whereas phosphorylation of AKAP-Lbc by the anchored PKA induces the recruitment of 14-3-3, which inhibits its GEF function. In the present report, using co-immunoprecipitation approaches, we demonstrated that AKAP-Lbc can form homo-oligomers inside cells. Mutagenesis studies revealed that oligomerization is mediated by two adjacent leucine zipper motifs located in the C-terminal region of the anchoring protein. Most interestingly, disruption of oligomerization resulted in a drastic increase in the ability of AKAP-Lbc to stimulate the formation of Rho-GTP in cells under basal conditions, suggesting that oligomerization maintains AKAP-Lbc in a basal-inactive state. Based on these results and on our previous findings showing that AKAP-Lbc is inactivated through the association with 14-3-3, we investigated the hypothesis that AKAP-Lbc oligomerization might be required for the regulatory action of 14-3-3. Most interestingly, we found that mutants of AKAP-Lbc impaired in their ability to undergo oligomerization were completely resistant to the inhibitory effect of PKA and 14-3-3. This suggests that 14-3-3 can negatively regulate the Rho-GEF activity of AKAP-Lbc only when the anchoring protein is in an oligomeric state. Altogether, these findings provide a novel mechanistic explanation of how oligomerization can regulate the activity of exchange factors of the Dbl family. Compartmentalization of signaling molecules through association with anchoring and scaffolding proteins is a mechanism that ensures specificity of transduction events involved in cellular regulation. A-kinase anchoring proteins (AKAPs) 1The abbreviations used are: AKAPs, A-kinase anchoring protein; PKA, cAMP-dependent protein kinase; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; DH, Dbl homology; RBD, Rho-binding domain; GTPγS, guanosine 5′-3-O-(thio)triphosphate; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; GST, glutathione S-transferase; GFP, green fluorescent protein; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; DMEM, Dulbecco's modified Eagle's medium. are a family of scaffolding proteins that compartmentalize the cAMP-dependent protein kinase (PKA) at precise subcellular sites in close proximity to its physiological substrates (1Michel J.J. Scott J.D. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 235-257Crossref PubMed Scopus (289) Google Scholar). Each AKAP contains a conserved amphipathic helix of 14–18 residues that binds to the regulatory subunit dimers of the PKA holoenzyme (2Carr 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, 3Newlon M.G. Roy M. Morikis D. Hausken Z.E. Coghlan V. Scott J.D. Jennings P.A. Nat. Struct. Biol. 1999; 6: 222-227Crossref PubMed Scopus (184) Google Scholar, 4Newlon M.G. Roy M. Hausken Z.E. Scott J.D. Jennings P.A. J. Biol. Chem. 1997; 272: 23637-23644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and displays a unique targeting motif that directs PKA·AKAP complexes to specific subcellular sites (5Wong W. Scott J.D. Nat. Rev. Mol. Cell. Biol. 2004; 12: 959-970Crossref Scopus (862) Google Scholar). Another fundamental role of AKAPs is to assemble signaling complexes by associating with multiple enzymes such as kinases, phosphatases, and other regulatory proteins. By simultaneously interacting with multiple signaling enzymes, AKAPs can integrate diverse transduction pathways that coordinately regulate the function of specific cellular substrates (5Wong W. Scott J.D. Nat. Rev. Mol. Cell. Biol. 2004; 12: 959-970Crossref Scopus (862) Google Scholar, 6Bauman A.L. Scott J.D. Nat. Cell Biol. 2002; 4: E203-E206Crossref PubMed Scopus (117) Google Scholar). Recently, we identified a novel member of the AKAP family, termed AKAP-Lbc, that functions as a type II PKA anchoring protein as well as guanine nucleotide exchange factor (GEF) for RhoA (7Diviani D. Soderling J. Scott J.D. J. Biol. Chem. 2001; 276: 44247-44257Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), a small GTP-binding protein of the Ras family that controls fundamental cell processes such as cell cycle progression, gene transcription, remodeling of the actin cytoskeleton, and cytokinesis (8Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3875) Google Scholar). AKAP-Lbc belongs to the Dbl family of GEFs, which all share a Dbl homology (DH) domain and an adjacent pleckstrin homology (PH) domain (9Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (987) Google Scholar). The DH domain is responsible for the guanine nucleotide exchange activity, whereas the PH domain regulates subcellular localization of Rho-GEFs or is implicated in the binding pocket for Rho-GT-Pases (8Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3875) Google Scholar). A truncated form of AKAP-Lbc missing the entire N-terminal and C-terminal regions, called Onco-Lbc, was originally isolated as an oncogene from myeloid leukemia patients and shown to represent a constitutively active Rho-GEF (10Zheng 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 (148) Google Scholar). The Rho-GEF activity of AKAP-Lbc can be strongly enhanced by the α-subunit of the heterotrimeric G protein G12 that is activated following the stimulation of G protein-coupled receptors that couple to G12 by serum or lysophosphatidic acid (7Diviani D. Soderling J. Scott J.D. J. Biol. Chem. 2001; 276: 44247-44257Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In the absence of activating stimuli, AKAP-Lbc is maintained in an inactive state through the association with 14-3-3. The recruitment of 14-3-3 to AKAP-Lbc is induced by the phosphorylation of serine 1565 located within the 14-3-3-binding site of the anchoring protein by the PKA holoenzyme anchored to AKAP-Lbc (11Diviani D. Abuin L. Cotecchia S. Pansier L. EMBO J. 2004; 23: 2811-2820Crossref PubMed Scopus (110) Google Scholar, 12Jin J. Smith F.D. Stark C. Wells C.D. Fawcett J.P. Kulkarni S. Metalnikov P. O'Donnell P. Taylor P. Taylor L. Zougman A. Woodgett J.R. Langeberg L.K. Scott J.D. Pawson T. Curr. Biol. 2004; 14: 1436-1450Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Many members of the Dbl family of GEF are maintained in a basal inactive conformation by intramolecular interactions involving the DH and PH domains as well as regulatory sequences. Such interactions have been proposed to block the access of Rho GTPases to the DH domain and/or suppress the GEF activity of the exchange factor (13Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Recent studies now demonstrate that the activity of Dbl family members can also be regulated through oligomerization. The functional role of this intermolecular interaction has been established only for a small number of them, including Ras GRF1 and Ras GRF2 (14Anborgh P.H. Qian X. Papageorge A.G. Vass W.C. DeClue J.E. Lowy D.R. Mol. Cell. Biol. 1999; 19: 4611-4622Crossref PubMed Scopus (70) Google Scholar), Dbl (15Zhu K. Debreceni B. Bi F. Zheng Y. Mol. Cell. Biol. 2001; 21: 425-437Crossref PubMed Scopus (40) Google Scholar), α- and β-Pix (16Feng Q. Baird D. Cerione R.A. EMBO J. 2004; 23: 3492-3504Crossref PubMed Scopus (74) Google Scholar, 17Kim S. Lee S.H. Park D. J. Biol. Chem. 2001; 276: 10581-10584Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), as well as p115-Rho-GEF, LARG, and PDZ-Rho-GEF (18Eisenhaure T.M. Francis S.A. Willison L.D. Coughlin S.R. Lerner D.J. J. Biol. Chem. 2003; 278: 30975-30984Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Chikumi H. Barac A. Behbahani B. Gao Y. Teramoto H. Zheng Y. Gutkind J.S. Oncogene. 2004; 23: 233-240Crossref PubMed Scopus (90) Google Scholar). Oligomerization of Ras GRF, Dbl, and β-Pix is required for the efficient execution of the exchange reaction (14Anborgh P.H. Qian X. Papageorge A.G. Vass W.C. DeClue J.E. Lowy D.R. Mol. Cell. Biol. 1999; 19: 4611-4622Crossref PubMed Scopus (70) Google Scholar, 15Zhu K. Debreceni B. Bi F. Zheng Y. Mol. Cell. Biol. 2001; 21: 425-437Crossref PubMed Scopus (40) Google Scholar), whereas oligomerization of p115-Rho-GEF, LARG, and PDZ-Rho GEF has been shown to negatively regulate the GEF activity (18Eisenhaure T.M. Francis S.A. Willison L.D. Coughlin S.R. Lerner D.J. J. Biol. Chem. 2003; 278: 30975-30984Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Chikumi H. Barac A. Behbahani B. Gao Y. Teramoto H. Zheng Y. Gutkind J.S. Oncogene. 2004; 23: 233-240Crossref PubMed Scopus (90) Google Scholar). Most interestingly, recent evidence suggests that oligomerization can also regulate the specificity of GEFs toward Rho GTPases. This mechanism was recently described for α-Pix, which can adopt a dimeric conformation that selectively activates Rac and a monomeric conformation that activates both Rac and Cdc42 (16Feng Q. Baird D. Cerione R.A. EMBO J. 2004; 23: 3492-3504Crossref PubMed Scopus (74) Google Scholar). Although it appears that oligomerization can affect the functional properties of Dbl family members, the molecular mechanisms through which oligomerization regulates the activity of these exchange factors are poorly understood. In the present study, by using co-immunoprecipitation approaches, we demonstrate that AKAP-Lbc can form homo-oligomers through a leucine zipper motif located in the C-terminal region of the anchoring protein. We found that disruption of oligomerization strongly enhances the basal Rho-GEF activity of AKAP-Lbc, suggesting that oligomerization maintains the anchoring protein in a basal inactive state. Most importantly, we also show that oligomerization maintains AKAP-Lbc in a conformation that can be regulated by 14-3-3, as shown by the fact that oligomerization-deficient mutants of AKAP-Lbc are completely resistant to the inhibitory effect of 14-3-3. These findings provide a molecular explanation for the functional role of oligomerization of Dbl family GEFs. Expression Constructs—The constructs encoding the FLAG-AKAP-Lbc and the FLAG-AKAP-Bc deletion mutant missing the first 1922 residues (FLAG-AKAP-Bc-ΔN-term) were described previously (11Diviani D. Abuin L. Cotecchia S. Pansier L. EMBO J. 2004; 23: 2811-2820Crossref PubMed Scopus (110) Google Scholar). The deletion mutant of FLAG-AKAP-Bc missing residues 2337–2817 (FLAG-AKAP-Bc-ΔC-term) was generated by subcloning a fragment excised from the AKAP-Lbc-(1923–2336)-pGEX4T1 construct, which contains a stop codon at position 2336, at Psp1406I/NotI into the FLAG-AKAP-Lbc construct. The coiled coil region included between residues 2573 and 2687 was deleted from FLAG-AKAP-Lbc (FLAG-AKAP-Lbc ΔCC) as well as from FLAG-tagged and GFP-tagged AKAP-Lbc fragments encompassing residues 1923–2817 (1923–2817 ΔCC) by using standard PCR techniques. cDNA fragments encoding amino acids 1–503, 504–1000, 1001–1387, 1388–1922, 1923–2336, 2337–2817, 1923–2817, 1923–2698, 1923–2589, and 2566–2698 of AKAP-Lbc were PCR-amplified from the AKAP-Lbc pEGFP vector and subcloned in the pFLAGCMV6 vector, to generate protein fragments fused with the FLAG epitope. AKAP-Lbc fragments encompassing residues 1–503, 504–1000, and 1001–1387 were subcloned at NotI/SalI; fragments 1388–1922 and 1923–2336 were subcloned at EcoRI/SalI, and fragments 2337–2817, 1923–2817, 1923–2698, 1923–2589, and 2566–2698 were subcloned at SalI/KpnI. Fragments 1923–2336, 1923–2817, 2337–2817, 1923–2698, 1923–2589, and 2566–2698 were also subcloned into the pEGFP vector to construct fusion proteins with GFP. For the mapping of the oligomerization site, different leucine and valine residues included in the putative leucine zipper motifs of AKAP-Lbc were substituted to alanine into the AKAP-Lbc-(1923–2817)-pFLAGCMV6 vector by PCR-directed mutagenesis using the Hot Star DNA polymerase (Qiagen). The mutants generated are the following: LZ mutant 1 (L216A, L2623A, and V2630A), LZ mutant 2 (L2637A and L2644A), LZ mutant 3 (L2658A and L2665A), LZ mutant 4 (L2672A and L2679A), LZ mutant 5 (L216A, L2623A, V2630A, L2637A, and L2644A), LZ mutant 6 (L2658A, L2665A, L2672A, and L2679A), and LZ mutant 7 (L216A, L2623A, V2630A, L2637A, L2644A, L2658A, and L2665A). The oligomerization-deficient mutant of AKAP-Lbc (FLAG-AKAP-Lbc LZm) was generated by subcloning a PCR fragment amplified from the LZ mutant 7-pFLAGCMV6c construct at Psp1406I/NotI into the FLAG-AKAP-Lbc construct. The rhotekin Rho-binding domain (RBD)-pGEX4T1 construct was a generous gift of Dr. Hitoshi Kurose (Fukuoka, Japan). Expression and Purification of Recombinant Proteins in Bacteria— GST fusion proteins of the RBD of rhotekin and RhoA were expressed using the bacterial expression vector pGEX4T1 in the BL21DE3 strain of Escherichia coli and were purified. To induce the expression of the GST-RBD fusion proteins, exponentially growing bacterial cultures were incubated 24 h at 16 °C with 1 mm isopropyl 1-thio-β-d-galactopyranoside and subsequently subjected to centrifugation. Pelleted bacteria were lysed in buffer A (50 mm Tris, pH 7.4, 500 mm NaCl, 10 mm MgCl2, 0.1% SDS, 0.5% sodium deoxycholate, 1% (w/v)Triton X-100, 1 mm PMSF, 1 mm benzamidine, 2 μg/ml leupeptin, 2 μg/ml pepstatin), sonicated, and centrifuged at 38,000 × g for 30 min at 4 °C. After incubating the supernatants with the glutathione-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C, the resin was washed five times with 10 volumes of buffer A. The protein content of the beads was assessed by Coomassie Blue staining of SDS-polyacrylamide gels. Beads were used immediately for rhotekin RBD pulldown assay. For the production of purified GST-RhoA, exponentially growing bacterial cultures were incubated 4 h at 37 °C with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside and subsequently subjected to centrifugation. Pelleted bacteria were lysed in buffer B (50 mm Tris, pH 7.6, 100 mm NaCl, 5 mm MgCl2, 1 mm DTT, 1% (w/v)Triton X-100, 1 mm PMSF, 1 mm benzamidine, 2 μg/ml leupeptin, 2 μg/ml pepstatin), sonicated, and centrifuged at 38,000 × g for 30 min at 4 °C. After incubating the supernatants with the glutathione-Sepharose beads (Amersham Biosciences) for 10 min at 4 °C, the beads were washed three times with 10 volumes of buffer B. GST-RhoA was eluted by incubating beads with 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm MgCl2, 1 mm DTT, 0.1% (w/v) Triton X-100, 20 mm reduced glutathione (Sigma), 1 mm PMSF, 1 mm benzamidine, 2 μg/ml leupeptin, 2 μg/ml pepstatin for 1 h at 4 °C with constant stirring. The eluted proteins were dialyzed three times with 10 mm Tris-HCl, pH 7.6, 50 mm NaCl, 2 mm MgCl2, 0.1 mm DTT and stored at -80 °C. The protein content of eluates was assessed by Coomassie Blue staining of SDS-polyacrylamide gels. Cell Culture and Transfections—HEK-293 were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and gentamycin (100 μg/ml) and transfected at 50–60% confluency in 100-mm dishes using the calcium-phosphate method. For the overexpression of constructs containing the full-length AKAP-Lbc, HEK-293 cells were transfected at 80% confluency in 100-mm dishes using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After transfection, cells were grown for 48 h in DMEM supplemented with 10% fetal calf serum before harvesting. The total amount of transfected DNA was 6 μg/100-mm dish for the FLAG-tagged AKAP-Lbc fragments, 12 μg/100-mm dish for the GFP-tagged AKAP-Lbc fragments, and 24 μg/100-mm dish for the full-length FLAG-AKAP-Lbc constructs. Immunoprecipitation Experiments—For co-immunoprecipitation experiments, cells were lysed in 1 ml of buffer C (20 mm Tris, pH 7.4, 150 mm NaCl, 1% (w/v) Triton X-100, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm PMSF). Cell lysates were incubated 6 h at 4°C on a rotating wheel and then centrifuged at 100,000 × g for 30 min at 4 °C. The supernatants were incubated overnight at 4 °C with 20 μl of anti-FLAG M2 beads (Sigma) to immunoprecipitate overexpressed FLAG-tagged AKAP-Lbc constructs. Following a brief centrifugation on a bench-top centrifuge, the pelleted beads were washed five times with buffer C and proteins eluted in SDS-PAGE sample buffer (65 mm Tris, pH 6.8, 2% SDS, 5% glycerol, 5% β-mercaptoethanol) by boiling samples for 3 min at 95 °C. Eluted proteins were analyzed by SDS-PAGE and by Western blotting. SDS-PAGE and Western Blotting—Samples denatured in SDS-PAGE sample buffer were separated on acrylamide gels and electroblotted onto nitrocellulose membranes. The blots were incubated with primary antibodies and horseradish-conjugated secondary antibodies (Amersham Biosciences) as indicated previously (11Diviani D. Abuin L. Cotecchia S. Pansier L. EMBO J. 2004; 23: 2811-2820Crossref PubMed Scopus (110) Google Scholar). The following affinity-purified primary antibodies were used for immunoblotting: mouse monoclonal anti-FLAG (Sigma, 4.9 mg/ml, 1:2000 dilution), mouse monoclonal anti-GFP (Roche Applied Science, 400 μg/ml, 1:500 dilution), mouse monoclonal anti-RhoA (Santa Cruz Biotechnology, 1:250 dilution), and rabbit polyclonal anti-14-3-3β (Santa Cruz Biotechnology, 1:250 dilution). GDP/GTP Exchange Assay—The exchange assays were performed as described previously (20Zheng Y. Hart M.J. Cerione R.A. Methods Enzymol. 1995; 256: 77-84Crossref PubMed Scopus (70) Google Scholar). A 2-μg portion of recombinant RhoA was incubated for 5 min in 60 μl of loading buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 2 mm EDTA, 0.2 mm DTT, 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 was continued for an additional 15 min. To initiate the exchange reaction, protein aliquots (20 μl) of GDP-loaded GTPases were mixed at room temperature with 80 μl of 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)) containing immunoprecipitated FLAG-AKAP-Lbc or FLAG-AKAP-Lbc LZ mutant. Aliquots (15 μl) of samples were taken at various time points and added to 10 ml of ice-cold phosphate-buffered saline. Bound and free nucleotides were separated by filtration through BA85 nitrocellulose filters. The amount of bound radioactivity was measured by liquid scintillation counting. Rhotekin Rho-binding Domain Pulldown Assay—HEK-293 cells grown in 100-mm dishes were transfected with 24 μg of FLAG-tagged AKAP-Lbc, AKAP-Lbc-ΔC-term, AKAP-Lbc-ΔCC, AKAP-Lbc LZm, or AKAP-Lbc S1565A constructs. At 24 h after transfection, cells were incubated in DMEM without serum for an additional 24 h. Cells were then treated for 1 h with 10% fetal calf serum in the absence or presence of 50 μm forskolin (Sigma) and lysed in RBD lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 30 mm MgCl2, 1 mm DTT, 10% glycerol, 1% (w/v) Triton X-100, 1 mm benzamidine, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm PMSF). Lysates were subjected to centrifugation at 100,000 × g for 10 min at 4 °C and incubated with 30 μg of RDB beads for 1 h at 4 °C. Beads were then washed three times with RBD buffer, resuspended in SDS sample buffer, and analyzed by SDS-PAGE. The C-terminal Region of AKAP-Lbc Negatively Regulates Basal Rho-GEF Activity—We have demonstrated recently that AKAP-Lbc displays a low basal Rho-GEF activity in serumstarved cells, which can be significantly enhanced by the deletion of the N-terminal region of the anchoring protein upstream of the DH domain. This suggested that inhibitory determinants located in the N-terminal sequence maintain AKAP-Lbc in an inactive state in the absence of external activating stimuli (7Diviani D. Soderling J. Scott J.D. J. Biol. Chem. 2001; 276: 44247-44257Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 11Diviani D. Abuin L. Cotecchia S. Pansier L. EMBO J. 2004; 23: 2811-2820Crossref PubMed Scopus (110) Google Scholar). On the other hand, our previous results could not clearly determine whether the C-terminal region downstream of the PH domain also regulates the function of AKAP-Lbc, because we found that the truncated form of AKAP-Lbc missing the N-terminal regulatory region displays a basal constitutive Rho-GEF activity comparable with that of a deletion mutant of AKAP-Lbc missing both N and C termini (7Diviani D. Soderling J. Scott J.D. J. Biol. Chem. 2001; 276: 44247-44257Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Therefore, in order to determine precisely the functional role of the C-terminal region, we assessed the Rho-activating properties of a deletion mutant of AKAP-Lbc missing only the C terminus. AKAP-Lbc as well as its deletion forms AKAP-Lbc Δ-Nterm and AKAP-Lbc Δ-Cterm, missing the N-terminal 1922 residues and the C-terminal 481 residues, respectively, were overexpressed in HEK-293 cells, and their basal Rho-GEF were activities assessed by using the rhotekin pulldown assay after 24 h of serum starvation (Fig. 1). Most interestingly, the C-terminal deletion mutant of AKAP-Lbc displayed a significant 4-fold higher basal Rho-GEF activity as compared with wild type AKAP-Lbc (Fig. 1B, top panel, lane 4). This activity is comparable with that of the N-terminal deletion mutant (Fig. 1B, top panel, lane 3), suggesting that both the N and C termini of the AKAP-Lbc contribute to maintain the basal Rho-GEF activity low in the absence of external activation stimuli. In order to determine whether the higher basal activity induced by N- and C-terminal deletions could be attributed to an increased association between AKAP-Lbc and RhoA, we determined the ability of RhoA to co-immunoprecipitate with wild type AKAP-Lbc, AKAP-Lbc Δ-Nterm, or AKAP-Lbc Δ-Cterm. As shown in Fig. 1C, both truncation mutants displayed a stronger interaction with endogenous RhoA under basal unstimulated conditions as compared with wild type AKAP-Lbc (Fig. 1C, middle panel, lanes 3 and 4). These findings strongly suggest that the C-terminal region of AKAP-Lbc, included between residues 2337 and 2817, negatively regulates the basal Rho-GEF activity by inhibiting the binding of RhoA to AKAP-Lbc. AKAP-Lbc Undergoes Homo-oligomerization through Its C-terminal Region—Recent evidence suggests that GEFs of the Dbl family can be maintained in a basal inactive conformation by intramolecular interactions between regulatory sequences and the GEF module (DH and PH domains) (13Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Such interactions have been proposed to modulate the function of the DH domain. In order to assess whether the C-terminal region of AKAP-Lbc included between amino acids 2337 and 2817 could establish intramolecular interactions with other domains of AKAP-Lbc, such as the N-terminal regulatory region or the GEF module, we generated a series of FLAG-tagged AKAP-Lbc fragments encompassing residues 1–503, 504–1000, 1001–1387, 1388–1922, 1923–2336 and 2337–1817, and we expressed them in HEK-293 cells in combination with either GFP or a GFP-tagged 2337–2817 fragment. Overexpressed FLAG-tagged proteins were immunoprecipitated using anti-FLAG antibodies, and the presence of associated 2337–2817 GFP fragment was assessed using anti-GFP antibodies. As shown in Fig. 2, whereas no intramolecular interactions could be detected between the 2337–2817 fragment of AKAP-Lbc and other fragments of the anchoring protein (Fig. 2B, middle panel, lanes 1–10), a strong association was observed between FLAG- and GFP-tagged 2337–2817 fragments (Fig. 2B, middle panel, lane 12). These findings suggest that the C-terminal region of AKAP-Lbc can undergo homo-oligomerization. Homo-oligomerization Occurs through Leucine Zipper Motifs Located in the Coiled Coil Region of AKAP-Lbc—To identify the oligomerization domain within the C-terminal region of AKAP-Lbc, we generated FLAG- and GFP-tagged AKAP-Lbc fragments encompassing residues 1923–2817, 1923–2336, 2337–2817, 1923–2589, and 1923–2698 (Fig. 3A), and we co-expressed them in HEK-293 cells. Overexpressed FLAG-tagged AKAP-Lbc fragments were immunoprecipitated using anti-FLAG antibodies, and the presence of associated GFP-tagged fragments was assessed using anti-GFP antibodies. Fragments encompassing residues 1923–2817, 2337–2817, and 1923–2698 retained the ability to undergo oligomerization (Fig. 3B, middle panel, lanes 4, 6, and 8), whereas fragments 1923–2589 and 1923–2336 did not (Fig. 3B, middle panel, lanes 2 and 10). This suggested that the oligomerization motif is located between residues 2589 and 2698. We found that this portion of AKAP-Lbc is entirely included in a coiled coil region that encompasses residues 2566–2698. Most interestingly, deletion of this region totally abolished oligomerization of the 1923–2817 fragment, whereas a fragment encompassing residues 2566–2698 retained the ability to form oligomers (Fig. 3B, middle panel, lanes 12 and 14). These results strongly suggest that the coiled coil region of AKAP-Lbc is necessary and sufficient for the homo-oligomerization process. Analysis of the primary sequence between residues 2566–2698 revealed the presence of two leucine zipper motifs encompassing residues 2616–2644 and 2658–2679, respectively (Fig. 4A). Based on this observation and on the fact that leucine zippers often function as protein-protein interaction motifs, we investigated the possibility that these motifs could mediate the oligomerization of AKAP-Lbc. We generated FLAG fusions of the 1923–2817 fragment of AKAP-Lbc in which valine 2630 as well as leucines 2616, 2623, 2637, 2644, 2658, 2665, 2672, and 2679 were substituted by alanine in different combinations (Fig. 4B). The different FLAG-tagged fragments were expressed in HEK-293 cells in combination with the GFP-tagged 1923–2817 fragment. Overexpressed FLAG-tagged proteins were immunoprecipitated using anti-FLAG antibodies, and the presence of associated GFP-tagged 1923–2817 fragment was assessed using anti-GFP antibodies. As shown in Fig. 4C the mutation of leucines 2616, 2623, and valine 2630 (LZ mutant 1), leucines 2637 and 2644 (LZ mutant 2), and leucines 2658 and 2665 (LZ mutant 3) reduced the homo-oligomerization of the 1923–2817 fragment of AKAP-Lbc by 50–60% (Fig. 4, C, middle panel, lanes 3–5, and D) whereas the substitution of leucines 2672 and 2679 (LZ mutant 4) had no effect (Fig. 4, C, middle panel, lane 6 and D). A 90% reduction in oligomerization could be observed after the substitution of all five leucines and valines included in the first leucine zipper (LZ mutant 5), whereas mutation of all four leucines of the second leucine zipper (LZ mutant 6) could inhibit the formation of oligomers only by 50% (Fig. 4, C, middle panel, lanes 7 and 8, and D). Finally, oligomerization was totally abolished after substitution of all leucines and valines of the first leucine zipper as well as leucines 2658 and 2665 of the second leucine zipper (LZ mutant 7) (Fig. 4, C, middle panel, lane 9, and D). Altogether these findings indicate that oligomerization requires the integrity of the entire leucine zipper motif encompassing residues 2616–2644, whereas the second leucine zipper is only partially involved. To assess the contribution of the leucine zipper motifs to the oligomerization of the full-length AKAP-Lbc, we overexpressed the FLAG-tagged forms of AKAP-Lbc and of its mutants AKAP-Lbc ΔCC (missing the entire coiled coil region) and AKAP-Lbc LZm (in which leucines 2616 to 2665 were mutated to alanine) in HEK-293 cells in combination with AKAP-Lbc-GFP. The FLAG-tagged proteins were immunoprecipitated by using anti-F" @default.
• W1973172146 created "2016-06-24" @default.
• W1973172146 creator A5026160799 @default.
• W1973172146 creator A5071606955 @default.
• W1973172146 creator A5079311926 @default.
• W1973172146 date "2005-04-01" @default.
• W1973172146 modified "2023-09-30" @default.
• W1973172146 title "Leucine Zipper-mediated Homo-oligomerization Regulates the Rho-GEF Activity of AKAP-Lbc" @default.
• W1973172146 cites W147106477 @default.
• W1973172146 cites W1484050792 @default.
• W1973172146 cites W1970170695 @default.
• W1973172146 cites W1977874249 @default.
• W1973172146 cites W1990283596 @default.
• W1973172146 cites W1997917368 @default.
• W1973172146 cites W2001234795 @default.
• W1973172146 cites W2013452582 @default.
• W1973172146 cites W2019731805 @default.
• W1973172146 cites W2032668695 @default.
• W1973172146 cites W2048168473 @default.
• W1973172146 cites W2058778918 @default.
• W1973172146 cites W2059059532 @default.
• W1973172146 cites W2060195091 @default.
• W1973172146 cites W2061126410 @default.
• W1973172146 cites W2062803349 @default.
• W1973172146 cites W2083240338 @default.
• W1973172146 cites W2110219256 @default.
• W1973172146 cites W2112769897 @default.
• W1973172146 cites W2126920991 @default.
• W1973172146 cites W2137096206 @default.
• W1973172146 cites W2144491728 @default.
• W1973172146 cites W2144604382 @default.
• W1973172146 cites W2144796569 @default.
• W1973172146 cites W2148910944 @default.
• W1973172146 cites W2154655439 @default.
• W1973172146 cites W2156511132 @default.
• W1973172146 cites W2165633817 @default.
• W1973172146 cites W2171432125 @default.
• W1973172146 doi "https://doi.org/10.1074/jbc.m414440200" @default.
• W1973172146 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15691829" @default.
• W1973172146 hasPublicationYear "2005" @default.
• W1973172146 type Work @default.
• W1973172146 sameAs 1973172146 @default.
• W1973172146 citedByCount "66" @default.
• W1973172146 countsByYear W19731721462012 @default.
• W1973172146 countsByYear W19731721462013 @default.
• W1973172146 countsByYear W19731721462014 @default.
• W1973172146 countsByYear W19731721462015 @default.
• W1973172146 countsByYear W19731721462016 @default.
• W1973172146 countsByYear W19731721462017 @default.
• W1973172146 countsByYear W19731721462018 @default.
• W1973172146 countsByYear W19731721462019 @default.
• W1973172146 countsByYear W19731721462020 @default.
• W1973172146 countsByYear W19731721462021 @default.
• W1973172146 countsByYear W19731721462022 @default.
• W1973172146 crossrefType "journal-article" @default.
• W1973172146 hasAuthorship W1973172146A5026160799 @default.
• W1973172146 hasAuthorship W1973172146A5071606955 @default.
• W1973172146 hasAuthorship W1973172146A5079311926 @default.
• W1973172146 hasBestOaLocation W19731721461 @default.
• W1973172146 hasConcept C104317684 @default.
• W1973172146 hasConcept C105782903 @default.
• W1973172146 hasConcept C11413529 @default.
• W1973172146 hasConcept C156860981 @default.
• W1973172146 hasConcept C185592680 @default.
• W1973172146 hasConcept C2776580952 @default.
• W1973172146 hasConcept C41008148 @default.
• W1973172146 hasConcept C515207424 @default.
• W1973172146 hasConcept C55493867 @default.
• W1973172146 hasConcept C86339819 @default.
• W1973172146 hasConcept C86803240 @default.
• W1973172146 hasConcept C95444343 @default.
• W1973172146 hasConceptScore W1973172146C104317684 @default.
• W1973172146 hasConceptScore W1973172146C105782903 @default.
• W1973172146 hasConceptScore W1973172146C11413529 @default.
• W1973172146 hasConceptScore W1973172146C156860981 @default.
• W1973172146 hasConceptScore W1973172146C185592680 @default.
• W1973172146 hasConceptScore W1973172146C2776580952 @default.
• W1973172146 hasConceptScore W1973172146C41008148 @default.
• W1973172146 hasConceptScore W1973172146C515207424 @default.
• W1973172146 hasConceptScore W1973172146C55493867 @default.
• W1973172146 hasConceptScore W1973172146C86339819 @default.
• W1973172146 hasConceptScore W1973172146C86803240 @default.
• W1973172146 hasConceptScore W1973172146C95444343 @default.
• W1973172146 hasIssue "15" @default.
• W1973172146 hasLocation W19731721461 @default.
• W1973172146 hasOpenAccess W1973172146 @default.
• W1973172146 hasPrimaryLocation W19731721461 @default.
• W1973172146 hasRelatedWork W1604014255 @default.
• W1973172146 hasRelatedWork W2005051736 @default.
• W1973172146 hasRelatedWork W2022380058 @default.
• W1973172146 hasRelatedWork W2051181331 @default.
• W1973172146 hasRelatedWork W2066101080 @default.
• W1973172146 hasRelatedWork W2125410766 @default.
• W1973172146 hasRelatedWork W2161371945 @default.
• W1973172146 hasRelatedWork W2164464592 @default.
• W1973172146 hasRelatedWork W2911806751 @default.
• W1973172146 hasRelatedWork W4236739251 @default.
• W1973172146 hasVolume "280" @default.

• next