A Functional Interaction between Sprouty Proteins and Caveolin-1
2006; Elsevier BV; Volume: 281; Issue: 39 Linguagem: Inglês
10.1074/jbc.m603921200
ISSN1083-351X
AutoresMiguel A. Cabrita, Fabienne Jäggi, Sandra P. Widjaja, Gerhard Christofori,
Tópico(s)Lipid metabolism and biosynthesis
ResumoGrowth factor-mediated signal transduction cascades can be regulated spatio-temporally by signaling modulators, such as Sprouty proteins. The four mammalian Sprouty family members are palmitoylated phosphoproteins that can attenuate or potentiate numerous growth factor-induced signaling pathways. Previously, we have shown that Sprouty-1 and Sprouty-2 associate with Caveolin-1, the major structural protein of caveolae. Like Sprouty, Caveolin-1 inhibits growth factor-induced mitogen-activated protein kinase activation. Here, we demonstrate that all four mammalian Sprouty family members physically interact with Caveolin-1. The C terminus of Caveolin-1 is the major Sprouty-binding site, whereas Sprouty binds Caveolin-1 via its conserved C-terminal domain. A single point mutation in this domain results in loss of Caveolin-1 interaction. Moreover, we demonstrate that the various Sprouty isoforms differ dramatically in their cooperation with Caveolin-1-mediated inhibition of mitogen-activated protein kinase activation and that such cooperation is also highly dependent on the type of growth factor investigated and on cell density. Together, the data suggest that the Sprouty/Caveolin-1 interaction modulates signaling in a growth factor- and Sprouty isoform-specific manner. Growth factor-mediated signal transduction cascades can be regulated spatio-temporally by signaling modulators, such as Sprouty proteins. The four mammalian Sprouty family members are palmitoylated phosphoproteins that can attenuate or potentiate numerous growth factor-induced signaling pathways. Previously, we have shown that Sprouty-1 and Sprouty-2 associate with Caveolin-1, the major structural protein of caveolae. Like Sprouty, Caveolin-1 inhibits growth factor-induced mitogen-activated protein kinase activation. Here, we demonstrate that all four mammalian Sprouty family members physically interact with Caveolin-1. The C terminus of Caveolin-1 is the major Sprouty-binding site, whereas Sprouty binds Caveolin-1 via its conserved C-terminal domain. A single point mutation in this domain results in loss of Caveolin-1 interaction. Moreover, we demonstrate that the various Sprouty isoforms differ dramatically in their cooperation with Caveolin-1-mediated inhibition of mitogen-activated protein kinase activation and that such cooperation is also highly dependent on the type of growth factor investigated and on cell density. Together, the data suggest that the Sprouty/Caveolin-1 interaction modulates signaling in a growth factor- and Sprouty isoform-specific manner. Receptor tyrosine kinases (RTKs) 3The abbreviations used are: RTK, receptor tyrosine kinase; Spry, Sprouty; Cav-1, Caveolin-1; aa, amino acids; ERK, extracellular-regulated kinase; m, mouse; h, human; Ad, adenovirus; EGFP, enhanced green fluorescent protein; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; EGF, epidermal growth factor; HEK, human embryonic kidney; TBST, Tris-buffered saline with 0.05% Tween 20; GST, glutathione S-transferase; HA, hemagglutinin; PBS, phosphate-buffered saline. are single membrane-spanning receptors that transduce signals from extracellular proteins, such as growth factors and hormones, thereby controlling numerous cellular processes, including migration, proliferation, differentiation, apoptosis, and survival (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6245) Google Scholar, 2Schlessinger J. 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Here, we have set out to characterize the biochemical interaction between Spry and Cav-1, explore its functional significance in the context of Spry activity (i.e. inhibition of p42/44 ERK activation), and systematically compare the four murine Spry proteins in the same cellular context. We demonstrate that all Spry isoforms physically interact with Cav-1 in a complex, multidomain-dependent manner. Yet, despite their binding to Cav-1, the various Spry family members exhibit differential cooperativity with Cav-1 in repressing p42/44 ERK activation. Moreover, dependent on the type of growth factor stimulation, Spry family members exert varying inhibitory activities on signal transduction. Thus, the interaction of the four Spry proteins with Cav-1 appears to play a complex, growth factor- and Spry isoform-dependent role in modulating signal transduction. Chemicals and Reagents—All chemicals used were obtained from Sigma or Merck unless otherwise indicated. Restriction enzymes were obtained from New England Biolabs (Frankfurt, Germany), Invitrogen, or Roche Diagnostics. Cloning and Plasmids—The four mSpry cDNAs were isolated as previously described (15Impagnatiello M.A. Weitzer S. Gannon G. Compagni A. Cotten M. Christofori G. J. Cell Biol. 2001; 152: 1087-1098Crossref PubMed Scopus (239) Google Scholar) and were cloned into pSG9M (58Green S. Walter P. Kumar V. Krust A. Bornert J.M. Argos P. Chambon P. Nature. 1986; 320: 134-139Crossref PubMed Scopus (1961) Google Scholar, 59Green S. Issemann I. Sheer E. Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (547) Google Scholar), which encodes a myc epitope on the N terminus. The cDNAs encoding the dominant-negative versions of all four myc-tagged mSpry proteins (mSpry1-Y53A, mSpry2-Y55A, mSpry3-Y27A, and mSpry4-Y53A) and the RD mutants (mSpry1-R249D, mSpry3-R221D, and mSpry4-R235D) were produced using the QuikChange Site-directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions and sequenced for confirmation. Full-length mSpry2 was cloned in-frame into pcDNA3.1/V5-His-TOPO (Invitrogen) using KpnI/XbaI by employing Pwo polymerase (Roche Diagnostics) and the following primers, 5′-CGCGGTACCGCCACCATGGAGGCCAGAGCTCAGAG-3′ and 5′-TGCTCTAGAAATGTCGGCTTTTCAAAGTTCCTG-3′, in a PCR amplification reaction. The N-terminal portion of mSpry2 (encoding aa 1-176) was cloned in-frame into pcDNA3.1/V5-His-TOPO using KpnI/XbaI by employing the following primers, 5′-CGCGGTACCGCCACCATGGAGGCCAGAGCTCAGAG-3′ and 5′-TGCTCTAGAAAGTAGGCATGGAGACCCAAATCATC-3′. The C-terminal portion of mSpry2 (encoding aa 177-315) was cloned in-frame into pcDNA3.1/V5-His-TOPO using BamHI/XbaI by employing the following primers, 5′-CGCGGATCCGCCACCATGAGGTGTGAGGACTGCGGC-3′ and 5′-TGCTCTAGAAATGTCGGCTTTTCAAAGTTCCTG-3′. The murine Cav-1 cDNA was isolated from NIH3T3 cells by reverse transcriptase-PCR. Briefly, RNA was isolated from NIH3T3 cells using TRIzol (Invitrogen), and random hexamers and Superscript II (Invitrogen) were used to generate single-stranded DNA. Pwo polymerase and primers 5′-CGGGATCCGCCACCATGTCTGGGGGCAAATAC-3′ and 5′-CCGCTCGAGTCATATCTCTTTCTGCGTGC-3′ were used for subsequent PCR amplification. The Cav-1 cDNA was cloned into pSG9M and pcDNA3.1 (Invitrogen) using BamHI/XhoI and sequenced for confirmation. The enhanced green fluorescent protein (EGFP) open reading frame from pEGFP-N2 (Clontech) was excised using BamHI/NotI and cloned into the respective sites of the pcDNA3.1 plasmid to generate the pcDNA3.1/EGFP construct. The human (h) Spry2 cDNA was amplified by PCR from human genomic DNA isolated (60Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1997Google Scholar) from HEK293T cells using the following primers, 5′-CGGGATCCGCCTGCTGGAGTGACCACAC-3′ and 5′-CCGCTCGAGTCCTCATTACTGTAATATTCCTGATT-3′. All cDNAs were cloned into pcDNA3.1 and sequenced. The pFLAG-hSpry2-R252D plasmid was a kind gift from Dr. Graeme Guy (Institute of Molecular and Cell Biology, Singapore) (61Lim J. Yusoff P. Wong E.S. Chandramouli S. Lao D.H. Fong C.W. Guy G.R. Mol. Cell Biol. 2002; 22: 7953-7966Crossref PubMed Scopus (69) Google Scholar). The hSpry2-R252D mutant cDNA was subcloned into pcDNA3.1 and sequenced for confirmation. Cell Culture—All reagents used for cell culture were obtained from Sigma. Human embryonic kidney cells (HEK293T), human breast carcinoma cells (T47D), African green monkey kidney cells (COS7), and human cervical carcinoma cells (HeLa) were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA). All cells were maintained at 37 °C with 5% CO2. HEK293T, HeLa, and COS7 cells were cultured in high glucose Dulbecco's modified Eagle's medium, containing 10% fetal bovine serum and 2 mm glutamine, whereas T47D cells were cultured in RPMI 1640 with the same supplements as above. Cells were passaged using trypsin-EDTA and counted manually or using a Coulter counter (Beckman Coulter, Nyon, Switzerland). Stable Transfections—The pcDNA3.1/Cav-1 and pcDNA3.1/EGFP plasmids were linearized with PvuI and transfected into T47D cells in a 6-well plate using PerFectin (Gene Therapy Systems, Inc., San Diego, CA) according to the manufacturer's instructions. 48 h post-transfection cells were trypsinized and plated onto 10-cm dishes. Stably transfected clones for pcDNA3.1/mCav-1 (T47D-Cav-1) were selected by addition of 0.5 mg/ml G418/Geneticin (Invitrogen). A few clones were selected and characterized by immunoblotting as described below and one clone was used for the experiments described herein. For the pcDNA3.1/EGFP transfectants (T47D-EFGP), cells cultured in 0.5 mg/ml G418/Geneticin were selected, pooled, and employed in the experiments described below. Transient Transfections—HEK293T cells were transiently transfected using PerFectin or Metafectene (Biontex, Munich, Germany). COS7 cells were transiently transfected using Metafectene. For co-transfections, the ratio of two plasmids was 1:1 and was normalized using empty vectors. HeLa cells were transiently transfected using FuGENE 6 (Roche Diagnostics). Transfection with pcDNA3.1/EGFP was used to monitor the transfection efficiency. Adenoviral Vectors and Stimulation Experiments—Adenoviral constructs encoding the mSpry cDNAs (AdmSpry1-4) and the firefly luciferase cDNA (AdLite) were generated as described previously (15Impagnatiello M.A. Weitzer S. Gannon G. Compagni A. Cotten M. Christofori G. J. Cell Biol. 2001; 152: 1087-1098Crossref PubMed Scopus (239) Google Scholar, 62Michou A.I. Lehrmann H. Saltik M. Cotten M. J. Virol. 1999; 73: 1399-1410Crossref PubMed Google Scholar). Amplification of the virus was carried out in HEK293 cells and virus particles were purified from cell lysates using cesium chloride gradients and gel filtration (62Michou A.I. Lehrmann H. Saltik M. Cotten M. J. Virol. 1999; 73: 1399-1410Crossref PubMed Google Scholar). Viral quantities were based on protein content using the conversion of 1 mg of viral protein/3.4 × 1012 virus particles. For viral infection of T47D cells, culture medium was replaced with starvation medium (RPMI 1640, 2 mm glutamine, no sera) containing 2,500 virus particles per cell. After 5 h, the medium was replaced with fresh growth medium. After viral infection, cells were allowed to recover for ∼5 h and then starved overnight (at least 14 h) in starvation medium. Next, cells were stimulated for the times indicated by adding 50 ng/ml of either recombinant human basic FGF (FGF2) or EGF (both from either Catalys AG/Promega, Wallisellen, Switzerland, or Sigma). Immunoprecipitations—Cells were lysed for 30 min on ice in lysis buffer (1% Triton X-100, 160 mm NaCl, 20 mm Tris, pH 8.0, 2mm Na3VO4, 10 mm NaF, and 1:200 dilution of stock protease inhibitor mixture for mammalian cells (Sigma)) and cleared by centrifugation (14,000 × g, 30 min, 4 °C). Protein concentration was determined using a modified Bradford protocol (Bio-Rad Protein Assay). For immunoprecipitations, equal amounts of lysates were incubated overnight (4 °C on rotator) with either preimmune sera or sera specific for one of the Spry isoforms. Protein G-Sepharose beads (Sigma) (10% v/v in lysis buffer) were added to each tube and allowed to incubate with the immune complexes for at least 1 h (4 °C on rotator). Immunoprecipitate-bead complexes were washed three times in cold lysis buffer, an equal volume of 2× SDS-PAGE loading buffer (20% glycerol, 4% SDS, 0.13 m Tris, bromphenol blue (1 mg/100 ml), 2% β-mercapthoethanol) was added to the washed beads followed by boiling of the samples. Immunoblotting—Total cell lysates and immunoprecipitates were resolved by 12% SDS-PAGE (63Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). SDS-PAGE gels were transferred to polyvinylidene difluoride (Millipore, Volketswil, Switzerland) by semi-dry transfer in Towbins buffer (20% methanol, 25 mm Tris, 192 mm glycine), blocked with either 4% bovine serum albumin or 5% skim milk powder in Tris-buffered saline with 0.05% Tween 20 (TBST). All primary and secondary antibodies were diluted in 5% skim milk powder in TBST. Detected proteins were visualized using enhanced chemiluminescence (GE Healthcare or Interchim, Montluçon, France). Membranes were stripped by incubation in 200 mm glycine, pH 2.5 (30 min, room temperature), followed by washing with TBST. Rabbit sera against mSpry1 and mSpry2 were generated as previously described (15Impagnatiello M.A. Weitzer S. Gannon G. Compagni A. Cotten M. Christofori G. J. Cell Biol. 2001; 152: 1087-1098Crossref PubMed Scopus (239) Google Scholar). Rabbit sera against mSpry3 were generated with a peptide corresponding to C-terminal residues (RKISSSSSPFPKAQEKSV) conjugated to keyhole limpet hemocyanin. Rabbit sera against mSpry4 were generated with peptides corresponding to either N-(PLLDSRAPHSRLQHP) or C-terminal (AASGDTKTSRSDKPF) residues conjugated to keyhole limpet hemocyanin. Antibody specificity
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