Novel Interaction Partners of the CD2BP2-GYF Domain

Abstract

The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B′ were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis. The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B′ were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis. The CD2BP2 protein is a cellular adapter protein that was originally identified as a binding partner of the T cell adhesion protein CD2 in the context of T cell signaling (1Nishizawa K. Freund C. Li J. Wagner G. Reinherz E.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14897-14902Crossref PubMed Scopus (91) Google Scholar). Subsequently, a nuclear role for CD2BP2 was suggested, based on reports that identified CD2BP2 as a protein associated with spliceosomal complexes and proteins involved in splicing (2Hartmuth K. Urlaub H. Vornlocher H.P. Will C.L. Gentzel M. Wilm M. Luhrmann R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16719-16724Crossref PubMed Scopus (242) Google Scholar, 3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). CD2 and SmB/B′ share the presence of (R/K/G)XXP-PGX(R/K) motifs that were shown to bind to CD2BP2 in vitro and in vivo (1Nishizawa K. Freund C. Li J. Wagner G. Reinherz E.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14897-14902Crossref PubMed Scopus (91) Google Scholar, 3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 4Freund C. Dotsch V. Nishizawa K. Reinherz E.L. Wagner G. Nat. Struct. Biol. 1999; 6: 656-660Crossref PubMed Scopus (79) Google Scholar). CD2BP2 contains a C-terminal fragment of ∼60 amino acids that confers binding to these proline-rich sequences. Structurally, this so-called GYF domain represents a small α/β protein that displays a set of aromatic residues from a unique bulge-helix-bulge motif creating a hydrophobic pocket accommodating the PPG core of the binding peptide (1Nishizawa K. Freund C. Li J. Wagner G. Reinherz E.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14897-14902Crossref PubMed Scopus (91) Google Scholar, 4Freund C. Dotsch V. Nishizawa K. Reinherz E.L. Wagner G. Nat. Struct. Biol. 1999; 6: 656-660Crossref PubMed Scopus (79) Google Scholar, 5Freund C. Kuhne R. Yang H. Park S. Reinherz E.L. Wagner G. EMBO J. 2002; 21: 5985-5995Crossref PubMed Scopus (69) Google Scholar). In addition to this core sequence, the positively charged arginine residues in the vicinity of the PPG motif contribute to binding of the CD2 and SmB/B′ ligand. Interestingly, the PPPPGHR motifs in CD2 can also be bound by the SH3 domain of the Fyn tyrosine kinase and show that proline-rich sequence recognition has ultimately to be viewed across domain borders (5Freund C. Kuhne R. Yang H. Park S. Reinherz E.L. Wagner G. EMBO J. 2002; 21: 5985-5995Crossref PubMed Scopus (69) Google Scholar). Functionally the protein-protein interactions mediated by the PPPPGHR motifs are associated with a modulation in CD2-mediated signaling events, as for example interleukin-2 production (6Chang H.C. Moingeon P. Pedersen R. Lucich J. Stebbins C. Reinherz E.L. J. Exp. Med. 1990; 172: 351-355Crossref PubMed Scopus (37) Google Scholar, 7Hahn W.C. Bierer B.E. J. Exp. Med. 1993; 178: 1831-1836Crossref PubMed Scopus (36) Google Scholar). In the case of the CD2BP2-SmB/B′ interaction, it was shown that both proteins colocalize to the nucleus of Jurkat and HeLa cells and that the GYF domain of CD2BP2 can coprecipitate SmB/B′ from cellular lysates (3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). However, CD2BP2 has not been detected in the active spliceosome (8Makarova O.V. Makarov E.M. Urlaub H. Will C.L. Gentzel M. Wilm M. Luhrmann R. EMBO J. 2004; 23: 2381-2391Crossref PubMed Scopus (151) Google Scholar), and its role in splicing or splicing-associated processes is still poorly understood. Because proline-rich sequence recognition is characterized by high off-rates and limited specificity (9Kay B.K. Williamson M.P. Sudol M. FASEB J. 2000; 14: 231-241Crossref PubMed Scopus (1044) Google Scholar), additional intracellular proteins are likely to interact with CD2BP2-GYF. Therefore, we attempted to find new binding partners for CD2BP2 based on the recognition code of its GYF domain. Our results show that in addition to CD2 and SmB/B′ several human proteins contain high affinity target sites for the CD2BP2-GYF domain. Yeast two-hybrid analyses confirm that two of these novel interactions, namely with NPWBP and PI31, can take place under cellular conditions. Constructs and Protein Expression—The recombinant expression of the GYF domain and the glutathione S-transferase (GST) 2The abbreviations used are: GST, glutathione S-transferase; NMR, nuclear magnetic resonance; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; snRNP, small nuclear ribonucleoprotein.-GYF fusion protein used in this study was performed according to published protocols (3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). For yeast two-hybrid analysis, full-length CD2BP2 or a fragment comprising residues 256–342 was cloned into pGADT7 (Clontech) via the NcoI-BamHI and the EcoRI-BamHI restriction sites, respectively. Fragments of the proteins AKNA-(795–894), MAGD1-(357–514), NEDD4-(267–328), NPWBP-(388–551), PI31-(214–271), SmB-(148–231), SWAN-(700–869), and WWP2-(293–491) were amplified from the following cDNA clones obtained from the Deutsches Ressourcenzentrum für Genomforschung GmbH: DKFZp667H017Q2 (AKNA), IRALp962H0828Q2 (MAGD1), IRAK-p961C1479Q2 (NED-D4), IRALp962P0114Q2 (NPWBP), IRAKp961G1751Q2 (PI31), IMA-Gp998D118415Q3 (SmB), IRAL-p962K1725Q2 (SWAN), and IRAK-p961B0119Q2 (WWP2). These fragments, comprising the proline-rich regions of the respective proteins, were inserted into pGBKT7 vector via NcoI-NotI restriction sites. The fragment coding for the cytoplasmic tail of CD2-(245–351) was inserted into the pGBKT7 via the EcoRI and BamHI restriction sites. Cloning of the focused library RKRSHRXXPPPXXXVQ into PC89 was similar to the procedure described elsewhere (10Felici F. Castagnoli L. Musacchio A. Jappelli R. Cesareni G. J. Mol. Biol. 1991; 222: 301-310Crossref PubMed Scopus (391) Google Scholar). PC89 and the PC89-nonapeptide-library (X9) were a gift from Gianni Cesareni (Dipartimento di Biologia, Universita di Roma). Phage Display—Phage displaying the nonapeptide (X9) or the focused peptide library fused to the major capsid protein was produced by transforming Escherichia coli XL-1 Blue cells with PC89 constructs followed by superinfection with the VCS-M13 helper phage (Stratagene). After overnight incubation in 2xYT medium in the presence of ampicillin and kanamycin (30 °C, 270 rpm), phage particles were purified by three polyethylene glycol/NaCl precipitations (11Yamamoto K.R. Alberts B.M. Benzinger R. Lawhorne L. Treiber G. Virology. 1970; 40: 734-744Crossref PubMed Scopus (961) Google Scholar). Library screening was performed as follows: 30–50 μl of GST-GYF-loaded glutathione-Sepharose 4B gel (Amersham Biosciences) were incubated with 5 × 109–5 × 1011 infectious particles at 4 °C overnight in phosphate-buffered saline. After washing three times with phosphate-buffered saline, bound phages were eluted with 100 mm glycine-HCl, pH 2.2. For phage amplification, E. coli XL-1 Blue cells were infected with eluted phage followed by superinfection with helper phage and subsequent incubation as described above. After three or six rounds of panning, the inserts of selected phage were sequenced to identify their displayed peptide. SPOT Analysis—Peptides were synthesized on Whatman 50 paper using an Auto-Spot Robot (ASP 222; Invartis AG, Köln, Germany). Peptide synthesis using Fmoc chemistry on β-alanine-functionalized cellulose membranes was performed according to standard protocols (12Kramer A. Schneider-Mergener J. Methods Mol. Biol. 1998; 87: 25-39PubMed Google Scholar). Membranes were probed with GST-GYF protein as described elsewhere (13Heuer K. Kofler M. Langdon G. Thiemke K. Freund C. Structure (Camb.). 2004; 12: 603-610Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Briefly, the membranes were incubated with GST-GYF (40 μg/ml) overnight. After washing, bound GST fusion protein was detected with rabbit polyclonal anti-GST antibodies (Z-5; Santa Cruz Biotechnology) and horseradish peroxidase-coupled anti-rabbit IgG antibodies (Rockland). An enhanced chemiluminescence substrate (SuperSignal West Pico; Pierce) was used for detection by a Lumi-Imager™ (Roche Applied Science). Preparation of Peptides—Peptides were synthesized automatically (ABI 433A) by the solid phase method using standard Fmoc chemistry in a batch-wise mode as described previously for the synthesis of CRF analogs (14Beyermann M. Fechner K. Furkert J. Krause E. Bienert M. J. Med. Chem. 1996; 39: 3324-3330Crossref PubMed Scopus (51) Google Scholar). After final cleavage/deprotection using trifluoroacetic acid/H2O (9/1), crude peptides were purified by preparative HPLC to give final products of 95% purity according to HPLC analysis. The expected masses of peptides were verified by mass spectrometry. NMR Titration Experiments—All NMR experiments were performed at 297 K using either Bruker DRX600 or DMX750 instruments equipped with standard triple resonance probes. Data processing and analysis were carried out with the XWINNMR (Bruker) and SPARKY (version 3.1, T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco) software packages. In the NMR titration experiments, increasing amounts of synthetic peptides were added to a 0.2-mm sample of the 15N-labeled CD2BP2-GYF domain. The gradual change of chemical shifts in the HSQC spectra allowed the resonances of the ligand-bound GYF domain to be unambiguously assigned. The sum of the chemical shift changes for 15N and 1H atoms in a sample with peptide was determined as [(Δ1H)2 + (Δ15N)2]1/2, where Δ1H is in units of 0.1 ppm, and Δ15N is in units of 0.5 ppm. Data were analyzed with the Microcal™ Origin™ program, assuming a simple two-state binding model. Yeast Two-hybrid Analysis—Yeast two-hybrid experiments were performed with the MATCHMAKER GAL4 two-hybrid system 3 according to the manufacturer's manual (Clontech). The yeast strain AH109 was cotransformed with the pGBKT7 bait constructs encoding the GAL4 DNA binding domain fused to the proline-rich regions of selected candidates and full-length CD2BP2 or its GYF domain in pGADT7. Colonies were grown on low, medium, or high stringency (synthetic drop-out) media without the amino acids Leu and Trp, or His, Leu, and Trp, or Ala, His, Leu, and Trp, respectively). Phage Display and Peptide Substitution Analysis—The sequence requirements for the binding of peptides to the CD2BP2-GYF domain were analyzed by screening either a focused library of the format X2PPPX3, or of a randomized X9 phage display library. For both libraries, similar results were obtained (TABLE ONE). Almost all sequences contain the PPG motif, in agreement with the previously defined recognition signature (R/K/G)XXPPGX(R/K) (3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and SPOT analysis confirmed that only peptides comprising the PPG motif conferred binding to the CD2BP2-GYF domain (TABLE ONE). A third, N-terminal proline is often present in peptides selected from the X9 library, but other amino acids are also found at this position, indicating that an extended stretch of PPII helix is not required for binding. Tryptophan is selected as the amino C-terminal to hydrophobic amino acids and were also at this position, the and were not sequences analyzed only sequence the CD2 motif Interestingly, for peptides containing the motif, for the presence of a positively charged amino was is in to the recognition signature of peptides from on CD2 and SmB/B′ and the CD2 of peptides in this study (1Nishizawa K. Freund C. Li J. Wagner G. Reinherz E.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14897-14902Crossref PubMed Scopus (91) Google Scholar, 3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Therefore, based on the phage display we a of for the CD2BP2-GYF the not on positively charged amino the In the case of the CD2 the within the motif from the GYF domain the arginine or at to at interactions via its its charged in to the charged of and of the GYF domain (5Freund C. Kuhne R. Yang H. Park S. Reinherz E.L. Wagner G. EMBO J. 2002; 21: 5985-5995Crossref PubMed Scopus (69) Google In for the a hydrophobic interaction the peptide following and of the domain is likely to take display results 3 and rounds of with the X9 and the The CD2BP2 GST-GYF fusion was used for The consensus is from results of both library of the of was from the with the library. The consensus is defined to all binding The stretch in the focused library as as of the binding motif are in with residues with presence in both are shown in with of selected sequences rounds of and the peptide were to SPOT analysis, performed as described in of the binding mode of the and of the GYF domain that the of the pocket are shown as with the conserved and residues in and the residues and as on The respective peptides are shown in and The the peptide arginine of the CD2 peptides and the charged of the GYF is by an In the case of the the of the domain and the of the peptide are to hydrophobic the from the phage display analysis, the peptide of the containing the motif was to SPOT analysis. the for a cellulose where of the binding peptide was by all other amino The motif PPG is for binding and a for Trp, and Leu at the to the PPG core be The also show and according to a from the binding mode to the CD2 binding mode for the motif for the binding. In with the results from phage we not a modulation of binding for the that as was for the CD2 not a role for the peptide. this the two of we performed analysis of where two were In the results of a analysis for peptides of the sequence are Although the of a in the peptides the peptides a of The and to a the of the arginine residues the core that the aromatic residues with the with for the interactions mediated by arginine in the CD2 of of by Data The phage display results the consensus as the signature that all high affinity binding motifs for the CD2BP2-GYF domain. In the presence of two motifs that are in sequence and likely interaction sites, as has been shown for the PPPPGHR motif in the CD2 cytoplasmic tail (5Freund C. Kuhne R. Yang H. Park S. Reinherz E.L. Wagner G. EMBO J. 2002; 21: 5985-5995Crossref PubMed Scopus (69) Google Scholar). The and were for proteins that contain at two of the consensus sites or a consensus motif the peptides from the respective proteins were synthesized on a cellulose and for binding to a GST-GYF fusion The results are in 3 and show that of the sequences are bound by CD2BP2-GYF. that the domain bound to or positively charged CD2 not In the of binding to a charged or following the PPG motif was in agreement with results from the analysis on the of the the peptides were and a set of candidates containing several binding sites, of at to the consensus were selected for analysis (TABLE candidates for yeast two-hybrid analysis from the human analysis were selected according to binding and of motifs present in the target of the proteins comprising the proline-rich sequences are The motifs to the phage display-derived consensus or to the CD2 consensus are shown in and respectively. motifs by the consensus are as candidates for yeast two-hybrid analysis from the human analysis were selected according to binding and of motifs present in the target of the proteins comprising the proline-rich sequences are The motifs to the phage display-derived consensus or to the CD2 consensus are shown in and respectively. motifs by the consensus are as of in confirm the binding of the CD2BP2-GYF domain to peptides identified as by phage display and SPOT analysis, NMR titration experiments were performed Because GYF domain and peptide are in with to the NMR the change of resonances in of be followed and a be determined a simple two-state binding was of the putative target peptides bound to the GYF and the for the phage display-derived peptide and for the CD2 peptide that the phage display method selected for a high affinity ligand. The NMR experiments also the binding for the peptides to be The in chemical shift and bound GYF domain for residues are shown in 4B and that the major interaction is by the bulge-helix-bulge motif of the as was previously shown in for the peptide The of of the GYF domain that display chemical shift changes is similar for all that an of the GYF domain interaction is not required for binding of the peptide Yeast Two-hybrid that the interactions can take place in the context of the proteins within a cellular full-length CD2BP2 or its GYF domain were cloned into the vector and of putative interaction partners were cloned into the bait vector of the yeast two-hybrid interaction partners selected for the analysis were and comprising interaction sites, the two interaction partners CD2 and to the CD2 and PI31, contains a binding of (TABLE In the results of this interaction analysis are of the putative binding namely and show as bait into interactions be analyzed by this to interact with of the CD2BP2 to only or interactions with the CD2BP2-GYF under its In these two of the the binding motifs these not be in yeast in to interactions with the CD2BP2-GYF domain. However, NPWBP and PI31, in addition to CD2 and in of of the interaction for the full-length CD2BP2 and the GYF that these interactions are mediated by the GYF domain. In this study we used phage display and peptide analysis to the recognition code for the CD2BP2-GYF domain. the were used to binding motifs within proteins by from these proteins comprising the binding motif were for binding on cellulose and proteins of were to a yeast two-hybrid analysis. Interestingly, the CD2 motif is a that with a affinity with the phage display motif However, the presence of two motifs the interaction and results in yeast two-hybrid interactions that to on media The for the SmB/B′ C-terminal but in this case GYF binding motifs are displayed by the to the results a of was the in addition to the previously described CD2 (3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The of is characterized by the motif and aromatic residues the interaction to The a two C-terminal to the PPG core sequence and is defined in this study as For this it was shown that to the presence of motifs the affinity of binding sites and in vivo interaction sites (3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 5Freund C. Kuhne R. Yang H. Park S. Reinherz E.L. Wagner G. EMBO J. 2002; 21: 5985-5995Crossref PubMed Scopus (69) Google Scholar). The of a defined set of GYF proteins in not the the of GYF proteins has been In CD2BP2 has been identified as an interaction partner of and other proteins associated with spliceosomal complexes also GYF domain binding sites (3Kofler M. Heuer K. Zech T. Freund C. J. Biol. Chem. 2004; 279: 28292-28297Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The yeast of was identified as an interaction partner of the splicing protein A. A. 2002; PubMed Scopus Google Scholar). The of this study two proline-rich sequences at the of to be for binding to the GYF domain of of these is also found at the of the yeast of Therefore, the interaction GYF of proteins and splicing-associated proteins, be conserved across The PI31 protein was characterized as a protein that the in vitro K. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the protein was shown to be a of the the of peptides by the major S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). Our analysis the PI31 protein to contain interaction sites for the CD2BP2-GYF domain in However, the are the interaction take place in cells and the two proteins to the in of and of the two proteins can be under cellular conditions. A that the of is the nuclear protein NPWBP or protein was described as a nuclear protein with binding that with the protein A. M. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). A subsequent study that the of NPWBP a cytoplasmic in with in human cells be G. D. J. T. S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). NPWBP has been described as a of the spliceosome R. 2002; PubMed Scopus Google and was identified as a protein of the (8Makarova O.V. Makarov E.M. Urlaub H. Will C.L. Gentzel M. Wilm M. Luhrmann R. EMBO J. 2004; 23: 2381-2391Crossref PubMed Scopus (151) Google Scholar). The proline-rich sequences of NPWBP that interact with CD2BP2-GYF were also shown to interact with the domain of A. M. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, both and the GYF domain of CD2BP2 were found to interact with S. Makarov E. Vornlocher H.P. D. T. Luhrmann R. PubMed Scopus Google Scholar, M. E. M. S. M. H. 2000; PubMed Scopus (69) Google Scholar), an splicing protein associated with the K. S. P. Luhrmann R. R. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, T. A. Sudol M. 2000; PubMed Scopus Google Scholar). Although CD2BP2 is not a of the active it is to be for the of the to the of the S. Makarov E. Vornlocher H.P. D. T. Luhrmann R. PubMed Scopus Google Scholar). Therefore, a interaction NPWBP and CD2BP2 to the context of experiments to the role of CD2BP2 and associated proteins in the of the the spliceosomal Gianni Cesareni for the vector and the randomized X9 phage library. also and Krause for peptide synthesis and and for peptide