Novel Interaction Partners of the CD2BP2-GYF Domain

摘要

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