Dynamic interaction of CD2 with the GYF and the SH3 domain of compartmentalized effector molecules

Abstract

Intracellular protein interaction domains are essential for eukaryotic signaling. In T cells, the CD2BP2 adaptor binds two membrane‐proximal proline‐rich motifs in the CD2 cytoplasmic tail via its GYF domain, thereby regulating interleukin‐2 production. Here we present the structure of the GYF domain in complex with a CD2 tail peptide. Unlike SH3 domains, which use two surface pockets to accommodate proline residues of ligands, the GYF domain employs phylogenetically conserved hydrophobic residues to create a single interaction surface. NMR analysis shows that the Fyn but not the Lck tyrosine kinase SH3 domain competes with CD2BP2 GYF‐domain binding to the same CD2 proline‐rich sequence in vitro. To test the in vivo significance of this competition, we used co‐immunoprecipitation experiments and found that CD2BP2 is the ligand of the membrane‐proximal proline‐rich tandem repeat of CD2 in detergent‐ soluble membrane compartments, but is replaced by Fyn SH3 after CD2 is translocated into lipid rafts upon CD2 ectodomain clustering. This unveils the mechanism of a switch of CD2 function due to an extracellular mitogenic signal.

Protein Interactions between CD2 and Lck Are Required for the Lipid Raft Distribution of CD2

“…10D). Lck and Fyn bind to nonoverlapping sequences of CD2 (21,22), but other CD2 partners compete for these sequences. The CD2 proline-rich motif PPPPGHR is complexed with the GYF domain of CD2BP2, but only outside lipid rafts, as upon translocation, the SH3 domain of Fyn displaces CD2BP2 (22).…”

“…Lck and Fyn bind to nonoverlapping sequences of CD2 (21,22), but other CD2 partners compete for these sequences. The CD2 proline-rich motif PPPPGHR is complexed with the GYF domain of CD2BP2, but only outside lipid rafts, as upon translocation, the SH3 domain of Fyn displaces CD2BP2 (22). The penultimate CD2 proline motif PPLPRPR has been reported to associate to the SH3 domains of both Lck and CD2BP1 (21,52), and so it is possible that the association between CD2 and Lck may be similarly regulated and confined to lipid rafts as well.…”

“…Human CD2 has been shown to translocate to lipid rafts upon CD2 Ab cross-linking or following binding to CD58 during conjugate formation (34), and the shift between phases may result in the replacement of CD2BP2 by raft-resident Fyn (22), which competes for the same proline-rich sequence of the cytoplasmic tail of CD2. In contrast to human CD2, which is reported to be entirely non-raftresident in resting cells (34), mouse CD2 contains a CIC motif at the membrane-proximal region that can putatively address CD2 to lipid rafts (32).…”

Proline-rich Sequence Recognition

“…5 l of the sample were injected and concentrated on a precolumn (PepMap C 18 , 5 m, 100 Å, 5 mm ϫ 300-m inner diameter, Dionex, Idstein, Germany). LC separations were performed on a capillary column (Atlantis dC 18 The processed MS/MS spectra and MASCOT server (version 2.0, Matrix Science Ltd., London, UK) were used to search in-house against the UniProt/Swiss-Prot database (release 50.0 of September 21, 2006, containing 234,112 sequence entries, comprising 85,963,701 amino acids). Finally all protein identities were updated using the latest UniProtKB/Swiss-Prot release (release 56.7 of January 20, 2009, containing 408,099 sequence entries, comprising 147,085,246 amino acids).…”

“…One interesting spliceosomal protein that mediates PRS interactions is CD2BP2/52K (14). It is a component of the U5 snRNP (15, 16) and contains a GYF adaptor domain that recognizes PRS via a set of conserved aromatic amino acids (17)(18)(19). A likely interaction partner of the GYF domain is the core splicing protein SmB/BЈ, which is present in all snRNPs.…”