Novel Interaction Partners of the CD2BP2-GYF Domain

Michael Kofler,Kathrin Motzny,Michael Beyermann,Christian Freund

doi:10.1074/jbc.m503989200

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.

Highlights

Results show that in addition to CD2 and SmB/BЈ several human proteins contain high affinity target sites for the CD2BP2-GYF domain
Fragments of the proteins AKNA-(795– 894), melanoma-associated antigen D1 (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 fur Genomforschung GmbH: DKFZp667H017Q2 (AKNA), IRALp962H0828Q2 (MAGD1), IRAK-p961C1479Q2 (NEDD4), IRALp962P0114Q2 (NPWBP), IRAKp961G1751Q2 (PI31), IMAGp998D118415Q3 (SmB), IRAL-p962K1725Q2 (SWAN), and IRAKp961B0119Q2 (WWP2)
Based on the phage display results, we suggest a second class of ligands for the CD2BP2-GYF domain, the PPGW class, which does not depend on positively charged amino acids, flanking the motif

Summary

MATERIALS AND METHODS

Constructs and Protein Expression—The recombinant expression of the GYF domain and the glutathione S-transferase (GST)2-GYF fusion protein used in this study was performed according to published protocols [3]. 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 fur Genomforschung GmbH: DKFZp667H017Q2 (AKNA), IRALp962H0828Q2 (MAGD1), IRAK-p961C1479Q2 (NEDD4), IRALp962P0114Q2 (NPWBP), IRAKp961G1751Q2 (PI31), IMAGp998D118415Q3 (SmB), IRAL-p962K1725Q2 (SWAN), and IRAKp961B0119Q2 (WWP2) These fragments, comprising the proline-rich regions of the respective proteins, were inserted into pGBKT7 vector via NcoI-NotI restriction sites. Library screening was performed as follows: 30 –50 ␮l of GST-GYF-loaded glutathioneSepharose 4B gel (Amersham Biosciences) were incubated with 5 ϫ 109–5 ϫ 1011 infectious particles at 4 °C overnight in phosphate-buffered saline. The CD2BP2-GYF Domain Recognition Code tide synthesis using Fmoc chemistry on ␤-alanine-functionalized cellulose membranes was performed according to standard protocols [12]. 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)

TABLE ONE

RESULTS

TABLE TWO

DISCUSSION