A Novel Pro-Arg Motif Recognized by WW Domains

Abstract

WW domains mediate protein-protein interactions through binding to short proline-rich sequences. Two distinct sequence motifs, PPXY and PPLP, are recognized by different classes of WW domains, and another class binds to phospho-Ser-Pro sequences. We now describe a novel Pro-Arg sequence motif recognized by a different class of WW domains using data from oriented peptide library screening, expression cloning, and in vitro binding experiments. Many signaling events in eukaryotic cells involve the assembly of large protein-protein complexes. These diverse associations are mediated through interactions of a limited number of modular signaling domains such as the SH2, 1 SH3, PDZ, and PTB domains and their respective ligands (1, 2). Particular families of domains occasionally share affinity for related ligands. For example, both SH2 and PTB domains can bind phosphotyrosine-containing substrates, while FHA domains, 14-3-3 proteins, and certain WW domains bind phosphoserine/ phosphothreonine-containing proteins (3). Recently, several unrelated signaling modules have been identified that specifically recognize proline-rich ligands. These include WW and EVH1 domains in addition to the canonical member of this group, the SH3 domain (4 -7). WW domains are encoded as 38 -40-amino acid long modules in single copy or tandem repeats in over 25 signaling proteins. WW domain-containing proteins include those implicated directly or indirectly in a variety of human diseases such as Liddle's syndrome, Duchenne muscular dystrophy, Huntington's disease, and Alzheimer's disease (6,8,9). Named after two highly conserved tryptophan residues characteristically spaced either 22 or 23 residues apart, WW domains participate in a variety of cellular processes, including ubiquitin-mediated protein degradation (10, 11), viral budding (12, 13), RNA splicing (14, 15), transcriptional co-activation (16 -18), and mitotic regulation (19,20). Recently another WW domain-containing protein, FBP30, was identified as one of the major genes up-regulated by p53 in thymocytes undergoing programmed cell death following ␥-irradiation (21). Despite their general importance in normal and disease states, most of the molecular targets recognized by different WW domains in vivo remain unidentified.Sudol and colleagues (22) have proposed a classification of WW domains based on their proline-based ligand specificity. In this scheme, Group I WW domains recognize and bind the motif PPXY, as typified by the prototype WW domain in Yes-associated protein (YAP), whereas Group II WW domains bind a PPLP motif, as observed for the WW domains in formin-binding protein (FBP)-11. A third class of WW domains bind sequences rich in Pro, Gly, and Met residues, although the exact amino acid sequence motif they recognize is not known. A fourth class of WW domain-containing proteins has recently been found that appears to bind Pro residues preceded by phosphoserine (23).In the present study we use oriented peptide library screening, phage-expression cloning, peptide binding studies, ...

Common Mechanism of Ligand Recognition by Group II/III WW Domains

“…The DNA encoding the second WW domain of Saccharomyces cerevisiae Rsp5 (Rsp5p(WW2)), human Fe65L2, and the first WW domain of mouse FBP30 (FBP30A) were amplified by PCR using their cDNAs as templates, which were kindly provided by Dr. H. Tanahashi and Dr. M. T. Bedford (16,24). The PCR products were inserted between the BamHI and EcoRI sites of the expression vector pGEX-4T-1 (Amersham Biosciences).…”

“…However, the classification between the Group II and III WW domains has been nebulous. For instance, the WW domain of Fe65 has been classified as Group II in one report (14) but as Group III in another report (16). In addition, there are a few proposed ligand motifs that can also bind some of the WW domains in these classes: the PGR motif and polyprolines (7,10,21,22).…”

“…Fe65 and ␤-amyloid precursor protein are involved in the reconstruction of actin cytoskeleton, which suggests the concerted action of these proteins with Mena and/or Abl (9,15). The WW domain of FBP30 has been reported to bind the Pro-Arg (PR) motif (10,16).…”

Regulation of p53 localization and transcription by the HECT domain E3 ligase WWP1

“…For example, the phosphorylation of Thr81 within this domain has been shown to be important in recruitment of the prolyl isomerase Pin1 (Zacchi et al, 2002), which contributes to p53 stability and activity following stress. In searching for E3 ligases that may associate with the proline-rich domains (Bedford et al, 2000;Verdecia et al, 2000), we have compared different members of the four WW-domains for their association with p53. Among those, WWP1 exhibited the strongest association (see data below).…”

WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins

“…WW domains are grouped according to their binding preference to a diverse set of prolinerich ligands (Bedford et al, 2000). There are four groups of WW domains: Group I binds PPXY ligands (Chen and Sudol, 1995), Group II binds PPLP (Chan et al, 1996), Group III binds polyproline sequences flanked by Arg or Lys (Bedford et al, 1998) and Group IV binds phospho-Ser-Pro or phospho-Thr-Pro (Lu et al, 1999).…”