A Novel Interaction between Atrophin-interacting Protein 4 and β-p21-activated Kinase-interactive Exchange Factor Is Mediated by an SH3 Domain

Abstract

Cross-talk between G protein-coupled receptors and receptor tyrosine kinase signaling pathways is crucial to the efficient relay and integration of cellular information. Here we identify and define the novel binding interaction of the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) with the GTP exchange factor ␤-p21-activated kinase-interactive exchange factor (␤PIX). We demonstrate that this interaction is mediated in part by the ␤PIX-SH3 domain binding to a proline-rich stretch of AIP4. Analysis of the interaction by isothermal calorimetry is consistent with a heterotrimeric complex with one AIP4-derived peptide binding to two ␤PIX-SH3 domains. We determined the crystal structure of the ␤PIX-SH3⅐AIP4 complex to 2.0-Å resolution. In contrast to the calorimetry results, the crystal structure shows a monomeric complex in which AIP4 peptide binds the ␤PIX-SH3 domain as a canonical Class I ligand with an additional type II polyproline helix that makes extensive contacts with another face of ␤PIX. Taken together, the novel interaction between AIP4 and ␤PIX represents a new regulatory node for G protein-coupled receptor and receptor tyrosine kinase signal integration. Our structure of the ␤PIX-SH3⅐AIP4 complex provides important insight into the mechanistic basis for ␤PIX scaffolding of signaling components, especially those involved in cross-talk.Two prominent transmembrane receptor families facilitate cellular communication with the extracellular environment: G protein-coupled receptors (GPCRs) 4 and receptor tyrosine kinases (RTKs). A wide array of ligands bind to these receptors to orchestrate signaling networks integral to many cellular functions. Internalization and degradation of receptor molecules from both families regulate signal duration and termination (1, 2). E3 ubiquitin ligases, which catalyze the ubiquitination of receptors, control intracellular sorting, recycling, and degradation (3-5). Representative E3 ligases include atrophininteracting protein 4 (AIP4), which ubiquitinates the GPCR chemokine receptor CXCR4 (6) and Cbl (Casitas B-lymphoma protein), which ubiquitinates the RTK epidermal growth factor (EGF) receptor (7-9). The importance of signal termination control is exemplified by recent work implicating prolonged signaling as a cause of cellular transformation (10, 11). In addition to mediating the ubiquitination and sorting of CXCR4, a number of recent studies detail the regulatory role AIP4 plays in developmental, immunological, and oncogenic signaling pathways (12-18). The AIP4/Itch protein is composed of an N-terminal C2 domain, followed by a proline-rich region, four WW domains, and a C-terminal catalytic HECT domain (3,4,12,13). AIP4 is abundantly expressed in most human tissues and displays tissue specificity similar to that of Cbl (19).␤-PAK-interactive exchange factor (␤PIX also referred to as Cool-1) is composed of modular domains, including an N-terminal SH3 domain followed by DH (Dbl, diffuse B-cell lymphoma homology) domain, PH (pleckstrin homology) domain, and a leucine...

Recognition of tandem PxxP motifs as a unique Src homology 3-binding mode triggers pathogen-driven actin assembly

“…Notably, the first Pro of such consensus peptides is accommodated by the specificity pocket of Abl SH3 analogously to the interaction of P 31 of R47 5 by W 378 and W 391 of IRTKS SH3. A similar interaction can be seen in an unrelated SH3/ligand interaction, namely, a typical intermediate affinity (7.4 μM) complex of βPIX SH3 with a class I ligand from atropin-interacting protein 4 (AIP4), which also involves a prolinedirected contact between βPIX SH3 and an extended PPII helical scaffold in the AIP4 peptide (32). However, in contrast to IRTKS: R47 5 , the βPIX:AIP4 complex involves a canonical polar interaction between an arginine residue of the AIP4 peptide and negatively charged residue in the specificity pocket of βPIX SH3, which accounts for much of the binding affinity.…”

SH3 domains: modules of protein–protein interactions

“…Noncanonical binding is observed in 53BP2, in which ankyrin repeats and the SH3 domain form a module that binds to p53 (Gorina and Pavletich 1996), immune cell adaptor SKAP55 interacting with the RKxxYxxY motif (Kang et al 2011), EPS8 recognizing PxxDY (Mongiovi et al 1999;Kesti et al 2007), GADS RxxK (Harkiolaki et al 2003), CIN85/CMS SH3 (Moncalián et al 2006) and βPIX SH3 (Janz et al 2007) interacting with PXXXPR. More sequences have been identified, including cortactin SH3 binding RxxPxxxP found in the cytoplasmic region of the calcium activated potassium (BK) channel (Tian et al 2006) and FUS1 binding Arg-Ser-rich sequences (Tong et al 2002).…”

“…Mutations in the SH3 domain of unconventional myosin VIIa have recently been shown to cause deafness in humans, with one mutation, A1628S, located directly in its SH3 domain (Wu et al 2011). The participation of modular protein domains in the regulation of signaling cascades of G protein-coupled receptors and receptor tyrosine kinases facilitates cross-talk between receptor families (Janz et al 2007). The acquisition of new SH3 domains leads to a new function (Jefferson et al 2007).…”

Distinct Peptide Binding Specificities of Src Homology 3 (SH3) Protein Domains Can Be Determined by Modulation of Local Energetics across the Binding Interface

“…The interaction of this "extended" region of target peptides with Surface II is complicated because this surface is relatively flat and displays little conservation across the SH3 domain family. In addition, the conformation of peptides interacting with this surface is variable (4), and different peptides binding the same domain may adopt distinct conformations when binding this surface (9,10).…”

“…These domains are ϳ60 residues long and are composed of five ␤-strands, which are sequentially joined by the RT, N-Src, and distal loops, and a short 3 10 -helix. SH3 domains generally recognize peptide sequences containing either ϩXXPXXP (class I) or PXXPXXϩ (class II) "core" motifs (where X can be variety of residues and ϩ represents either a Lys or Arg residue).…”