The Phosphotyrosine Peptide Binding Specificity of Nck1 and Nck2 Src Homology 2 Domains

Abstract

Nck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nck␣) and Nck2 (Nck␤ and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1.Dynamic processes in eukaryotic cells, such as cellular movement, changes in cell shape, and transport of vesicles, rely on constant remodeling of the actin cytoskeleton. Adapter proteins, essential in transmitting and modulating corresponding stimuli, frequently contain SH2 3 domains to recognize and bind tyrosine-phosphorylated motifs. Nck1 (Nck␣) and Nck2 (Nck␤ or Grb4) are two such adapter proteins (1-3), both bearing three SH3 domains and a C-terminal SH2 domain (4). Mice lacking both Nck genes are not viable, underscoring the importance of these adapters (1). A high sequence identity (68% overall and 82% for the SH2 domains) and single gene knockouts of Nck1 and Nck2(1) indicate that the function of the proteins may substantially overlap. Both bind receptor tyrosine kinases such as the PDGFR (5) and other tyrosine-phosphorylated proteins via their SH2 domains (3). However, Nck1 or Nck2 has also been reported to bind distinct targets. Exclusive Nck2 binders include EphrinB1 (6, 7), EphrinB2 (8), and Disabled-1 (Dab-1) (9), all involved in neuronal signaling. In the case of the PDGFR, Tyr (P) 751 is reported to be Nck1-specific (5), whereas Tyr(P) 1009 isNck2-specific (10). Furthermore, Nck1 and Nck2 have both been implicated in the infection process of enteropathogenic Escherichia coli (EPEC) (11), a frequent cause of severe infant diarrhea (12). EPEC adheres tightly to the membrane of intestinal enterocytes inducing massive remodeling of the microfilament system and suppression of microvilli (13,14). This involves the "translocated intimin receptor" (Tir), introduced into the host cell by a type III secretion system (11). Insertion of Tir into the host cell membrane (15) provides a binding site to the bacterial outer membrane protein intimin (16). Tir clustering induces phosphoryla...

EphA2 Engages Git1 to Suppress Arf6 Activity Modulating Epithelial Cell–Cell Contacts

“…It has been reported that Tyr392 of Git1 can be phosphorylated and that this phosphorylation enables its binding to the SH2 domain of Nck (Frese et al, 2006). We however found that mutation of Tyr392 into phenylalanine (Git1Y392F-FLAG) does not affect its complex formation with EphA2-HA ( Figure 6D).…”

“…In contrast, Git1 has been shown to bind to several adaptor proteins, such as Nck (Frese et al, 2006). Among the Nck isoforms, MDCK cells predominantly express Nck1 (Supplemental Figure S1).…”

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

“…Nck1 and Nck2 display 68% identity at the amino acid level and are regarded to be functionally redundant in many aspects although some Nck1-or Nck2-specific interactions/functions have been reported (reviewed in [2]). Nonetheless, binding specificities of the isolated SH2 domains of Nck1 and Nck2 are nearly the same and both recognize a YDEV consensus-binding sequence [35]. Although both Y 378 (YEDV) and Y 397 (YEEV) of HS1 closely resemble this consensus motif, Nck specifically binds to phosphorylated Y 378 but not to Y 397 .…”

“…Although both Y 378 (YEDV) and Y 397 (YEEV) of HS1 closely resemble this consensus motif, Nck specifically binds to phosphorylated Y 378 but not to Y 397 . This specificity might rely on a leucine residue in the +4 position of Y 397 , since it has been reported to be disfavored in this position and to impair Nck SH2 domain binding [35]. In contrast the Y 378 motif harbors a glutamic acid in the +4 position that is rather tolerated.…”

“…Nck1 and Nck2 SH2 domains (YDEV) [35]. To investigate the role of these phospho-sites for the interaction with the SH2 domain of Nck, we analyzed the interaction of transiently overexpressed EGFP-tagged Nck1 with WT HS1 and HS1 variants carrying tyrosine/phenylalanine substitutions in individual or both phosphorylation motifs (Y 378 F, Y 397 F, Y 378/397 F) by performing coimmunoprecipitation experiments from pervanadate-treated 293T cells.…”