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Sci. STKE, 26 April 2005
Vol. 2005, Issue 281, p. tw154
[DOI: 10.1126/stke.2812005tw154]

EDITORS' CHOICE

INTERACTION DOMAINS Another Phosphotyrosine Binding Domain

Phosphorylated tyrosine residues have multiple roles in cell signaling, including mediating protein-protein interaction through specific domains that can recognize phosphotyrosines in specific contexts. Benes et al. added C2 (for conserved domain 2) to the two well-known phosphotyrosine binding domains SH2 and PTB. The authors found that PKC{delta} interacted directly with CDCP1 (CUB domain-containing protein 1, also known as SIMA135) in response to stimuli that activated the tyrosine kinase Src. The interaction between PKC{delta} and CDCP1 required only the C2 domain of the regulatory domain of PKC{delta} and correlated with phosphorylation of CDCP1. Screening of a degenerate phosphotyrosine peptide library identified a consensus sequence for the recognition of phosphorylated tyrosine by a glutathione S-transferase (GST)-PKC{delta} regulatory domain fusion protein. Competition experiments confirmed the specificity of the interaction, and isothermal calorimetry titration experiments demonstrated that the dissociation constant for the C2 domain with the phosphopeptide was 250 nM, with a stoichiometry of 1 phosphopeptide per C2 domain. CDCP1 contains a tyrosine (Tyr762) within a sequence that resembles the predicted consensus binding motif. Analysis of the crystal structure of the C2 domain complexed with an optimized phosphotyrosine peptide revealed a potential mechanism for regulation of PKC{delta} by tyrosine phosphorylation. The residue that is phosphorylated (Tyr64) on PKC{delta} lies just outside the binding pocket for the phosphorylated tyrosine of the bound peptide, and phosphorylation of Tyr64 may inhibit binding to phosphorylated peptides, thus blocking protein interactions through the C2 domain. Coexpression experiments in transfected cells revealed that both Src and PKC{delta} interacted with CDCP1, and the interaction of PKC{delta} with CDCP1 required the kinase activity of Src. CDCP1 appears to bind both PKC{delta} and Src simultaneously through phosphorylated tyrosine motifs (Tyr734 for Src and Tyr762 for PKC{delta}). Mutation of Tyr734 blocked both Src and PKC{delta} binding, whereas mutation of Tyr762 only blocked the association of PKC{delta}. This is consistent with Src binding first to CDCP1 and phosphorylating Tyr762, thereby creating the PKC{delta} binding site.

C. H. Benes, N. Wu, A. E. H. Elia, T. Dharia, L. C. Cantely, S. P. Soltoff, The C2 domain of PKC{delta} is a phosphotyrosine binding domain. Cell 121, 271-280 (2005). [Online Journal]

Citation: Another Phosphotyrosine Binding Domain. Sci. STKE 2005, tw154 (2005).



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