Interaction Domains

Another Phosphotyrosine Binding Domain

Science's STKE  26 Apr 2005:
Vol. 2005, Issue 281, pp. tw154
DOI: 10.1126/stke.2812005tw154

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 PKCδ 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 PKCδ and CDCP1 required only the C2 domain of the regulatory domain of PKCδ 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)-PKCδ 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 PKCδ by tyrosine phosphorylation. The residue that is phosphorylated (Tyr64) on PKCδ 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 PKCδ interacted with CDCP1, and the interaction of PKCδ with CDCP1 required the kinase activity of Src. CDCP1 appears to bind both PKCδ and Src simultaneously through phosphorylated tyrosine motifs (Tyr734 for Src and Tyr762 for PKCδ). Mutation of Tyr734 blocked both Src and PKCδ binding, whereas mutation of Tyr762 only blocked the association of PKCδ. This is consistent with Src binding first to CDCP1 and phosphorylating Tyr762, thereby creating the PKCδ binding site.

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