Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin

Abstract

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as Ecadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.

Early events in cell adhesion and polarity during epithelial-mesenchymal transition

“…In HGF-induced EMT, Met co-localizes with Ecadherin and they are both co-endocytosed (Kamei et al, 1999) through the upregulation of endogenous ADP-ribosylation factor 6 (ARF6) GTPase (Palacios et al, 2001). This leads to the phosphorylation of E-cadherin on two tyrosine residues and its subsequent ubiquitination by the E3-ubiquitin ligase HAKAI (also known as CBLL1) (Fujita et al, 2002;Mukherjee et al, 2012). EGF also induces EMT in non-transformed cells, such as normal ovarian surface epithelium (Salamanca et al, 2004) and in various human carcinoma cells that overexpress EGFR (Barr et al, 2008).…”

Cell Regulation by Phosphotyrosine-Targeted Ubiquitin Ligases

“…However, we now know that Hakai and Cbl have different mechanisms of pY substrate binding. Hakai dimerization creates an unusual HYB domain that binds pY substrates (20). The RING domains are directly adjacent to the HYB domain, allowing positioning of E2 and ubiquitin close to the pY residues in the substrate.…”

“…The LHR linker between the SH2 and the RING domains is represented by a green noodle, with Tyr371 highlighted in red. (D) Homodimeric structure of the Hakai HYB domain (PDB code 3VK6 [20]). The dimer coordinates six zinc ions (gray balls), two of which are located behind ␣1 and ␣1= helices and are thus invisible in this figure.…”

“…Hakai targets a cytoplasmic NVYYY motif in E-cadherin when it is phosphorylated on the second tyrosine (19,20). Interestingly, the same motif is also the binding site for the Numb PTB domain, although Numb binds when the motif is nonphosphorylated (41).…”

The regulation of cell-cell adhesion during epithelial-mesenchymal transition, motility and tumor progression

“…Src targets the tyrosine residues, Tyr755 and 756, at the juxtamembranous domain of Ecadherin displacing p120 catenin, 70,71 thereby destabilizing AJs. E-cadherin is then degraded by the ubiquitin ligases Hakai, or MDM2 [72][73][74] ( Fig. 1).…”