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

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 E-cadherin, 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.

Sprouty2 interacts with protein kinase C delta and disrupts phosphorylation of protein kinase D1.

The Sprouty (Spry) proteins act as inhibitors of the Ras/ERK pathway downstream of receptor tyrosine kinases. In this study, we report a novel interaction between protein kinase C delta (PKCdelta) and Spry2. Endogenous PKCdelta and Spry2 interact in cells upon basic fibroblast growth factor stimulation, indicating a physiological relevance for the interaction. This interaction appeared to require the full-length Spry2 protein and was conformation-dependent. Conformational constraints were released upon FGFR1 activation, allowing the interaction to occur. Although this interaction did not affect the phosphorylation of PKCdelta by another kinase, it reduced the phosphorylation of a PKCdelta substrate, protein kinase D1 (PKD1). Spry2 was found to interact more strongly with PKCdelta with increasing amounts of PKD1, which indicated that instead of competing with PKD1 for binding with PKCdelta, it was more likely to form a trimeric complex with both PKCdelta and PKD1. Formation of the complex was found to be dependent on an existing PKCdelta-PKD1 interaction. By disrupting the interaction between PKCdelta and PKD1, Spry2 was unable to associate with PKCdelta to form the trimeric complex. As a consequence of this trimeric complex, the existing interaction between PKCdelta and PKD1 was increased, and the transfer of phosphate groups from PKCdelta to PKD1 was at least partly blocked by Spry2. The action of Spry2 on PKCdelta resulted in the inhibition of both ERK phosphorylation and invasion of PC-3 cells via PKCdelta signaling. By disrupting the capacity of PKCdelta to phosphorylate its cognate substrates, Spry2 may serve to modulate PKCdelta signaling downstream of receptor tyrosine kinases and to regulate the physiological outcome.