Sprouty proteins are involved in organogenesis, particularly during the branching of endothelial tubes, and existing evidence suggests that Sprouty's point of action lies downstream of receptor signaling to inhibit the activation of the central Ras/Erk pathway. How Sprouty proteins accomplish their inhibitory action and whether they interact with other signaling pathways are significant questions. Sprouty proteins are devoid of any recognizable protein interaction domain, and clues as to how they function have been mainly derived from screening for interacting partners. Conserved across all the Sprouty proteins are three sequences: a Cbl-tyrosine kinase-binding (TKB) binding motif centered on an obligatorily phosphorylated tyrosine (Y55 in Sprouty2), a serine-rich motif (SRM) and a cysteine-rich domain (CRD). With the exception of a handful of proteins that bind to the N-terminus, most of the binding to Sprouty occurs via the CRD, predominantly by serine/threonine kinases that target sites within the SRM on Sprouty. Some of the resultant increase in phosphorylation is opposed by activated protein phosphatase 2A that binds to the N-terminal Cbl-TKB binding motif. Significantly, two ubiquitin E3 ligases also bind to the N-terminus of Sprouty: c-Cbl binds with high affinity to the TKB binding motif and SIAH2 binds constitutively to a different site; both proteins are able to direct the ubiquitination of Sprouty proteins and its destruction. The collective evidence points to Sprouty proteins as being substantially covalently-modified to control its location, stability, association, and destruction. With such stringent control of the Sproutys, the main question is what key proteins does this facilitator bring together?

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.

No abstract

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 ␦ (PKC␦) and Spry2. Endogenous PKC␦ 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 conformationdependent. Conformational constraints were released upon FGFR1 activation, allowing the interaction to occur. Although this interaction did not affect the phosphorylation of PKC␦ by another kinase, it reduced the phosphorylation of a PKC␦ substrate, protein kinase D1 (PKD1). Spry2 was found to interact more strongly with PKC␦ with increasing amounts of PKD1, which indicated that instead of competing with PKD1 for binding with PKC␦, it was more likely to form a trimeric complex with both PKC␦ and PKD1. Formation of the complex was found to be dependent on an existing PKC␦-PKD1 interaction. By disrupting the interaction between PKC␦ and PKD1, Spry2 was unable to associate with PKC␦ to form the trimeric complex. As a consequence of this trimeric complex, the existing interaction between PKC␦ and PKD1 was increased, and the transfer of phosphate groups from PKC␦ to PKD1 was at least partly blocked by Spry2. The action of Spry2 on PKC␦ resulted in the inhibition of both ERK phosphorylation and invasion of PC-3 cells via PKC␦ signaling. By disrupting the capacity of PKC␦ to phosphorylate its cognate substrates, Spry2 may serve to modulate PKC␦ signaling downstream of receptor tyrosine kinases and to regulate the physiological outcome.