A Drosophila protein-interaction map centered on cell-cycle regulators
A Drosophila protein-protein interaction map was constructed using the LexA system, complementing a previous map using the GAL4 system and adding many new interactions.
Abstract Background: Maps depicting binary interactions between proteins can be powerful starting points for understanding biological systems. A proven technology for generating such maps is highthroughput yeast two-hybrid screening. In the most extensive screen to date, a Gal4-based twohybrid system was used recently to detect over 20,000 interactions among Drosophila proteins. Although these data are a valuable resource for insights into protein networks, they cover only a fraction of the expected number of interactions.
The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.
Cyclin Y is one of the most highly conserved members of the cyclin superfamily of proteins, which are famous for their crucial roles in regulating the cell cycle and transcription. Despite this high degree of conservation, very little was known about Cyclin Y function prior to a handful of studies published in this past year. Cyclins typically function by activating cyclin-dependent kinases (Cdks) and one insight has come from the identification of a Cdk that is activated by Cyclin Y. Yeast two-hybrid data first linked Cyclin Y with Cdk14, known as Eip63E in Drosophila or PFTAIRE1 in vertebrates. In Drosophila, both Cyclin Y and Eip63E are essential at many stages of development, from embryogenesis to metamorphosis and null mutants show a similar spectrum of developmental defects. In cultured cells, Cyclin Y and Eip63E were shown to phosphorylate the Wg/Wnt co-receptor Arrow/LRP6 in a ligand-independent manner. Eip63E is recruited to LRP6 at the plasma membrane by interacting with Cyclin Y, which is tethered to the membrane through an N-terminal myristoylation. Cyclin Y-dependent LRP6 phosphorylation appears to prime the receptor for subsequent ligand-dependent phosphorylation and activation of the canonical Wnt signaling pathway. Interestingly, Wnt receptor phosphorylation and signaling is maximal in G₂/M when Cyclin Y is at its highest levels, suggesting that Cyclin Y may serve to entrain Wnt signaling to the cell cycle. Given the wide range of roles for Wnt signaling during development, these studies may help explain why Cyclin Y is required at several developmental stages and in turn why these proteins are so well conserved in metazoans.
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