Natural adaptor proteins act as molecular scaffolds to bring two proteins in proximity to promote their interaction. This mechanism provides efficiency and specificity to cellular signaling pathways. We have created a fully synthetic adaptor protein which allows us to connect two signaling pathways that do not normally interact - Src family kinase (SFK) signaling pathways and the p53-binding protein hDM2. Our synthetic adaptor protein is constructed through the fusion of two miniature proteins - small, well-folded proteins that can display key epitopes involved in protein-protein interactions. Here, we have fused one miniature protein that binds and activates SFKs, with another that binds hDM2. We created several fusions with varied linker lengths and found them to bind both hDM2 and SFKs. We show that the presence of this fusion protein leads to an increase in phosphorylation of hDM2 by SFK relative to either of the unfused miniature proteins alone. Our fusion protein thus appears to act as an adaptor protein, bringing hDM2 in close proximity to the SFK and promoting its phosphorylation. This fully synthetic adaptor protein may be used to probe the implications of hDM2 phosphorylation in the inhibition of the p53•hDM2 interaction and programmed cell death.
This preliminary program is subject to change.
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Modulation of the Phosphorylation Level of hDM2 by a Synthetic Adaptor Protein
Natural adaptor proteins act as molecular scaffolds to bring two proteins in proximity to promote their interaction. This mechanism provides efficiency and specificity to cellular signaling pathways. We have created a fully synthetic adaptor protein which allows us to connect two signaling pathways that do not normally interact - Src family kinase (SFK) signaling pathways and the p53-binding protein hDM2. Our synthetic adaptor protein is constructed through the fusion of two miniature proteins - small, well-folded proteins that can display key epitopes involved in protein-protein interactions. Here, we have fused one miniature protein that binds and activates SFKs, with another that binds hDM2. We created several fusions with varied linker lengths and found them to bind both hDM2 and SFKs. We show that the presence of this fusion protein leads to an increase in phosphorylation of hDM2 by SFK relative to either of the unfused miniature proteins alone. Our fusion protein thus appears to act as an adaptor protein, bringing hDM2 in close proximity to the SFK and promoting its phosphorylation. This fully synthetic adaptor protein may be used to probe the implications of hDM2 phosphorylation in the inhibition of the p53•hDM2 interaction and programmed cell death.