Caged phosphopeptides and phosphoproteins : probes to dissect the role of phosphorylation in complex signaling pathways

Abstract

Protein phosphorylation is a central regulatory mechanism in signal transduction pathways and cellular migration. Current genetic strategies for the study of phosphorylation, including gene knockout and point mutation, are limited in providing temporal information. As a complement to these techniques, the synthesis and semisynthesis of probes that enable researchers to observe the downstream effects of kinase-mediated phosphorylation in "real time" are presented in this thesis. The release of a physiologically-relevant concentration of a phosphopeptide with temporal and spatial control is accomplished by the photolysis of a photolabile precursor, a caged phosphopeptide. The synthesis and application of NI-Fmoc-protected 1-(2-nitrophenyl) ethyl (NPE) caged phosphothreonine, serine, and tyrosine building blocks facilitate the straightforward assembly of any caged phosphopeptide through Fmoc-based solid phase peptide synthesis. Removal of the NPE caging group by irradiation with long-wavelength UV light generates a concentration burst of the corresponding phosphopeptide. In addition, the installation of a caged phosphoamino acid into a full-length, multi-domain protein, the cellular migration protein paxillin, is described. A strategy, which is applicable to any expressible protein target, is detailed for the semisynthesis of a paxillin variant with a caged phosphorylated tyrosine at residue 31 of the 557-residue protein using native chemical ligation.

(cont.) Paxillin is a 61-kDa protein known to orchestrate the interaction of signaling proteins involved in cell migration by acting as a molecular adaptor, with the creation of specific binding sites dependent on paxillin phosphorylation. Therefore, the semisynthetic probe comprises the entire paxillin macromolecule, including all other binding and localization domains, which are essential for creating a native-like system to probe the effect of phosphorylation. The comprehensive biochemical characterization of the paxillin probe and quantification of uncaging following irradiation with long-wavelength UV light are also described. Additionally, the strategy developed for the paxillin semisynthesis was applied to incorporate a different unnatural amino acid, the fluorescent chemosensing residue Sox, into a protein-domain sensor for ERK2 kinase activity. The protein domain sensor demonstrated significantly improved sensitivity for ERK2 phosphorylation over the corresponding peptide probe.