Current Chemical Biology Tools for Studying Protein Phosphorylation and Dephosphorylation

Abstract

Amongst different posttranslational events involved in cellular-signaling pathways, phosphorylation and dephosphorylation of proteins are the most prevalent. Aberrant regulations in the cellular phosphoproteome network are implicated in most major human diseases. Consequently, kinases and phosphatases are two of the most important groups of drug targets in medicinal research today. A major challenge in the understanding of protein phosphorylation and dephosphorylation is the sheer complexity of the phosphoproteome network and the lack of tools capable of studying protein phosphorylation and dephosphorylation as they occur in cells. We highlight herein various chemical biology tools that have emerged in the last decade for such studies. First, we discuss the use of small-molecule mimics of phosphoamino acids and their use in elucidating the function of protein phosphorylation and dephosphorylation. We also introduce recent advances in the field of activity-based protein profiling (ABPP) for proteome-wide detection of protein phosphorylation and dephosphorylation. We next discuss the key concepts in the design of peptide- and protein-based biosensors capable of real-time reporting of phosphorylation/dephosphorylation events. Finally, we highlight the application of peptide and small-molecule microarrays (SMMs), and their applications in high-throughput screening and discovery of new compounds related to phosphorylation/dephosphorylation.

Activity-Based Probe for Histidine Kinase Signaling

“…11 Unlike the study of phosphorylation at serine, threonine and tyrosine that has enjoyed great advancement in the last decade, 12,13 detection of phosphorylated histidine residues has lagged far behind. Some success has been achieved by application of Phos-tag, a phosphate-binding molecule embedded in a polyacrylamide gel matrix, to facilitate the separation and quantitation of phosphorylated from non-phosphorylated HKs.…”

Activity‐Based Probes for Studying the Activity of Flavin‐Dependent Oxidases and for the Protein Target Profiling of Monoamine Oxidase Inhibitors

“…[1] The application of small-molecule activity-based probes to interrogate enzyme activity on the cell level has led to the identification and functional characterization of proteins involved in cancer,[2] signaling pathways,[3] microbial pathogenesis and virulence,[4] host–virus interactions,[5] and other biological processes. However, up to now most ABPP studies have aimed at enzyme classes with well-established catalytic mechanisms and nucleophilic active-site residues participating in the formation of a covalent bond to activity-based probes (e.g.…”

“…serine hydrolases,[6] cysteine[7] and threonine proteases[8]). Thus, one important challenge in ABPP is expanding the pool of probe molecules to enzyme classes with more complex catalytic activities such as kinases,[3, 9] transferases,[10] and oxidoreductases[11] to extend the proteome coverage. Here, we introduce unprecedented activity-based probes for an important group of oxidoreductases, namely flavin-dependent oxidases.…”

In-Situ Generation of Differential Sensors that Fingerprint Kinases and the Cellular Response to Their Expression

“…8–10 Efforts in this area have focused on the development of receptors whose responses are sensitive, as well as being functional in physiological environments. 11,12 For instance, the chelation-enhanced fluorescence strategy popularized by Imperiali using a sulfonamido oxime (SOX) fluorophore is a leading approach. 13 Phosphorylation of Ser/Thr residues enhances the affinity of SOX with magnesium, and as a result increases the fluorescence of SOX.…”