The chemical biology of protein phosphorylation

Abstract

The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain.

Figure 1

Molecules for protein kinase modulation. (a) Kinase inhibitors: staurosporine, quercetin, SB202190, BIRB 796, imatinib, lapatinib, fmk, tetrafluorotyrosine, and bisubstrate analog. (b) Analog-sensitive phosphoryltransferase substrates/inhibitors. The naturally occurring nucleotide triphosphate substrate, ATP (left), and its unnatural analog, xanthosine triphosphate (middle). The inhibitor N⁶-benzyl-PP1 (right), which is selective and specific for mutant kinases that can accommodate xanthosine triphosphate.

Figure 2

Site-specific incorporation of phosphoamino acids and mimics in proteins. (a,b) Strategies used for protein semisynthesis involving native chemical ligation. (a) Expressed protein ligation for attaching a synthetic peptide to the C terminus of a protein. (b) Proteolytic generation of N-terminal Cys for attaching a synthetic peptide thioester to the N terminus of a protein. (c) Phosphoamino acids and their mimics. (top) Phosphoamino acids and mimics that can be incorporated into proteins through semisynthesis. (bottom) Genetically encodable mimics for phosphoamino acids that can be incorporated into proteins through point mutations or by using nonsense codon suppression technology.

Figure 3

Peptide biosensors. (a) Environmentally sensitive biosensor. (b) Deep-quench biosensor. (c) Self-reporting biosensors. (d ) Metal chelation-enhanced fluoroscence biosensor.

Figure 4

Forster (fluorescence) resonance energy transfer (FRET)-based genetically encoded biosensor. Basic design of biosensor consists of two fluorescent proteins (FPs) that are fused with a phosphoamino acid-binding domain (gray) and a substrate peptide (green) between them. Upon phosphorylation by a kinase or dephosphorylation of the substrate on the biosensor, there is a conformational change that results in a change in FRET.