In the 1950s, it was discovered that the metabolic enzyme phosphorylase, responsible for the conversion of glycogen to glucose-1-phosphate, existed in two stable forms, an inactive (phosphorylase b) and an active (phosphorylase a) state (1
). A search for the molecular mechanism responsible for the conversion of the inactive state to the active revealed that a protein kinase, phosphorylase kinase, could catalyze the attachment of a phosphate to phosphorylase and render it fully active (2
). It was subsequently shown that phosphorylase kinase was itself activated by a protein kinase, protein kinase A (PKA, also called cyclic AMP-dependent protein kinase), and the concept of protein phosphorylation as a key regulatory mark was born (3
). In the ensuing years, protein phosphorylation networks have been understood as undergirding most physiologic processes ranging from the cardiovascular system, gastrointestinal action, neurologic mechanisms and behavior, immune response, endocrine action, and musculoskeletal regulation (4
). Moreover, the linkage of protein phosphorylation to pathogenic mechanisms, involving the aforementioned physiologic systems as well as cancer, have heightened interest in the phosphate posttranslational modification (PTM) within the academic and private sectors of the biomedical research community. The concept that protein kinases are “druggable” began to take hold by the mid-1990s and was placed on firm footing when it was shown that the Abl tyrosine kinase inhibitor imatinib (gleevec) () could effect dramatic remissions in more than 90% of preblast phase patients with chronic myelogenous leukemia (CML) (5
). Moreover, patients treated with imatinib are generally free of serious side effects, refuting concerns of severe toxicity associated with targeting protein kinase enzymes considered to be essential to many cellular pathways. These findings have not gone unnoticed in the pharmaceutical industry, and over the past decade about a dozen protein phosphorylation-linked drugs have been launched, and many more are in the pipeline for a host of indications (6
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 (more ...)
Despite these successes and the substantial enthusiasm for furthering protein kinase inhibitor development, many fundamental challenges remain in the protein phosphorylation field. Although imatinib has been a great success for controlling CML (5
), most drugs that modulate protein phosphorylation have been less effective in disease treatment. It can be argued that we still have a primitive understanding of the function of protein kinases, phosphatases, and their substrates and effectors. The complexity of the phosphoproteome is daunting. There are about 500 mammalian protein kinases (7
), 100 protein phosphatases (8
), and hundreds of proteins containing domains (SH2, PTB, 14-3-3, BRCT, etc.), which interact with phosphorylated proteins (9
). We have also learned recently that precise timing (within minutes) and spatial aspects of protein phosphorylation are crucial to cell functioning. Genetic and conventional biochemical approaches, including the powerful RNAi methodologies (11
), have made enormous contributions to our understanding of protein kinase and phosphatase actions. However, these methods have had limitations in pinpointing kinase contributions to signaling, revealing rapid kinetic changes, clarifying functional effects of specific phosphorylation events, and relating cellular localization to kinase/phosphatase activity. In response to these obvious limitations, over the past 10–15 years a range of technologies have been introduced and applied to fill in the missing details in our understanding of phosphorylation networks. In this review, we outline a number of these approaches, which have at their heart the merging of chemistry and biology.
We discuss three general areas where chemical biological methods have been applied to sort out kinase action. First, we describe methods to modulate the action of kinases in vitro and cellular systems. Here, chemical design and screening have been used with wild-type and mutant kinases to gain specificity that has allowed insights into the rapid changes in signaling cascades. Second, we discuss methods to site specifically introduce phosphoamino acids or their mimics at known sites of phosphorylation in proteins. Protein semisynthesis and unnatural amino acid mutagenesis are the main vehicles for this new age protein engineering. Third, we highlight the recently introduced fluorescent reporters that allow for high-resolution imaging of phosphoryl transfer in cells and lysates. Such molecular imaging is providing unprecedented insights into the timing and cellular localization of signaling networks.