The phosphorylation and dephosphorylation of proteins is a mechanism widely used in nature for the activation and deactivation of enzymes, protein trafficking and the transmission of extracellular signals in cells, from prokaryotic to eukaryotic life. A large body of work has been carried out in the last several decades unraveling the intricate signaling pathways mediated by protein phosphorylation and dephosphorylation events. In prokaryotic systems extracellular messages such as chemosensing lead to intracellular signaling mediated by protein phosphorylation leading to chemotaxis. Growth factor signaling is mediated by tyrosine kinase activity associated with their receptors, recruitment of SH2 (Src homology 2) domain proteins to the receptors, and serine/threonine phosphorylation of several proteins in the pathway from the cell surface to the nucleus. Cell–cell contact signaling mediated by the integrins is mediated by focal adhesion kinase and a series of other tyrosine kinases and SH2–phosphoprotein interactions. The importance of these events is illustrated by the fact that a search of the Medline database on the term “protein phosphorylation” yielded 533 citations of review articles alone. Understanding and studying any biological system requires highly specialized reagents. In the case of protein phosphorylation and dephosphorylation, phosphopeptides have proven to be very valuable. Among other applications, these reagents have been used to probe SH2 domain specificity and PTB (phosphotyrosine binding) domains, substrate specificity of phosphatases, as well as aspects of the structures of milk casein and the τ protein found in paired helical filaments in Alzheimer's disease patients. In this issue we discuss various aspects of the generation and use of phosphopeptides. Whereas today the synthesis of phosphopeptides is considered routine, such was not always the case. In the last century, and especially in the 1980s and 1990s, the synthesis of phosphopeptides has evolved from the phosphorylation of simple amino acid with chlorophosphates to the advances of phosphoramidites and the protected building blocks currently in use. The efforts of several laboratories from around the globe have provided us these tools. In this issue, McMurray et al. have reviewed the synthesis of phosphopeptides. The development of bioorganophosphate chemistry is briefly discussed and the synthesis of Nα-protected building blocks and global phosphorylation is discussed in detail. Other aspects such as side reactions, cyclic phosphopeptides, and phosphopeptide prodrugs are reviewed as well. One of the liabilities of phosphopeptides is the susceptibility of the phosphate to cleavage by phosphatases. In drug design efforts targeted to SH2 domains and phosphatases, this is a major problem. Terry Burke and his colleagues have developed a variety of nonhydrolyzable phosphotyrosine analogues and has applied them in the development of inhibitors of phosphotyrosine specific enzymes such as the Grb2 SH2 domain and PTP-1B. In this issue Dr. Burke has reviewed the state of the art in nonhydrolyzable phosphotyrosine mimics. By and large, protein phosphorylation and dephosphorylation occurs within the cell. The study of these events using phosphopeptides is complicated by the fact that in general peptides do not readily cross cellular membranes due to the polarity of the peptide bond. The large energy penalty that must be paid to strip the water molecules from the peptide bond before the peptide can enter and pass through the lipid environment of the cell membrane severely impedes passive diffusion. The addition of a negatively charged phosphate group exacerbates the problem. However, membrane transporter peptides such as those derived from the antennapedia homeodomain (also called Penetratin), the HIV TAT protein, and polyarginines have been shown to allow the passive diffusion of peptides and other cargo into the interior of cells. In this issue, Dunican and Doherty describe their work on the use of these transporter peptides to deliver phosphopeptides into cells to study the effect of impeding SH2 domain mediated signal transduction pathways. Finally, sequence-specific antiphosphopeptide antisera are very valuable reagents in the study of signal transduction mechanisms. These antibodies allow the detection of phosphorylation at select sites of proteins of interest—information that can be used to precisely delineate their roles in cellular events. Arlinghaus et al. briefly review antiphosphopeptide antibodies. They then describe the development of antisera directed against phosphoserine peptides derived from Bcr–Abl and their use in delineating the aberrant signaling pathways in which this oncogenic protein participates in cancer cell lines.
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May 18, 2022
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