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| Protein biochemistry: The tags that bind – a new way to label PTPs
A newly developed chemical probe could considerably simplify the process of determining changes in the activity of tyrosine phosphatase enzymes, both globally and for individual proteins within the cell. The processes by which cellular functions are regulated are, of course, exceedingly complex, and rely on a diverse array of pathways. Many systems rely on the phosphorylation of key tyrosine residues on specific proteins as a means to control signaling. This phosphorylation state is in turn regulated by the activity of two types of proteins: protein tyrosine kinases (PTKs), which phosphorylate the tyrosine residues, and protein tyrosine phosphatases (PTPs), which remove the phosphate group. There are more than a hundred known PTPs in humans, involved in the regulation of such essential functions as cell metabolism, growth, and differentiation. There is therefore great interest in developing a universal reagent for the monitoring of PTP activity, although no reagent has yet been developed that is suitable for such detection in vivo. Recent efforts generated a suicide inhibitor compound capable of covalently modifying PTPs (see Lo et al.), but closer examination determined that it lacked specificity, and was in fact capable of acting as a substrate for a number of unrelated phosphatases. "There really were no viable alternatives," explains Zhong-Yan Zhang, a researcher at Albert Einstein College of Medicine (Bronx, NY), even though such a reagent would be of considerable interest to a large number of investigators. Zhang and his colleagues set out to identify a new class of activity-based probes, small molecule compounds capable of highly sensitive and specific detection of PTP activity. Although all PTP family members share a common catalytic mechanism, the individual phosphatases participate in a wide variety of functions; as a result, the challenge was to generate a reagent that could interact specifically with this common catalytic site, while still offering the capability to track changes in individual proteins. "Our goal," says Zhang, "was to quickly profile the activity of the entire PTP family in both normal and disease states, so that we can identify those PTP family members that are abnormally regulated in terms of activity." His team derived two probes with The probes also proved effective for the screening of a variety of cellular lysates. A comparative analysis of two human-derived cell lines, hTERT and MCF-7, revealed significant differences in the activity levels of several different PTPs, which were readily detectable by immunoblotting for biotin. Zhang and his colleagues are currently looking into methods for the rapid affinity purification of biotin-tagged PTPs from treated lysates, as a means to simplify the process of large-scale analysis of phosphatase activity. Having successfully demonstrated the proof of concept behind this system for generating PTP-tagging compounds, Zhang's team intends to explore further modifications that can improve quality and sensitivity of detection, such as the replacement of biotin with a fluorescent tag, or using isotope labeled compounds for mass spectrometry ICAT experiments, which would allow for the simultaneous comparison of PTP profiles in normal and diseased extracts within a single experiment. "In this paper we developed a synthetic strategy to generate a common intermediate," says Zhang, "and now we can basically hook it onto whatever we want, in terms of tags." Michael Eisenstein References
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-bromobenzylphosphonate (BBP), a compound known to have affinity for PTP active sites, conjugated via a specialized linker to biotin. Initial tests confirmed that these probes bound effectively and irreversibly to a variety of PTPs in vitro in an active site-directed manner. Furthermore, the extent of PTP labeling was found to correlate directly with the level of activity observed for a given PTP. On the other hand, these probes had no apparent affinity for any of the non-PTP enzymes screened by Zhang's group.