Global Analysis of Protein Tyrosine Phosphatase Activity with Ultra-Sensitive Fluorescent Probes

Abstract

Protein tyrosine phosphatases (PTPs) consist of a large family of enzymes known to play important roles in controlling virtually all aspects of cellular processes. However, assigning functional significance of PTPs in normal physiology and in diseases remains a major challenge in cell signaling. Since the function of a PTP is directly associated with its intrinsic activity, which is subject to post-translational regulation, new tools are needed to monitor the dynamic activities of PTPs, rather than mere abundance, on a global scale within the physiologically relevant environment of cells. To meet this objective, we report the synthesis and characterization of two rhodamine-conjugated probes that covalently label the active site of the PTPs in an activity-dependent manner, thus providing a direct readout of PTP activity and superior sensitivity, robustness, and quantifiability to previously reported biotinylated probes. We present evidence that the fluorescent probes can be used to identify new PTP markers and targets for potential diagnosis and treatment of human diseases. We also show that the fluorescent probes are capable of monitoring H(2)O(2)-mediated PTP inactivation, which should facilitate the study of regulated H(2)O(2) production as a new tier of control over tyrosine phosphorylation-dependent signal transduction. The ability to profile the entire PTP family on the basis of changes in their activity is expected to yield new functional insights into pathways regulated by PTPs and contribute to the discovery of PTPs as novel therapeutic targets.

Development of an Activity‐Based Probe for Autotaxin

“…This trapping device has previously been used to probe protein tyrosine phosphatase activity. [23][24][25][26][27] Upon ATXmediated cleavage of the phosphodiester bond in the probe, the released intermediate undergoes rapid 1,6-elimination of the fluoride and generates a reactive quinone methide species that, in turn, traps nearby nucleophiles in the ATX active site; this results in covalent labelling of ATX in an activity-dependent manner. Incorporation of a fluorescent dye allows visualisation of active ATX.…”

Cellular Biochemistry Methods for Investigating Protein Tyrosine Phosphatases

“…These probes have been linked to either a biotin tag (66) that allows for purification and/or visualization, or to fluorescent lissamine rhodamine B (67). BBP probes irreversibly inactivate a variety of PTPs, including cytosolic PTPs YopH, PTP1B, HePTP, PTPH1, and SHP-2, transmembrane PTPs LAR, PTPa, and DEP-1, dual-specificity PTPs (DUSPs) VHR, PRL-3, and CDC14, and the low-molecular-weight PTPs (LMPTP or LMW-PTP), but do not bind to alkaline phosphatases, prostatic acid phosphatase, Ser/Thr phosphatases, SH2 or PTB domains, SRC kinase, serine proteases, metalloprotease thermolysin, the cysteine proteases calpain and papain, glyceradehyde-3-phosphate dehydrogenase, GST, lysozyme, or the entire E. coli proteome (66,67). Covalent binding of the probes to PTPs was confirmed by western blotting for biotinylated PTPs or by in-gel imaging of PTP fluorescence, and by mass spectrometry.…”

“…For example, BBP probes were used to demonstrate that four PTPs showed anomalous activity/expression in lysates of the MCF-7 breast cancer cell line compared with lysates of the human telomerase reverse transcriptase immortalized mammary epithelial cell line (67). Interestingly, when a sideby-side comparison of a fluorescently imaged versus a Coomassie blue-stained gel of the same lysates treated with the fluorescent BBP probe was performed to assess PTP activities in a panel of cancer cell line lysates, differences in PTP activity were revealed that were not evident in the Coomassie blue staining of total protein in the crude lysates.…”

Current Chemical Biology Tools for Studying Protein Phosphorylation and Dephosphorylation

“…However, the highly unstable nature of the probe renders it impractical for widespread applications in ABPP. [28] Probes based on phenyl vinyl sulfone/sulfonates (20) are another class of PTP probes that were recently developed, [29] but they are likely cross-reactive toward other classes of enzymes that also possess nucleophilic cysteine residues, that is, cysteine proteases. In a most recent example, we developed peptidebased ABPs for PTP by incorporating a novel pTyr mimic, 2-FMPT (21), into suitable peptide sequences.…”