pCAP-based peptide substrates: The new tool in the box of tyrosine phosphatase assays

Abstract

Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.

T-helper signals restore B-cell receptor signaling in autoreactive anergic B cells by upregulating CD45 phosphatase activity

“…38,39 This increased activity was found by using a highly specific and robust assay that takes advantage of the fluorogenic properties of pCAP, an analog of phosphotyrosine that can be incorporated into peptides. 22,40,41 Using the pCAP-SP1 peptide, we demonstrated that T H 2 cell factors significantly increased CD45 phosphatase activity in both B ND and naive B cells. Moreover, on T-cell help, CD45 inhibition led to a decrease in levels of the active Lyn-pTyr396 form, further reinforcing the central role of CD45 in transmitting T-cell help to B cells, which is fully in line with data supporting a positive regulatory role for CD45 in B cells.…”

Covalent Inhibition of the Lymphoid Tyrosine Phosphatase

“…Several covalent, irreversible inhibitors of PTPs have been reported as activity-based probes for proteomic profiling, including non-hydrolyzable α-bromobenzyl phosphonates [28, 29] and phenyl vinyl sulfones and sulfonates. [30] While covalent inhibitors have significant advantages as mechanism-based probes for proteomic profiling [31, 32] and several successful drugs are known to function through a covalent mechanism, irreversible inhibitors have largely been avoided in drug development due to the disadvantage of potential off-target reactivity.…”

“…In a recent high-throughput screen of the National Institutes of Health Molecular Libraries for compounds capable of inhibiting LYP activity (PubChem AID:1779), [28] we identified a few compounds that contained electrophilic moieties and could, potentially, inhibit LYP activity through a covalent, irreversible inhibition mechanism. One compound in particular, compound 1 (Figure 1), was identified for further study.…”

Interrogating Protein Phosphatases with Chemical Activity Probes

“…The resulting pCAP-based sensors have been employed to monitor PTP activity in living cells, in campaigns to identify PTP inhibitors, and in efforts to develop optimal substrate sequences for PTPs. [63] …”