Specific Inhibition of Sensitized Protein Tyrosine Phosphatase 1B (PTP1B) with a Biarsenical Probe

Abstract

Protein tyrosine phosphatase 1B (PTP1B) is a key regulator of the insulin-receptor and leptin-receptor signaling pathways, and it has therefore emerged as a critical anti-type-II-diabetes and anti-obesity drug target. Toward the goal of generating a covalent modulator of PTP1B activity that can be used for investigating its roles in cell signaling and disease progression, we report that the biarsenical probe FlAsH-EDT2 can be used to inhibit PTP1B variants that contain cysteine point mutations in a key catalytic loop of the enzyme. The site-specific cysteine mutations have little effect on the catalytic activity of the enzyme in the absence of FlAsH-EDT2. Upon addition of FlAsH-EDT2, however, the activity of the engineered PTP1B is strongly inhibited, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. We show that the cysteine-rich PTP1B variants can be targeted with the biarsenical probe in either whole-cell lysates or intact cells. Together, our data provide an example of a biarsenical probe controlling the activity of a protein that does not contain the canonical tetra-cysteine biarsenical-labeling sequence CCXXCC. The targeting of “incomplete” cysteine-rich motifs could provide a general means for controlling protein activity by targeting biarsenical compounds to catalytically important loops in conserved protein domains.

Activation of Engineered Protein Tyrosine Phosphatases with the Biarsenical Compound AsCy3‐EDT2

“…The plasmids encoding His 6 -tagged PTP1B [22] and TCPTP [23] have been previously described. Site-directed mutations were introduced using the Quikchange mutagenesis kit (Stratagene) according to the manufacturer’s instructions.…”

Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

“…The plasmids encoding His 6 -tagged catalytic domains of Shp2 (pJGO0001), 24 Shp1 (pACB149), 24 PTP1B (pOBD0002), 21 PTPH-1 (pERB047), 26 HePTP (pBAD-HePTP), 16 and FAP-1 (pAML001) 16 have been previously described. All site-directed mutations were introduced using the Quikchange mutagenesis kit (Stratagene) according to the manufacturer’s instructions.…”

“…The His 6 -tagged catalytic domains of Shp2, 24 Shp1, 24 PTP1B, 21 PTPH-1, 26 HePTP, 16 and FAP-1 16 were expressed and purified as described previously.…”

“…19–20 Targeting engineered cysteine-enriched WPD loops with biarsenicals has proven to be a general approach for PTP inhibition. 16, 21–22 However, the WPD loop constitutes part of the PTP active site (in its closed state) and plays a key role in the catalytic mechanism of PTPs, presenting the possibility that mutations on the loop itself could alter the inherent kinetic activity or substrate specificity of the enzyme. Moreover, the presence of catalytically essential residues on the WPD loop significantly constrains the options for its engineering in a manner that is functionally silent ( e.g.…”

Small Molecule Gated Split-Tyrosine Phosphatases and Orthogonal Split-Tyrosine Kinases

“…31 They also developed an elegant allosteric strategy, where the insertion of a specific tetra-cysteine sequence within a PTP that binds a biarsenical probe, can enhance inhibition of a desired PTP. 32,33 We sought to complement these two existing avenues for the immediate turn-off of a PTP, by designing a potentially general turn-on approach for controlling the temporal activity of one or more PTPs.…”