Abstract
Strategies for the direct chemical activation of specific signaling proteins could provide powerful tools for interrogating cellular signal transduction. However, targeted protein activation is chemically challenging, and few broadly applicable activation strategies for signaling enzymes have been developed. Here we report that classical protein tyrosine phosphatase (PTP) domains from multiple subfamilies can be systematically sensitized to target-specific activation by the cyanine-based biarsenical compounds AsCy3 and AsCy5. Engineering of the activatable PTPs (actPTPs) is achieved by the introduction of three cysteine residues within a conserved loop of the PTP domain, and the positions of the sensitizing mutations are readily identifiable from primary sequence alignments. In the current study we have generated and characterized actPTP domains from three different subfamilies of both receptor and non-receptor PTPs. Biarsenical-induced stimulation of the actPTPs is rapid and dose-dependent, and is operative with both purified enzymes and complex proteomic mixtures. Our results suggest that a substantial fraction of the classical PTP family will be compatible with the act-engineering approach, which provides a novel chemical-biological tool for the control of PTP activity and the study of PTP function.
Highlights
Strategies for the direct chemical activation of specific signaling proteins could provide powerful tools for interrogating cellular signal transduction
Guided by protein tyrosine phosphatase (PTP)-domain primary-sequence alignments, we show that WPD-loop engineering generates three new PTP-domain constructs—actHePTP, actPTPκ, and actSHP2—whose enzymatic activities are potently augmented by administration of the cyanine-based biarsenicals AsCy3 and/or AsCy5
We have previously shown that the biarsenical compound AsCy3, which does not affect the activities of wild-type PTP1B or T-cell PTP (TCPTP), successfully binds to and activates the tri-cysteine mutants actPTP1B and actTCPTP (Fig. 1A)[31]
Summary
Strategies for the direct chemical activation of specific signaling proteins could provide powerful tools for interrogating cellular signal transduction. Proteins that have been suitably engineered, either through fusion with other protein domains or through site-directed mutagenesis, can potentially be activated using optogenetics tools[17,18,19], chemical inducers of dimerization[20,21], or chemical rescue[22,23,24,25,26,27,28,29] By and large, these activation approaches utilize light or small molecules to convert a target engineered protein from an “off ” state to an “on” state. We have attempted to develop methods for engineering activatable PTPs that retain wild-type-like enzymatic activities and regulatory-control mechanisms until a small-molecule activator is administered[30,31] In this vein, we recently reported that the phosphatase PTP1B can be rendered activatable by targeting the enzyme’s
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