Substrate-trapping techniques in the identification of cellular PTP targets

doi:10.1016/j.ymeth.2004.07.007

Methods

Volume 35, Issue 1, January 2005, Pages 44-53

https://doi.org/10.1016/j.ymeth.2004.07.007 Get rights and content

Abstract

Tyrosine phosphorylation is negatively regulated by the protein-tyrosine phosphatases (PTPs). In order to find the physiological substrates of these enzymes, diverse PTP mutants that do not possess any catalytic activities but appear to bind tightly to their tyrosine phosphorylated substrates have been designed. Hence, they can be used as tools to pull out their respective substrates from heterogeneous extracts. Named PTP “substrate-trapping” mutants by the Tonks laboratory, they represent a diverse variety of defective PTPs that are epitomized by the Cys to Ser mutant (C/S) where the active cysteine residue of the signature motif is mutated to a serine residue. In addition, new mutants have been developed which are expected to help characterize novel and less abundant substrates. In this article, we review and describe all the different substrate-trapping mutants that have successfully been used or that hold interesting promises. We present their methodology to identify substrates in vivo (co-immunoprecipitation) and in vitro (GST pulldown), and provide a current list of substrates that have been identified using these technologies.

Introduction

Protein-tyrosine phosphorylation is a reversible post-translational modification that is essential for eukaryotic cells. The counteracting activities of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulate the level of cellular tyrosine phosphorylation. Because of the complexity of these gene families, to decipher the signaling in which each of the kinases and phosphatases are involved can be very challenging. One obvious step is to characterize their physiological substrates. Intuitively, it seems easier to find a substrate for a kinase than for a phosphatase. Kinases will act directly on their targets, which results on the addition of a phosphate group that can directly point to the substrates of the kinases (radioactively or using chemiluminescence). However, to uncover the phosphatase’s substrates, one requires the detection of such phosphate removal from previously phosphorylated proteins. Furthermore, the promiscuous in vitro activity of PTP complicates the identification of genuine PTP substrates. Fortunately, the PTP field obtained a valuable tool with the generation of mutant PTPs that could act as substrate-trapping mutants.

Typically, in PTP substrate-trapping mutants, the ongoing PTP catalysis is blocked. As a consequence, the substrate is trapped in the catalytic pocket of the PTP. Such enzyme–substrate interaction is sufficiently stable so that the complex can be purified. Substrate-trapping mutants have been largely used to characterize (or confirm) physiological substrates and consequently the signaling pathway in which a PTP is involved. These mutants became a very useful and important biochemical tool. The characteristic of a good substrate-trapping mutant are (i) to be inactive or barely active (lowest k_cat), (ii) to bind efficiently to its physiological substrate (low k_m), and (iii) to keep its structural integrity as much as possible.

The PTP substrate-trapping mutants have been largely used in different set up. They have been used to find PTP specificity (this issue, Espanel et al.), to isolate specific inhibitor (this issue, Kumar et al.) and to screen cDNA library in a modified two-hybrid system (this issue, Fukada et al.). In this article, we review and describe all the different substrate-trapping mutants that have successfully been used or that hold interesting promises. We present the substrate-trapping methodology for in vivo (co-immunoprecipitation) and in vitro (pulldown) studies. We also listed the substrates that have been identified using these technologies.

Section snippets

Substrate-trapping mutants

The development of substrate-trapping mutants is a direct consequence of understanding the PTP’s catalytic mechanism. Enzymatic and structural characterization of PTP1B, in particular, pinpointed to critical residues for phosphotyrosine catalysis whose mutations would generate a defective enzyme that could fulfill the criteria required to generate a substrate-trapping mutant. Experimentally, the substrate-trapping mutants the most used so far have been the mutants in which the signature motif

Principle and advantages

In vitro substrate trapping is performed by mixing cell lysates containing tyrosine phosphorylated proteins with bacterially expressed and purified GST–PTP substrate-trapping mutant. After incubation and washing, the tyrosine phosphorylated proteins bound to the substrate-trapping mutant are detected by anti-phosphotyrosine antibody on a western blot. The main advantage of this method is that it can easily be scaled-up, eventually leading to the purification of enough substrate to identify it

Principle and advantages

The purification of GST–PTP, the use of iodoacetic acid and DTT is sometime laborious and more prone to artifactual results. Another approach to trap substrates is through co-immunoprecipitation with the PTP substrate-trapping mutant directly expressed in mammalian cells. After the transfection and immunoprecipitation of the tagged PTP, the samples are analyzed by western blotting using anti-phosphotyrosine antibodies. In this case, the phosphotyrosine-proteins that are co-immunoprecipitated

Vanadate competition

Any tyrosine phosphorylated protein that forms a complex with the substrate-trapping mutant is not necessarily a substrate. It can be a protein interacting directly or indirectly to the PTP outside of its catalytic pocket. Experimentally, these PTP interacting proteins are often found to be substrate as well, but not necessarily. One way to validate that a substrate binds to the catalytic site of the substrate-trapping mutant is to use vanadate as a competitor of the substrate. Vanadate (1–10

Concluding remarks

The plethora of substrate-trapping mutant becomes larger with mutants showing increased affinity for their substrates. These mutants will be important tool to isolate new, less abundant substrates. In all cases, the potential substrate(s) (phosphotyrosine-proteins) binding to substrate-trapping mutants must be validated as physiological substrates by other methods. Ideally, several conditions should be fulfilled to prove that a phosphoprotein is a physiological substrate: (i) a substrate should

Acknowledgments

This work was supported by a Human Frontier Science Program (HFSP) long term fellowship (to C.B.), an Alexander McFee Memorial Fellowship (to M.C.), a Canadian Institutes of Health Research doctoral award (to N.D.), an Arthur S. Hawkes Fellowship (to M.H.), and CIHR research grant (#MOP 12466, to M.L.T.). M.L.T. is a scientist of the CIHR.

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