Substrate-trapping techniques in the identification of cellular PTP targets.
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.
Blanchetot
C
C
McGill Cancer Centre, McGill University, 3655 Sir William-Osler, Room 715, Montreal, Quebec, H3G1Y6 Canada. christophe.blanchetot@mcgill.ca
Chagnon
M
M
Dubé
N
N
Hallé
M
M
Tremblay
M L
ML
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D014638:Vanadates / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D014638:Vanadates; D012694:Serine; D017027:Protein Tyrosine Phosphatases; D005978:Glutathione; D003545:Cysteine; D000409:Alanine