Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z.
Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.
Fukada
Masahide
M
Division of Molecular Neurobiology, National Institute for Basic Biology, 5-1 Higashiyama, Myodaiji-cho, Okazaki 444-8787, Japan.
Kawachi
Hiroyuki
H
Fujikawa
Akihiro
A
Noda
Masaharu
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology; D020798:Two-Hybrid System Techniques
D017027:Protein Tyrosine Phosphatases