Andrew Johns
Columbia University
In situ Identification of Substrates of the Protein Tyrosine Phosphatase PTP1B Using Photo-crosslinking Chemistry
Protein tyrosine phosphorylation is critical for cellular function, and aberrant phosphorylation is tied to a wide range of human diseases. However, there are few tools to identify tyrosine phosphatase substrates. The state-of-the-art method for tyrosine phosphatase substrate identification requires the use of mutations that kill catalytic activity and modestly increase the lifetime of the enzyme-substrate complexes. While these “substrate-trapping” mutants are useful tools, they only work effectively on high-affinity substrates. We have developed a photo-crosslinking strategy that maintains catalytic activity and covalently captures enzyme-substrate complexes in situ in response to UV irradiation. Using Amber codon suppression, we introduced photo-crosslinker amino acids into the model phosphatase PTP1B at eight different minimally perturbing sites around the active site. These were expressed in mammalian cells, irradiated, and enriched by immunoprecipitation. Crosslinked proteins were identified by MS proteomics. From these experiments, we found that PTP1B likely regulates key proteins that mediate ER-PM contacts. Notably, our approach can detect signal-dependent interactions. For example, we found that PTP1B crosslinked the known substrate EGFR in an EGF stimulation-dependent manner. And suggests other EGF-dependent PTP1B substrates. For example, we found that PTP1B likely dephosphorylates Hexokinase-1 in response to EGF stimulation, dampening the cells shift to glycolysis in response to growth signals. This technology has been used to elucidate previously unknown roles of PTP1B in signaling systems and can be applied to other tyrosine phosphatases of the same family.
Protein tyrosine phosphorylation is critical for cellular function, and aberrant phosphorylation is tied to a wide range of human diseases. However, there are few tools to identify tyrosine phosphatase substrates. The state-of-the-art method for tyrosine phosphatase substrate identification requires the use of mutations that kill catalytic activity and modestly increase the lifetime of the enzyme-substrate complexes. While these “substrate-trapping” mutants are useful tools, they only work effectively on high-affinity substrates. We have developed a photo-crosslinking strategy that maintains catalytic activity and covalently captures enzyme-substrate complexes in situ in response to UV irradiation. Using Amber codon suppression, we introduced photo-crosslinker amino acids into the model phosphatase PTP1B at eight different minimally perturbing sites around the active site. These were expressed in mammalian cells, irradiated, and enriched by immunoprecipitation. Crosslinked proteins were identified by MS proteomics. From these experiments, we found that PTP1B likely regulates key proteins that mediate ER-PM contacts. Notably, our approach can detect signal-dependent interactions. For example, we found that PTP1B crosslinked the known substrate EGFR in an EGF stimulation-dependent manner. And suggests other EGF-dependent PTP1B substrates. For example, we found that PTP1B likely dephosphorylates Hexokinase-1 in response to EGF stimulation, dampening the cells shift to glycolysis in response to growth signals. This technology has been used to elucidate previously unknown roles of PTP1B in signaling systems and can be applied to other tyrosine phosphatases of the same family.
