A Generalizable Photo-Crosslinking Strategy to Identify Tyrosine Phosphatase Substrates

PROJECT SUMMARY Protein tyrosine phosphorylation is a common mechanism for relaying essential information in animal cells, and many cancers are driven by aberrant tyrosine phosphorylation events. This protein modification is mediated by two classes of enzymes: tyrosine kinases and tyrosine phosphatases, which phosphorylate and dephosphorylate, respectively, thousands of different proteins. Many tyrosine kinases are extremely well- characterized and are the targets of numerous clinically-approved cancer therapies. By contrast, most tyrosine phosphatases are not biochemically well-characterized, and in particular, we do not know the specific proteins that many of these enzymes dephosphorylate. Despite this dearth of knowledge, we known that a few tyrosine phosphatases directly contribute to cancer signaling, and genetic evidence suggests that many more are likely to be important cancer drivers, tumor suppressors, or modulators of the immune response against cancer cells. In order to elucidate the precise roles of individual tyrosine phosphatases in cellular processes, we need tools to rapidly and reliable identify their substrates. Here, we propose a strategy to identify direct substrates of tyrosine phosphatases in live cells, by combining protein engineering and mass spectrometry-based proteomics. A major challenge in the identification of tyrosine phosphatase substrates is that their interactions with their cognate enzymes are weak and transient, making them difficult to isolate from complex proteomic mixtures. To solve this problem, we will use genetic code expansion to introduce light-activatable crosslinkers into tyrosine phosphatases. In Aim 1, we will combine structural and evolutionary information to identify ideal positions on tyrosine phosphatases to place photo-crosslinkers, such that they do not perturb phosphatase function but can irreversibly capture a substrate when exposed to UV light. We will test out candidate positions on a model tyrosine phosphatase, PTP1B, with three different photo-crosslinkers. In Aim 2, we will examine the generality of our best designs by testing them on several tyrosine phosphatases and an array substrates. In Aim 3, we will establish methods to express the engineered tyrosine phosphatases in live mammalian cells, induce the capture of substrates with UV light, and identify those substrates using mass spectrometry. We will compare our strategy to the current state-of-the-art method, which relies on mutations in tyrosine phosphatases that significantly disrupt their function and only modestly stabilize their interactions with substrates. Successful development of our photo-crosslinking method will enable efficient identification of the substrates of any tyrosine phosphatases in virtually any cell line of interest. This technique could be used to delineate the roles of individual tyrosine phosphatases in cancer signaling and cancer-relevant immunity.