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Protein tyrosine phosphorylation is critical for cellular function, and aberrant phosphorylation is tied to a wide range of human diseases. Identifying the substrates of protein tyrosine phosphatases, the enzymes that erase this modification, is critical to understanding human biology and disease states. The state-of-the-art method for tyrosine phosphatase substrate identification requires the use of mutations that modestly increase the lifetime of enzyme-substrate complexes by kill catalytic activity. While these “substrate-trapping” mutants are useful tools, they work best for high-affinity or abundant substrates that remain phosphatase-bound through cell lysis and enrichment. Here, we use site-specific photo-crosslinking to covalently capture the substrates of tyrosine phosphatases in situ. We identify eight different positions around the active site of the phosphatase PTP1B where photo-crosslinker amino acids can be incorporated via amber codon suppression without dramatically disrupting catalytic activity. We then conduct photo-crosslinking experiments in mammalian cells and identify crosslinked proteins by mass spectrometry proteomics, revealing that our approach can capture known PTP1B interactors and substrates. We then show that PTP1B photo-crosslinking in situ is sensitive to enzyme localization and identify new PTP1B substrates that regulate contacts between the endoplasmic reticulum and plasma membrane. We also demonstrate that photo-crosslinking can capture signal-dependent interactions. For example, we observe PTP1B crosslinking to the epidermal growth factor (EGF) receptor, a known substrate, in an EGF-dependent manner, and we identify other potential EGF-dependent substrates. Overall, our approach reveals previously unknown roles of PTP1B in signaling systems and could be readily extended to other tyrosine phosphatases in the same family.
RNA-binding proteins (RBPs) are key regulators of gene expression that shape cellular function in health and disease. However, the roles of RBPs in immune cells within the central nervous system (CNS) remain poorly understood. Here, we identify ARID5A as an RBP highly expressed in microglia and uncover its RNA-mediated regulatory functions using integrated multi-omics analyses of its RNA, DNA, and protein interactions. ARID5A regulates the splicing and translation of its RNA targets, many of which are integral to lysosomal, immune, and iron metabolism pathways. We confirm the functional relevance of this ARID5A-dependent RNA regulatory network by demonstrating that ARID5A modulates lysosomal activity, cytokine secretion, iron accumulation, and ferroptosis in iPSC-derived microglia. We further demonstrate that knockdown of microglial ARID5A reduces neuronal ferroptosis in co-cultures, underscoring the interconnected nature of these pathways. Moreover, in microglia harboring the TREM2-T66M mutation, ARID5A depletion restores dysregulated lysosomal and metabolic functions. Our results highlight the importance of protein-RNA interactions in regulating microglial cell biology.