Mapping of PTP1B, TCPTP, SHP2, and Putative Substrates Reveals Novel Networks in Glomerular Podocytes

Preservation of the insulin-sensitive glomerular podocyte is imperative for normal kidney function. The protein tyrosine phosphatases (PTPs), protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), and Src homology phosphatase 2 (SHP2) are established regulators of insulin signaling in vivo and implicated in renal function. However, knowledge gaps exist regarding the roles of these enzymes and their integrated modulation of signaling in podocytes. Accordingly, uncovering the mediators of PTP function is critical to elucidate their modes of action and help develop mechanism-based interventions for podocytopathies. We generated E11 podocyte cell lines expressing the substrate-trapping mutants of these PTPs and then used immunoprecipitation and mass spectrometry to identify their putative substrates. Bioinformatic analyses were used to decipher the pathways affected by these enzymes in the insulin-stimulated podocytes. We identified known and novel targets, some common across the three PTPs, others shared between two PTPs, and others unique to a single phosphatase. Additionally, cytoskeleton and cellular junction-associated pathways were significantly enriched among the phosphatases and their putative substrates. Moreover, we uncovered a signaling node that is likely key to the action of these PTPs, comprising the protein tyrosine kinase Src, cortactin, and lamin A/C, interconnected via vimentin. To further validate this, we demonstrated that vimentin is a substrate of SHP2 in podocytes. The current findings suggest that PTP1B, TCPTP, and SHP2 act coordinately and engage numerous targets to orchestrate an integrated response to insulin in podocytes. Notably, these enzymes are components of a crucial signaling node that modulates cytoskeletal and junctional proteins, thereby influencing podocyte function.