(P23-045-23) Phospho-Proteomic Analysis of Insulin Signaling in Cultured Podocytes Reveals Novel Signaling Networks

Objectives: The objective of this study is to uncover novel substrates of protein tyrosine phosphatase 1-B (PTP1B), Src homology phosphatase-2 (Shp2), and T-cell protein tyrosine phosphatase (TCPTP) during insulin signaling in the glomerular podocyte. Podocytes are insulin sensitive cells within the kidney and crucial regulators of kidney function.

Methods: We generated isogenic E11 podocyte cells with the knockdown of PTP1B, Shp2, or TCPTP. Cells were reconstituted with the corresponding substrate trapping mutant of the phosphatase to identify putative substrates. Cells were stimulated with insulin and PTPs were immunoprecipitated then immunoblotted for phospho-tyrosine residues. Hyper-phosphorylated bands were excised and processed via LC-MS/MS Mass Spectrometry. Protein lists were analyzed by DAVID functional annotation and STRING using the KEGG Pathway Database. Advanced pathway analysis was performed with Qiagen Ingenuity Pathway Analysis (IPA).

Results: The top 50 putative substrates were identified for each PTP, including unique and redundant proteins. DAVID analysis indicated adherens junction and focal adhesion as functional groups significantly associated between all three PTP’s putative substrates. STRING analysis suggests direct (physical) and indirect (functional) interactions of putative substrates relevant to significantly associated functional groups.

Conclusions: We suggest PTP1B, Shp2, and TCPTP participate in the coordinated regulation of insulin signaling through redundant and unique putative substrates. These PTPs appear to impart effects on filament and junctional proteins thereby affecting cell motility and actin cytoskeleton organization.

Funding Sources: Research in the Haj laboratory was funded by the National Institute of Diabetes and Digestive and Kidney Diseases grants R01DK095359 and R01DK090492, the National Institute of Environmental Health Sciences grant P42ES04699 and NIFA grant CA-D*-NTR-7836H. Dr. Hsu was supported by National Institute on Alcohol Abuse and Alcoholism grant R21AA027633. Research in the Meng laboratory is funded by the Academia Sinica Next-generation Pathway of Taiwan Cancer Precision Medicine Program Grant AS-KPQ-107-TCPMP.