Increased kinase signaling may play an important role in the abnormal cellular proliferation, angiogenesis, and immune dysregulation associated with pulmonary arterial hypertension (PAH).
We performed an unbiased phosphoproteomic analysis of lung tissue obtained from subjects with idiopathic PAH and compared it to control donor lung tissue using UPLC MS/MS on an LTQ Orbitrap ELITE System. Data were analysed with Progenesis LCMS software. NetworKin and Phosphonet were used to predict kinases responsible for phosphorylation. STRING was used to build regulatory kinase networks implicated in PAH. Immunohistochemistry and Western blot analyses were performed to confirm the presence of the most upregulated phosphoproteins in iPAH lung.
Several dramatically increased and decreased phosphoproteins were identified in iPAH lungs compared to controls. The most increased phosphoproteins included S378-IKZF3, S73-HMHA1, S709-BCAS3, T442-RHG25, S2047-NUMA1, Y900-AQR, S710-ASPP1, T180/Y182-MK14, T231-ZN404, and S107-HDGF. The most decreased phosphoproteins included S119-PTPRB, Y48-SNG2L, S624-HTSF1, S275-PSIP1, S294-ANXA2, S422-HDAC2, and T113-S10A9. Kinase networks identified from phosphopeptide analysis revealed CSNK2A1 (casein kinase 2 subunit alpha), CDK1, MAPK12, MAPK14, PRKs, PDKs, DYRK2, AKT1, AurkA, CAMK2A, CAMK2B, CAMK2D, CAMK2G, CAMK4, GSK3A, GSK3B, HIPK2, HYRC, and PAKs as potentially important kinases in PAH. In vitro phosphorylation assays indicated that GSK3α and/or GSK3β could phosphorylate IKZF3 (Aiolos) at S378. Western blot analysis confirmed that pIKZF3 (S378), a zinc finger transcription factor that plays a key role in lymphocyte regulation, was increased in iPAH compared to control lung lysates. Immunohistochemistry demonstrated pIKZF3 in lymphocytes surrounding severely hypertrophied pulmonary arterioles which corresponded predominantly to perivascular T cells.
An unbiased phosphoproteomic analysis demonstrated several novel targets regulated by kinase networks, and reinforced the role of dysregulated immune, angiogeneic, and proliferative pathways in iPAH. The identified up and down-regulated phosphoproteins have potential as biomarkers to guide targeted therapy for PAH and provide new insights for therapeutic strategies.