Epithelial Systems Biology Laboratory

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Phosphoproteomic Profiling of Renal Epithelial Cells

Transporters in renal epithelia can be regulated via a variety of mechanisms including post-translational modification of amino acid side chains at specific sites. Such modifications can result in changes in the transport properties of the target protein or in changes in the ability of the modified protein to interact with other proteins or membrane lipids. Among the many types of post-translational modification known, phosphorylation is of premier importance, playing central roles in virtually every signaling process affecting epithelial cell function. To generate new hypotheses about regulation of transport in kidney epithelia, we have carried out LC-MS/MS-based phosphoproteomics in native inner medullary collecting ducts (11, 8, 4), thick ascending limbs of Henle's loops (10), and proximal tubules (7) after biochemical isolation from rat kidneys. Studies have been extended to cultured mpkCCD collecting duct cells (9, 5, 2, 1), yielding new knowledge about pathways targeted by vasopressin. Data is made available via online databases.

Modeling vasopressin signaling. A major computational objective is to use the accrued phosphoproteomic data to create candidate signaling-network models that can explain how vasopressin drives the regulation of aquaporin-2 by effecting changes in Aqp2 gene transcription and membrane trafficking (6, 3).

References:

  1. Limbutara K, Kelleher A, Yang CR, Raghuram V, Knepper MA. Phosphorylation Changes in Response to Kinase Inhibitor H89 in PKA-Null Cells. Sci Rep. 2019; 9:2814. PMID: 30808967.

  2. Isobe K, Jung HJ, Yang CR, Claxton J, Sandoval P, Burg MB, Raghuram V, Knepper MA. Systems-level identification of PKA-dependent signaling in epithelial cells. Proc Natl Acad Sci USA. 2017; 114:E8875-E8884. PMID: 28973931.

  3. Bradford D, Raghuram V, Wilson JL, Chou CL, Hoffert JD, Knepper MA, Pisitkun T. Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256. Am J Physiol Cell Physiol. 2014; 307:C123-39. PMID: 24598363.

  4. Hoffert JD, Pisitkun T, Saeed F, Wilson JL, Knepper MA. Global analysis of the effects of the V2 receptor antagonist satavaptan on protein phosphorylation in collecting duct. Am J Physiol Renal Physiol. 2014; 306: 410-21. PMID: 24259510.

  5. Bolger SJ, Hurtado PA, Hoffert JD, Saeed F, Pisitkun T, Knepper MA. Quantitative phosphoproteomics in nuclei of vasopressin-sensitive renal collecting duct cells. Am J Physiol Cell Physiol. 2012; 303:C1006-20. PMID: 22992673.

  6. Knepper MA. Systems biology in physiology: the vasopressin signaling network in kidney. Am J Physiol Cell Physiol. 2012; 303:C1115-24. PMID: 22932685.

  7. Feric M, Zhao B, Hoffert JD, Pisitkun T, Knepper MA. Large-scale phosphoproteomic analysis of membrane proteins in renal proximal and distal tubule. Am J Physiol Cell Physiol. 2011; 300: C755-70. PMID: 21209370.

  8. Bansal AD, Hoffert JD, Pisitkun T, Hwang S, Chou CL, Boja ES, Wang G, Knepper MA. Phosphoproteomic profiling reveals vasopressin-regulated phosphorylation sites in collecting duct. J Am Soc Nephrol. 2010; 21:303-15 PMID: 20075062.

  9. Rinschen MM, Yu MJ, Wang G, Boja ES, Hoffert JD, Pisitkun T, Knepper MA. Quantitative phosphoproteomic analysis reveals vasopressin V2-receptor-dependent signaling pathways in renal collecting duct cells. Proc Natl Acad Sci USA. 2010; 107: 3882-7. PMID: 20139300.

  10. Gunaratne R, Braucht DW, Rinschen MM, Chou CL, Hoffert JD, Pisitkun T, Knepper MA. Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells. Proc Natl Acad Sci USA. 2010; 107: 15653-8. PMID: 20713729.

  11. Hoffert JD, Pisitkun T, Wang G, Shen RF, Knepper MA. Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites. Proc Natl Acad Sci USA. 2006; 103: 7159-64. PMID: 16641100.