Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

Abstract

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulusexpressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.

Casein Kinase 1 α Phosphorylates the Wnt Regulator Jade-1 and Modulates Its Activity

“…Mass Spectrometry and Phosphoproteomic Analysis-Phosphoproteomic analysis of whole cell lysates was performed as described previously with slight modifications (35,36). Harvested HEK 293T cells were lysed in a buffer containing 8 M urea and 50 mM ammonium bicarbonate with added Pierce protease and phosphatase inhibitor mixture (Thermo Scientific).…”

“…Thermo raw files were analyzed using the MaxQuant software suite, as described previously, with the label-free quantitation option enabled (36,39,40). Briefly, carbamidomethylation of cysteine was put as a fixed modification, and phosphorylation and N-terminal acetylation was put as a variable modification.…”

Jade-1S phosphorylation induced by CK1α contributes to cell cycle progression

“…56,57 Harvested HEK 293T cells were lysed in a buffer containing 8 M urea and 50 mM ammonium bicarbonate including 1x Pierce protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). 400 mg of total protein (determined with a commercial BCA assay kit) was reduced using DTT and alkylated using Iodoacetamide as previously described and peptides were digested using trypsin at a w:w ratio of 1:50 at 37 C for 16 hours.…”

Insights into autosomal dominant polycystic kidney disease by quantitative mass spectrometry-based proteomics

“…Moreover, previously undescribed phosphorylation sites on the slit diaphragm proteins KIRREL, NPHS1, NPHS2, CD2AP and TRPC6 were discovered. The sites were found exclusively in acidic amino acid motifs indicating that acidic kinases such as casein kinases are responsible (Rinschen et al 2014b). To prioritize and identify physiologically meaningful phosphorylation sites, a cross-species comparability of glomerular phosphorylation sites between cow and rat was performed (Rinschen et al 2015).…”