Dataset Information

Comparative phosphoproteomic analysis of mammalian renal glomeruli

ABSTRACT: Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Recent, in-depth analysis of the mouse glomerular phosphoproteome by tandem mass spectrometry confidently identified thousands of phosphorylation sites providing a unique opportunity to study signaling in vivo. However, the multitude of phosphorylated residues demands for additional systems biology approaches to separate physiologically important sites from non-functional phosphorylated residues. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of additional phosphoproteomic analyses of the glomerular phosphoproteome in cow and rat to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. Given the pivotal role of podocin for glomerular biology we studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Rattus Norvegicus (rat) Bos Taurus (bovine)

TISSUE(S): Epithelial Cell, Kidney

SUBMITTER: Markus Rinschen

LAB HEAD: Markus Rinschen

PROVIDER: PXD001005 | Pride | 2015-01-16

REPOSITORIES: Pride

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			Action	DRS
	0089-120927MaRiS01Tscx01pD.RAW	Raw
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	0089-120927MaRiS01Tscx03pD.RAW	Raw
	0089-120927MaRiS01Tscx04pD.RAW	Raw
	0089-120927MaRiS01Tscx05pD.RAW	Raw

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Publications

Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C-terminal phosphorylation as a determinant of slit diaphragm complex architecture.

Rinschen Markus M MM Pahmeyer Caroline C Pisitkun Trairak T Schnell Nicole N Wu Xiongwu X Maaß Martina M Bartram Malte P MP Lamkemeyer Tobias T Schermer Bernhard B Benzing Thomas T Brinkkoetter Paul T PT

Proteomics 20150119 7

Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We disco ...[more]

PMID: 25420462

Similar Datasets

Project description:The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the lipid-binding protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore now analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and RNA levels of podocinwildtype and podocinΔexon. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes’ foot process morphology. Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 hours after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas RNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes’ slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.