Tyrosine Phosphorylation of CD2AP Affects Stability of the Slit Diaphragm Complex

Significance Statement The connection between the slit diaphragm and actin network of podocytic foot processes involves complex signaling between slit diaphragm proteins and multiple signaling pathways of the actin machinery. CD2AP, a slit diaphragm–associated scaffolding protein, is considered a “stabilizer” of the complex that connects the slit diaphragm protein nephrin to the cell’s cytoskeleton. In this study, the authors define CD2AP as a phosphorylation target of receptor tyrosine kinases stimulated by VEGF-A in podocytes. They demonstrate that phosphorylation of tyrosine at position Y10 of the SH3-1 domain of CD2AP can change the affinity of CD2AP to nephrin and is indispensable for CD2AP function and slit diaphragm functionality in vivo . These findings implicate CD2AP phosphorylation as a molecular target in proteinuric kidney diseases. Background CD2-associated protein (CD2AP), a slit diaphragm–associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a “stabilizer” of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. Methods We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in Cd2ap KD zebrafish and in Drosophila cindrRNAi mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. Results We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo . We demonstrated that tyrosine in position Y10 of the SH3–1 domain of CD2AP is indispensable for CD2AP function in vivo . We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. Conclusions Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.