Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination.

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.