Large‐scale iTRAQ‐based quantification of phosphorylation changes during vasopressin signaling

Abstract

Protein phosphorylation plays a critical role in the signaling pathways regulating water transport in kidney collecting duct. A central mediator in this process is the hormone arginine vasopressin (AVP), which regulates phosphorylation of the water channel aquaporin-2 (AQP2), although the exact mechanisms are not fully understood. Here we utilized a multiplexed, isotopic label-based quantitative phosphoproteomic approach in order to explore the temporal dynamics of phosphorylation events triggered by vasopressin across multiple timepoints. Briefly, rat inner medullary collecting duct (IMCD) samples were incubated in the presence or absence of 1nM AVP for 0.5, 2, 5, and 15 min (n=3). Each sample was labeled with a different iTRAQ reagent, mixed and processed for shotgun phosphoproteomic analysis. Of the 12,533 phosphopeptides identified, 3,298 were found in at least 2 out of 3 time courses and had quantifiable iTRAQ ratios. Clustering this subset of peptides using a mapping-based temporal pattern mining algorithm identified 30 clusters of phosphopeptides with distinct temporal profiles. Phosphopeptides for Rap1 GTPase activating protein, kinesin 13B, Bcl-2-related ovarian killer (Bok), Pigb, Lrrfip1, and Tbc1d1 were increased in abundance within 0.5 min and displayed similar temporal dynamics to those of AQP2 peptides phosphorylated at residue Ser-256, a site which is critical for AQP2 trafficking. Phosphopeptides for two pore calcium channel protein 1, septin 9, EGFR pathway substrate 8-like protein 1, Ahnak, and actin binding LIM protein 1 were decreased in abundance within 5–15 min of AVP exposure. These data provide the foundation for development of a network model for vasopressin signaling.