Phosphorylation is a reversible post-translational modification that is commonly involved in regulation of protein function and distribution. In the kidney, water and NaCl absorption along the nephron is regulated by vasopressin (AVP); thus understanding of AVP-regulated protein phosphorylation is desirable to gain a comprehensive systems-level understanding of water and NaCl absorption mechanisms. We have recently identified several novel phosphorylation sites in the water channel aquaporin-2 (AQP2) and the thiazide-sensitive NaCl-cotransporter (NCC). AQP2 contains four conserved phosphorylation sites at the COOH-terminal tail, namely Ser-256, Ser-261, Ser-264 and Ser-269 (pS256, pS261, pS264, pS269), which play a complex role in regulation of AQP2 trafficking and cellular distribution. The V2 receptor-specific AVP analog dDAVP increases phosphorylation at S256, S264 and S269 within minutes (S261 decreases), but the dynamics of phosphorylation differ; phosphorylation at S264 and S269 occurrs at a much slower rate than the corresponding increase in S256 phosphorylation. AVP-mediated changes in phosphorylation at all sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays demonstrated that PKA phosphorylates S256, but not S264 or S269. Phosphorylation of AQP2 at S264 or S269 cannot occur when S256 is replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in MDCK cells expressing an S256A mutant. These results suggest that the complicated process of COOH-terminal polyphosphorylation of AQP2 occurs as a hierarchal event. Manipulation of the AVP axis in animals, followed by examination of the intracellular distribution of phosphorylated AQP2 has provided clues to cellular functions. For example, in contrast to all other phosphorylated forms of AQP2, pS269-AQP2 is localized exclusively in the apical plasma membrane of collecting duct principal cells and is not internalized, suggesting a particular role for this phosphorylation site in AQP2 trafficking or function. Studies in Xenopus laevis oocytes demonstrated that phosphorylation of S256, S261, S264 and S269 do not directly alter the water transport function of AQP2. Additionally, the relative unit water permeability of phosphorylation deficient mutant forms of AQP2 were identical; suggesting that the water permeability of AQP2 is not regulated by COOH-terminal phosphorylation dependent gating. We have used numerous stably transfected MDCK cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2 and (iii) AQP2 protein-protein interactions. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Forskolin significantly increased the membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. In addition to AQP2, we have begun to examine the role of vasopressin in modulating phosphorylation of NCC. Immunogold electron microscopy demonstrated that, unlike total NCC, pNCC was localized only in the apical plasma membrane and was not observed within the cell. In rats, acute dDAVP exposure for 15 min significantly increased pNCC abundance at the apical plasma membrane approximately 300%, whereas total NCC abundance and distribution were not significantly altered. dDAVP significantly increased the abundance of phosphorylated STE20 (sterile 20)-like kinase SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1). Early and late DCTs increased their intracellular Ca2+ levels in response to 1 min of superfusion of dDAVP (2 x 10(-10) M), confirming that the DCT is an AVP responsive segment. In rats fed a high salt diet alongside AT1-R blockade by candesartan, similar increases in pNCC, p-SPAK and p-OSR1 abundance were observed following chronic or acute dDAVP, indicating that the effects of AVP can occur independently of ANGII. In conclusion, the post-translational mechanism of protein phosphorylation is a dynamic event that is able to regulate channels and transporters by various mechanisms.