Epithelial Systems Biology Laboratory (ESBL)

Phosphopeptides from proteins involved in membrane trafficking that are altered in abundance in response to dDAVP in rat IMCD suspensions* (See below for narrative)

*Peptides satisfied dual criteria: |Mean log2(dDAVP/Control)| > 0.342 and FDR < 0.005. The value 0.342 defines the 95% confidence interval for control:control comparisons.

§, Phosphorylated amino acid indicated by *; #, deamidated asparagine or glutamine; †, False Discovery Rate; ‡ Imported from "FUNCTION" field of UniProt record



Gene Symbol UniProt no. Residue(s) Peptide§ Mean log2(dDAVP/Vehicle) SD log2(dDAVP/Vehicle) FDR† Membrane Trafficking Function ‡

Vasopressin-mediated increase in AQP2 exocytosis and decrease in AQP2 endocytosis. In collecting duct cells, addition of vasopressin stimulates a rapid increase in AQP2 exocytosis [(Nielsen, 1993), (Knepper, 1993), (Nielsen, 1995), (Brown, 2003)] followed by a partially compensating increase AQP2 endocytosis [(Nielsen, 1993), (Knepper, 1993), (Nielsen, 1995), (Brown, 2003)]. These responses are sustained until a new steady-state is reached with increased occupancy of AQP2 in the apical plasma membrane, resulting increase in water permeability (Nielsen, 1995) (Figure 0). In the present study, we identified a number of phosphopeptides whose abundances were altered by vasopressin and whose molecular functions are compatible with roles in AQP2 trafficking. Three of the differentially phosphorylated proteins in the above table are regulators of the SNARE-mediated vesicle fusion necessary for the exocytic process, namely tomosyn (Stxbp5), PCTAIRE kinase 1 (Cdk16) and synaptotamin-17 (Syt17). SNARE complexes have been proposed to play essential roles in AQP2 exocytosis (Valenti, 2005). Protein kinase A has been shown to directly phosphorylate tomosyn, causing it to dissociate from the SNARE complex thereby enhancing exocytic vesicle fusion (Baba, 2005). PCTAIRE kinase 1/Cdk16 also regulates exocytosis in part by phosphorylating NSF, an AAA-ATPase that catalyzes SNARE-mediated vesicle fusion (Liu, 2006) and has been reported to play a role in the exocytic release of insulin in pancreatic beta cells (Chen, 2012). Synaptotagmin-17, also called Brain/Kidney (B/K) protein, is a calcium-sensor protein implicated in regulation of SNARE-mediated vesicle fusion. Protein kinase A has been shown to directly phosphorylate it and, when expressed in LLC-PK1 cells, the vasopressin analog dDAVP increased its phosphorylation (Chin, 2006). Beyond this, a protein kinase D1 increases trafficking from the trans-Golgi network to the plasma membrane, i.e. exocytosis, indirectly by phosphorylating and activating phosphatidylinositol 4-kinase beta (Pi4kb) [(Bossard, 2007), (Hausser, 2005)]. In the present study, Pi4kb was also found to undergo a marked increase in phosphorylation at Ser511, previously identified as a direct PKA phosphorylation site (Isobe, 2017) (https://hpcwebapps.cit.nih.gov/ESBL/PKANetwork/Exocytosis.html)

Vasopressin also caused phosphorylation changes in several proteins involved in endosomal sorting and recycling to the plasma membrane, namely Myo5b (Myosin Vb), Usp10, Snx1 (sorting nexin 1), Ndrg1, Vps50, Washc2 and Vipas39. With the exception of Vipas39, all of these proteins underwent marked increases in phosphorylation at sites consistent with phosphorylation by a basophilic protein kinase such as PKA. Myosin Vb (Myo5b) has been previously implicated in vasopressin-mediated exocytosis of AQP2-containing intracellular vesicles (Nedvetsky, 2007). It interacts with recycling endosomes via Rab11a and Lima1 (Zhang, 2018). In previous studies, Lima1 was shown to be regulated by vasopressin in collecting duct, not by changes in phosphorylation, but rather through long-term increases in protein abundance (Khositseth, 2011), resulting from increased mRNA levels and translation rate (Sandoval, 2016).