Data resource: vasopressin-regulated protein phosphorylation sites in the collecting duct

Abstract

Vasopressin controls renal water excretion through actions to regulate aquaporin-2 (AQP2) trafficking, transcription, and degradation. These actions are in part dependent on vasopressin-induced phosphorylation changes in collecting duct cells. Although most efforts have focused on the phosphorylation of AQP2 itself, phosphoproteomic studies have identified many vasopressin-regulated phosphorylation sites in proteins other than AQP2. The goal of this bioinformatics-based review is to create a compendium of vasopressin-regulated phosphorylation sites with a focus on those that are seen in both native rat inner medullary collecting ducts and cultured collecting duct cells from the mouse (mpkCCD), arguing that these sites are the best candidates for roles in AQP2 regulation. This analysis identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. We provide resource web pages at https://esbl.nhlbi.nih.gov/Databases/AVP-Phos/ and https://esbl.nhlbi.nih.gov/AVP-Network/, listing the phosphorylation sites and describing annotated functions of each of the vasopressin-targeted phosphoproteins. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. The remaining sites are predicted to be phosphorylated by other kinases, most notably ERK1/2, which accounts for decreased phosphorylation at sites with a X-p(S/T)-P-X motif. Additional protein kinases that undergo vasopressin-induced changes in phosphorylation are Camkk2, Cdk18, Erbb3, Mink1, and Src, which also may be activated directly or indirectly by PKA. The regulated phosphoproteins are mapped to processes that hypothetically can account for vasopressin-mediated control of AQP2 trafficking, cytoskeletal alterations, and Aqp2 gene expression, providing grist for future studies.NEW & NOTEWORTHY Vasopressin regulates renal water excretion through control of the aquaporin-2 water channel in collecting duct cells. Studies of vasopressin-induced protein phosphorylation have focused mainly on the phosphorylation of aquaporin-2. This study describes 44 phosphoproteins other than aquaporin-2 that undergo vasopressin-mediated phosphorylation changes and summarizes potential physiological roles of each.

Keywords: V2 vasopressin receptor; aquaporin-2; kidney; phosphoproteomics; protein kinases.

Graphical abstract

Figure 1.

Consensus vasopressin-regulated protein phosphorylation sites in collecting duct cells. Two separate datasets, one from the cultured collecting duct cells from the mouse (mpkCCD) cell line treated with 0.1 nM desmopressin [1-deamino-8-d-arginine vasopressin (dDAVP)] for 30 min and one from an inner medullary collecting duct (IMCD) suspension treated with 1 nM dDAVP for 30 min, were combined to identify the phosphorylation sites that changed significantly in response to vasopressin in both studies (27, 61). 51 consensus dDAVP-responsive phosphosites are identified.

Figure 2.

Relational graph showing the vasopressin-regulated phosphoproteins mapped to molecular functions. An annotated version with popups showing descriptions of each node can be found online at https://esbl.nhlbi.nih.gov/AVP-Network/. The 51 phosphosites are categorized into 11 vasopressin-associated cellular processes (see Table 1). Each cellular process is known to play roles in the regulation of collecting duct water permeability.

Figure 3.

Role of protein kinase A (PKA) in vasopressin-mediated phosphorylation. A: correlation between the response to vasopressin for the sites shown in Fig. 1 and response to deletion of both PKA catalytic genes in cultured collecting duct cells from mouse (mpkCCD) cells. The plot shows a strong negative correlation (P < 0.01). B: volcano plot showing changes in phosphorylation for all sites in mpkCCD cells in response to deletion of both PKA catalytic genes, highlighting the phosphorylation sites shown in Fig. 1. C: desmopressin [1-deamino-8-d-arginine vasopressin (dDAVP)]-responsive phosphosites show differing responses to PKA-Cα single knockout (KO) versus PKA-Cβ single KO mpkCCD cells. Many of the dDAVP-responsive phosphosites showed opposite phosphorylation changes in PKA-Cα KO versus PKA-Cβ KO cells, suggesting that the two PKA catalytic subunits have different functions in the cell. Green indicates sites with increased phosphorylation in response to dDAVP; pink indicates sites with decreased phosphorylation in response to dDAVP. Ctrl, control.

Figure 4.

Dynamics of phosphorylation responses to vasopressin for different phosphorylation sites in native inner medullary collecting duct (IMCD) cells. Data are replotted from Leo et al. (97). A: sites associated with vesicle-mediated transport: Bin1 (Ser³⁰⁴), Exoc7 (Ser²⁵⁰), Nsfl1c (Ser¹⁷⁶), and Sec22b (Ser¹³⁷). B: sites associated with the regulation of GTPase activity/actin cytoskeleton/microtubule cytoskeleton: Agfg1 (Ser¹⁸¹), Arfgef1 (Ser¹⁵⁶⁶), Arhgef2 (Ser⁸⁸⁵), Specc1l (Ser³⁸⁵), and Rmdn3 (Ser⁴⁶). C: sites associated with the regulation of transcription: Ctnnb1 (Ser⁵⁵² and Thr⁵⁵¹), Lrrfip1 (Ser⁸⁸), and Tsc22d4 (Thr²²³). D: sites associated with protein phosphorylation: Camkk2 (Ser⁴⁹⁵ and Ser⁵¹¹), CDK18 (Ser⁶⁶), Erbb3 (Ser⁹⁸⁰), and Src (Ser¹⁷). E: sites associated with cAMP-dependent signaling/Ca²⁺ signaling: Prkar2a (Ser⁹⁶), Itpr1 (Ser¹⁵⁸⁸), Itpr3 (Ser¹⁸³²), and Stim1 (Ser⁵⁷⁵). F: sites associated with cell polarity: Igsf5 (Ser³³⁵), Luzp (Ser²⁶¹), and Slc9a3r1 (Ser²⁷⁵). Some sites shown in Fig. 1 did not yield time course data in Leo et al. (97). In general, phosphorylation responses were faster than observed water permeability responses, indicating that phosphorylation is not rate limiting for the overall response (see text).

Figure 5.

Vasopressin-regulated phosphoproteins with hypothetical roles in “vesicle-mediated transport.” Listed phosphoproteins are likely to be involved in three biological processes, “SNARE-mediated vesicle fusion,” “endosomal biogenesis,” and “interaction with dynamins,” which could regulate vasopressin-mediated aquaporin-2 (AQP2) trafficking. Cdk18, cyclin-dependent kinase 18; PKA, protein kinase A.

Figure 6.

Vasopressin-regulated phosphoproteins with hypothetical roles in the regulation of the “actin cytoskeleton,” “microtubule cytoskeleton,” and “GTPase activity.” The phosphorylation responses could potentially be involved in the regulation of aquaporin-2 (AQP2) trafficking. PKA, protein kinase A.

Figure 7.

Vasopressin-regulated phosphoproteins with hypothetical roles in the “regulation of transcription” and “RNA processing.” The phosphorylation responses could potentially be involved in the regulation of the abundance of aquaporin-2 (Aqp2) mRNA and secondarily AQP2 protein. PKA, protein kinase A.