Pablo C. Sandoval

Proteome-Wide Measurement of Protein Half-Lives and Translation Rates in Vasopressin-Sensitive Collecting Duct Cells

Vasopressin regulates water excretion, in part, by controlling the abundances of the water channel aquaporin-2 (AQP2) protein and regulatory proteins in the renal collecting duct. To determine whether vasopressin-induced alterations in protein abundance result from modulation of protein production, protein degradation, or both, we used protein mass spectrometry with dynamic stable isotope labeling in cell culture to achieve a proteome-wide determination of protein half-lives and relative translation rates in mpkCCD cells. Measurements were made at steady state in the absence or presence of the vasopressin analog, desmopressin (dDAVP). Desmopressin altered the translation rate rather than the stability of most responding proteins, but it significantly increased both the translation rate and the half-life of AQP2. In addition, proteins associated with vasopressin action, including Mal2, Akap12, gelsolin, myosin light chain kinase, annexin-2, and Hsp70, manifested altered translation rates. Interestingly, desmopressin increased the translation of seven glutathione S-transferase proteins and enhanced protein S-glutathionylation, uncovering a previously unexplored vasopressin-induced post-translational modification. Additional bioinformatic analysis of the mpkCCD proteome indicated a correlation between protein function and protein half-life. In particular, processes that are rapidly regulated, such as transcription, endocytosis, cell cycle regulation, and ubiquitylation are associated with proteins with especially short half-lives. These data extend our understanding of the mechanisms underlying vasopressin signaling and provide a broad resource for additional investigation of collecting duct function (http://helixweb.nih.gov/ESBL/Database/ProteinHalfLives/index.html).

Vasopressin inhibits apoptosis in renal collecting duct cells

The peptide hormone arginine vasopressin (AVP) plays a critical role in regulating salt and water transport in the mammalian kidney. Recent studies have also demonstrated that AVP can promote cell survival in neuronal cells through V1 receptors. The current study addresses whether AVP can inhibit apoptosis in kidney collecting duct cells via V2 receptors and also explores the downstream signaling pathways regulating this phenomenon. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis and caspase cleavage assays demonstrated that 1-desamino-8-d-arginine vasopressin (dDAVP) inhibited apoptosis induced by various agents (staurosporine, actinomycin D, and cycloheximide) in cultured mouse cortical collecting duct cells (mpkCCD). Incubation with dDAVP also inhibited apoptosis induced by the phosphatidylinositol 3-kinase (PI3K) pathway inhibitor LY294002, suggesting that the antiapoptotic effects of dDAVP are largely independent of PI3K signaling. The V2 receptor antagonist SR121463 completely abolished the antiapoptotic effects of dDAVP. In addition, incubation with 8-cpt-cAMP, a cell-permeable analog of cAMP, reproduced the antiapoptotic effects of dDAVP. Both dDAVP and 8-cpt-cAMP increased phosphorylation of proapoptotic Bcl-2 family members Bad and Bok. Bad phosphorylation at Ser-112 and Ser-155 is known to inhibit its proapoptotic activity. Preincubation with H89 blocked dDAVP-induced phosphorylation of both Bad and Bok, suggesting dependence on protein kinase A (PKA). This study provides evidence that AVP can inhibit apoptosis through the V2 receptor and downstream cAMP-mediated pathways in mammalian kidney. The antiapoptotic action of AVP may be relevant to a number of physiological and pathophysiological conditions including osmotic tolerance in the inner medulla, escape from AVP-induced antidiuresis, and polycystic kidney disease.

Systems-level identification of PKA-dependent signaling in epithelial cells

Significance Maintenance of homeostasis is dependent on intercellular communication via secreted hormones that bind G protein-coupled receptors. Many of these receptors activate an enzyme called protein kinase A (PKA) that modifies cell function by covalently attaching phosphate groups to proteins. To comprehensively identify PKA substrates, we used genome editing (CRISPR-Cas9) to delete PKA from kidney epithelial cells followed by large-scale mass spectrometry to measure phosphorylation changes throughout the proteome; 229 PKA target sites were identified, many previously unrecognized. Surprisingly, PKA deletion caused seemingly paradoxical phosphorylation increases at many sites, indicating secondary activation of one or more mitogen-activated kinases. The data, coupled with transcriptomics and standard proteomics, identified a signaling network that explains the effects of PKA that regulate cellular functions. G protein stimulatory α-subunit (Gαs)-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a “multiomic” analysis in mouse kidney epithelial cells expressing the Gαs-coupled V2 vasopressin receptor. RNA-seq (sequencing)–based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA targets are thus far unannotated in public databases. Surprisingly, 1,915 phosphorylation sites with the motif x-(S/T)-P showed increased phosphooccupancy, pointing to increased activity of one or more MAP kinases in PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is upstream of JNK1 activation. The datasets were integrated to identify a causal network describing PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA activation, vasopressin stimulates Aqp2 transcription through induction of nuclear translocation of the acetyltransferase EP300, which increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq).

A knowledge base of vasopressin actions in the kidney

Biological information is growing at a rapid pace, making it difficult for individual investigators to be familiar with all information that is relevant to their own research. Computers are beginning to be used to extract and curate biological information; however, the complexity of human language used in research papers continues to be a critical barrier to full automation of knowledge extraction. Here, we report a manually curated knowledge base of vasopressin actions in renal epithelial cells that is designed to be readable either by humans or by computer programs using natural language processing algorithms. The knowledge base consists of three related databases accessible at https://helixweb.nih.gov/ESBL/TinyUrls/Vaso_portal.html. One of the component databases reports vasopressin actions on individual proteins expressed in renal epithelia, including effects on phosphorylation, protein abundances, protein translocation from one subcellular compartment to another, protein-protein binding interactions, etc. The second database reports vasopressin actions on physiological measures in renal epithelia, and the third reports specific mRNA species whose abundances change in response to vasopressin. We illustrate the application of the knowledge base by using it to generate a protein kinase network that connects vasopressin binding in collecting duct cells to physiological effects to regulate the water channel protein aquaporin-2.

Systems-level analysis reveals selective regulation of Aqp2 gene expression by vasopressin.

Vasopressin-mediated regulation of renal water excretion is defective in a variety of water balance disorders in humans. It occurs in part through long-term mechanisms that regulate the abundance of the aquaporin-2 water channel in renal collecting duct cells. Here, we use deep DNA sequencing in mouse collecting duct cells to ask whether vasopressin signaling selectively increases Aqp2 gene transcription or whether it triggers a broadly targeted transcriptional network. ChIP-Seq quantification of binding sites for RNA polymerase II was combined with RNA-Seq quantification of transcript abundances to identify genes whose transcription is regulated by vasopressin. (View curated dataset at https://helixweb.nih.gov/ESBL/Database/Vasopressin/). The analysis revealed only 35 vasopressin-regulated genes (of 3659) including Aqp2. Increases in RNA polymerase II binding and mRNA abundances for Aqp2 far outstripped corresponding measurements for all other genes, consistent with the conclusion that vasopressin-mediated transcriptional regulation is highly selective for Aqp2. Despite the overall selectivity of the net transcriptional response, vasopressin treatment was associated with increased RNA polymerase II binding to the promoter proximal region of a majority of expressed genes, suggesting a nearly global positive regulation of transcriptional initiation with transcriptional pausing. Thus, the overall net selectivity appears to be a result of selective control of transcriptional elongation.

Endogenous Carbamylation of Renal Medullary Proteins

Protein carbamylation is a post-translational modification that can occur in the presence of urea. In solution, urea is in equilibrium with ammonium cyanate, and carbamylation occurs when cyanate ions react with the amino groups of lysines, arginines, protein N-termini, as well as sulfhydryl groups of cysteines. The concentration of urea is elevated in the renal inner medulla compared with other tissues. Due to the high urea concentration, we hypothesized that carbamylation can occur endogenously within the rat inner medulla. Using immunoblotting of rat kidney cortical and medullary homogenates with a carbamyl-lysine specific antibody, we showed that carbamylation is present in a large number of inner medullary proteins. Using protein mass spectrometry (LC-MS/MS) of rat renal inner medulla, we identified 456 unique carbamylated sites in 403 proteins, including many that play important physiological roles in the renal medulla [Data can be accessed at https://helixweb.nih.gov/ESBL/Database/Carbamylation/Carbamylation_peptide_sorted.html]. We conclude that protein carbamylation occurs endogenously in the kidney, modifying many physiologically important proteins.