A resource database for protein kinase substrate sequence-preference motifs based on large-scale mass spectrometry data.

BACKGROUND: Protein phosphorylation is one of the most prevalent posttranslational modifications involved in molecular control of cellular processes, and is mediated by over 520 protein kinases in humans and other mammals. Identification of the protein kinases responsible for phosphorylation events is key to understanding signaling pathways. Unbiased phosphoproteomics experiments have generated a wealth of data that can be used to identify protein kinase targets and their preferred substrate sequences. METHODS: This study utilized prior data from mass spectrometry-based studies identifying sites of protein phosphorylation after in vitro incubation of protein mixtures with recombinant protein kinases. PTM-Logo software was used with these data to generate position-dependent Shannon information matrices and sequence motif 'logos'. Webpages were constructed for facile access to logos for each kinase and a new stand-alone application was written in Python that uses the position-dependent Shannon information matrices to identify kinases most likely to phosphorylate a particular phosphorylation site. RESULTS: A database of kinase substrate target preference logos allows browsing, searching, or downloading target motif data for each protein kinase ( https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/ ). These logos were combined with phylogenetic analysis of protein kinase catalytic sequences to reveal substrate preference patterns specific to particular groups of kinases ( https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/KinaseTree.html ). A stand-alone program, KinasePredictor, is provided ( https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/KinasePredictor.html ). It takes as input, amino-acid sequences surrounding a given phosphorylation site and generates a ranked list of protein kinases most likely to phosphorylate that site. CONCLUSIONS: This study provides three new resources for protein kinase characterization. It provides a tool for prediction of kinase-substrate interactions, which in combination with other types of data (co-localization, etc.), can predict which kinases are likely responsible for a given phosphorylation event in a given tissue. Video Abstract.

Collecting duct water permeability inhibition by EGF is associated with decreased cAMP, PKA activity, and AQP2 phosphorylation at Ser269.

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-d-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser264 and Ser269. This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at Ser256, Ser264, and Ser269. None of the other kinases tested phosphorylated Ser269. In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser269. These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser269, a site previously shown to regulate AQP2 endocytosis.NEW & NOTEWORTHY The authors used native rat collecting ducts to show that inhibition of vasopressin-stimulated water permeability by epidermal growth factor involves a reduction of aquaporin 2 phosphorylation at Ser269, a consequence of reduced cAMP production and PKA activity.

Vasopressin V2 receptor, tolvaptan, and ERK1/2 phosphorylation in the renal collecting duct.

Tolvaptan, a vasopressin antagonist selective for the V2-subtype vasopressin receptor (V2R), is widely used in the treatment of hyponatremia and autosomal-dominant polycystic kidney disease (ADPKD). Its effects on signaling in collecting duct cells have not been fully characterized. Here, we perform RNA-seq in a collecting duct cell line (mpkCCD). The data show that tolvaptan inhibits the expression of mRNAs that were previously shown to be increased in response to vasopressin including aquaporin-2, but also reveals mRNA changes that were not readily predictable and suggest off-target actions of tolvaptan. One such action is activation of the MAPK kinase (ERK1/ERK2) pathway. Prior studies have shown that ERK1/ERK2 activation is essential in the regulation of a variety of cellular and physiological processes and can be associated with cell proliferation. In immunoblotting experiments, we demonstrated that ERK1/ERK2 phosphorylation in mpkCCD cells was significantly reduced by vasopressin, in contrast to the increases seen in non-collecting-duct cells overexpressing V2R in prior studies. We also found that tolvaptan has a strong effect to increase ERK1/ERK2 phosphorylation in the presence of vasopressin and that tolvaptan's effect to increase ERK1/ERK2 phosphorylation is absent in mpkCCD cells in which both protein kinase A (PKA)-catalytic subunits have been deleted. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and is not due to an off-target effect of tolvaptan. We conclude that in cells expressing V2R at endogenous levels: 1) vasopressin decreases ERK1/ERK2 activation; 2) in the presence of vasopressin, tolvaptan increases ERK1/ERK2 activation; and 3) these effects are PKA-dependent.NEW & NOTEWORTHY Vasopressin is a key hormone that regulates the function of the collecting duct of the kidney. ERK1 and ERK2 are enzymes that play key roles in physiological regulation in all cells. The authors used collecting duct cell cultures to investigate the effects of vasopressin and the vasopressin receptor antagonist tolvaptan on ERK1 and ERK2 phosphorylation and activation.

Using CRISPR-Cas9/phosphoproteomics to identify substrates of calcium/calmodulin-dependent kinase 2δ.

Ca2+/Calmodulin-dependent protein kinase 2 (CAMK2) family proteins are involved in the regulation of cellular processes in a variety of tissues including brain, heart, liver, and kidney. One member, CAMK2δ (CAMK2D), has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water excretion through regulation of the water channel aquaporin-2 (AQP2). To identify CAMK2D target proteins in renal collecting duct cells (mpkCCD), we deleted Camk2d and carried out LC-MS/MS-based quantitative phosphoproteomics. Specifically, we used CRISPR/Cas9 with two different guide RNAs targeting the CAMK2D catalytic domain to create multiple CAMK2D KO cell lines. AQP2 protein abundance was lower in the CAMK2D KO cells than in CAMK2D-intact controls. AQP2 phosphorylation at Ser256 and Ser269 (normalized for total AQP2) was decreased. However, trafficking of AQP2 to and from the apical plasma membrane was sustained. Large-scale quantitative phosphoproteomic analysis (TMT-labeling) in the presence of the vasopressin analog dDAVP (0.1 nM, 30 min) allowed quantification of 11,570 phosphosites of which 169 were significantly decreased, while 206 were increased in abundance in CAMK2D KO clones. These data are available for browsing or download at https://esbl.nhlbi.nih.gov/Databases/CAMK2D-proteome/. Motif analysis of the decreased phosphorylation sites revealed a target preference of -(R/K)-X-X-p(S/T)-X-(D/E), matching the motif identified in previous in vitro phosphorylation studies using recombinant CAMK2D. Thirty five of the significantly downregulated phosphorylation sites in CAMK2D KO cells had exactly this motif and are judged to be likely direct CAMK2D targets. This adds to the list of known CAMK2D target proteins found in prior reductionist studies.

Circadian gene expression in mouse renal proximal tubule.

Circadian variability in kidney function is well recognized but is often ignored as a potential confounding variable in physiological experiments. Here, we have created a data resource consisting of expression levels for mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. Small-sample RNA sequencing was applied to microdissected S1 proximal convoluted tubules and S2 proximal straight tubules. After stringent filtering, the data were analyzed using JTK-Cycle to detect periodicity. The data set is provided as a user-friendly webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox2/. In proximal convoluted tubules, 234 transcripts varied in a circadian manner (4.0% of the total). In proximal straight tubules, 334 transcripts varied in a circadian manner (5.3%). Transcripts previously known to be associated with corticosteroid action and with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day [zeitgeber time (ZT)14-22], corresponding to peak levels of corticosterone and glomerular filtration rate in mice. To ask whether there is a time-of-day dependence of protein abundances in the kidney, we carried out LC-MS/MS-based proteomics in whole mouse kidneys at ZT12 and ZT0. The full data set (n = 6,546 proteins) is available at https://esbl.nhlbi.nih.gov/Databases/Circadian-Proteome/. Overall, 293 proteins were differentially expressed between ZT12 and ZT0 (197 proteins greater at ZT12 and 96 proteins greater at ZT0). Among the regulated proteins, only nine proteins were found to be periodic in the RNA-sequencing analysis, suggesting a high level of posttranscriptional regulation of protein abundances.NEW & NOTEWORTHY Circadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-sequencing transcriptomics and LC-MS/MS-based proteomics to identify gene products expressed in a periodic manner. The data were used to construct user-friendly web resources.

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

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.

Bayesian analysis of dynamic phosphoproteomic data identifies protein kinases mediating GPCR responses.

BACKGROUND: A major goal in the discovery of cellular signaling networks is to identify regulated phosphorylation sites ("phosphosites") and map them to the responsible protein kinases. The V2 vasopressin receptor is a G-protein coupled receptor (GPCR) that is responsible for regulation of renal water excretion through control of aquaporin-2-mediated osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on protein kinase A (PKA), but events downstream from PKA are still obscure. METHODS: Here, we used: 1) Tandem mass tag-based quantitative phosphoproteomics to experimentally track phosphorylation changes over time in native collecting ducts isolated from rat kidneys; 2) a clustering algorithm to classify time course data based on abundance changes and the amino acid sequences surrounding the phosphosites; and 3) Bayes' Theorem to integrate the dynamic phosphorylation data with multiple prior "omic" data sets covering expression, subcellular location, known kinase activity, and characteristic surrounding sequences to identify a set of protein kinases that are regulated secondary to PKA activation. RESULTS: Phosphoproteomic studies revealed 185 phosphosites regulated by vasopressin over 15 min. The resulting groups from the cluster algorithm were integrated with Bayes' Theorem to produce corresponding ranked lists of kinases likely responsible for each group. The top kinases establish three PKA-dependent protein kinase modules whose regulation mediate the physiological effects of vasopressin at a cellular level. The three modules are 1) a pathway involving several Rho/Rac/Cdc42-dependent protein kinases that control actin cytoskeleton dynamics; 2) mitogen-activated protein kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling. CONCLUSIONS: Our findings identify a novel set of downstream small GTPase effectors and calcium/calmodulin-dependent kinases with potential roles in the regulation of water permeability through actin cytoskeleton rearrangement and aquaporin-2 trafficking. The proposed signaling network provides a stronger hypothesis for the kinases mediating V2 vasopressin receptor responses, encouraging future targeted examination via reductionist approaches. Furthermore, the Bayesian analysis described here provides a template for investigating signaling via other biological systems and GPCRs. Video abstract.

Systems Biology of the Vasopressin V2 Receptor: New Tools for Discovery of Molecular Actions of a GPCR.

Systems biology can be defined as the study of a biological process in which all of the relevant components are investigated together in parallel to discover the mechanism. Although the approach is not new, it has come to the forefront as a result of genome sequencing projects completed in the first few years of the current century. It has elements of large-scale data acquisition (chiefly next-generation sequencing-based methods and protein mass spectrometry) and large-scale data analysis (big data integration and Bayesian modeling). Here we discuss these methodologies and show how they can be applied to understand the downstream effects of GPCR signaling, specifically looking at how the neurohypophyseal peptide hormone vasopressin, working through the V2 receptor and PKA activation, regulates the water channel aquaporin-2. The emerging picture provides a detailedframework for understanding the molecular mechanisms involved in water balance disorders, pointing the way to improved treatment of both polyuric disorders and water-retention disorders causing dilutional hyponatremia.

"ADPKD-omics": determinants of cyclic AMP levels in renal epithelial cells.

The regulation of cyclic adenosine monophosphate (cAMP) levels in kidney epithelial cells is important in at least 2 groups of disorders, namely water balance disorders and autosomal dominant polycystic kidney disease. Focusing on the latter, we review genes that code for proteins that are determinants of cAMP levels in cells. We identify which of these determinants are expressed in the 14 kidney tubule segments using recently published RNA-sequencing and protein mass spectrometry data ("autosomal dominant polycystic kidney disease-omics"). This includes G protein-coupled receptors, adenylyl cyclases, cyclic nucleotide phosphodiesterases, cAMP transporters, cAMP-binding proteins, regulator of G protein-signaling proteins, G protein-coupled receptor kinases, arrestins, calcium transporters, and calcium-binding proteins. In addition, compartmentalized cAMP signaling in the primary cilium is discussed, and a specialized database of the proteome of the primary cilium of cultured "IMCD3" cells is provided as an online resource (https://esbl.nhlbi.nih.gov/Databases/CiliumProteome/). Overall, this article provides a general resource in the form of a curated list of proteins likely to play roles in determination of cAMP levels in kidney epithelial cells and, therefore, likely to be determinants of progression of autosomal dominant polycystic kidney disease.

Bayesian identification of candidate transcription factors for the regulation of Aqp2 gene expression.

Aquaporin-2 (Aqp2) gene transcription is strongly regulated by vasopressin in the renal collecting duct. However, the transcription factors (TFs) responsible for the regulation of expression of Aqp2 remain largely unknown. We used Bayes' theorem to integrate several -omics data sets to stratify the 1,344 TFs present in the mouse genome with regard to probabilities of regulating Aqp2 gene transcription. Also, we carried out new RNA sequencing experiments mapping the time course of vasopressin-induced changes in the transcriptome of mpkCCD cells to identify TFs that change in tandem with Aqp2. The analysis identified 17 of 1,344 TFs that are most likely to be involved in the regulation of Aqp2 gene transcription. These TFs included eight that have been proposed in prior studies to play a role in Aqp2 regulation, viz., Cebpb, Elf1, Elf3, Ets1, Jun, Junb, Nfkb1, and Sp1. The remaining nine represent new candidates for future studies (Atf1, Irf3, Klf5, Klf6, Mef2d, Nfyb, Nr2f6, Stat3, and Nr4a1). Conspicuously absent is CREB (Creb1), which has been widely proposed to mediate vasopressin-induced regulation of Aqp2 gene transcription (Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, Knepper MA. Physiol Rev 82: 205-244, 2002; Kortenoeven ML, Fenton RA. Biochim Biophys Acta 1840: 1533-1549, 2014; Bockenhauer D, Bichet DG. Nat Rev Nephrol 11: 576-588, 2015; Pearce D, Soundararajan R, Trimpert C, Kashlan OB, Deen PM, Kohan DE. Clin J Am Soc Nephrol 10: 135-146, 2015). Instead, another CREB-like TF, Atf1, ranked fourth among all TFs. RNA sequencing time-course experiments showed a rapid increase in Aqp2 mRNA, within 3 h of vasopressin exposure. This response was matched by an equally rapid increase in the abundance of the mRNA coding for Cebpb, which we have shown by chromatin immunoprecipitation-sequencing studies to bind downstream from the Aqp2 gene. The identified TFs provide a roadmap for future studies to understand regulation of Aqp2 gene expression.NEW & NOTEWORTHY Abetted by the advent of systems biology-based ("-omics") techniques in the 21st century, there has been a massive expansion of published data relevant to virtually every physiological question. The authors have developed a large-scale data integration approach based on the application of Bayes'' theorem. In the current work, they integrated 12 different -omics data sets to identify the transcription factors most likely to mediate vasopressin-dependent regulation of transcription of the aquaporin-2 gene.

"SLC-omics" of the kidney: solute transporters along the nephron.

The fluid in the 14 distinct segments of the renal tubule undergoes sequential transport processes that gradually convert the glomerular filtrate into the final urine. The solute carrier (SLC) family of proteins is responsible for much of the transport of ions and organic molecules along the renal tubule. In addition, some SLC family proteins mediate housekeeping functions by transporting substrates for metabolism. Here, we have developed a curated list of SLC family proteins. We used the list to produce resource webpages that map these proteins and their transcripts to specific segments along the renal tubule. The data were used to highlight some interesting features of expression along the renal tubule including sex-specific expression in the proximal tubule and the role of accessory proteins (ß-subunit proteins) that are thought to be important for polarized targeting in renal tubule epithelia. Also, as an example of application of the data resource, we describe the patterns of acid-base transporter expression along the renal tubule.

Landscape of GPCR expression along the mouse nephron.

Kidney transport and other renal functions are regulated by multiple G protein-coupled receptors (GPCRs) expressed along the renal tubule. The rapid, recent appearance of comprehensive unbiased gene expression data in the various renal tubule segments, chiefly RNA sequencing and protein mass spectrometry data, has provided a means of identifying patterns of GPCR expression along the renal tubule. To allow for comprehensive mapping, we first curated a comprehensive list of GPCRs in the genomes of mice, rats, and humans (https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/) using multiple online data sources. We used this list to mine segment-specific and cell type-specific expression data from RNA-sequencing studies in microdissected mouse tubule segments to identify GPCRs that are selectively expressed in discrete tubule segments. Comparisons of these mapped mouse GPCRs with other omics datasets as well as functional data from isolated perfused tubule and micropuncture studies confirmed patterns of expression for well-known receptors and identified poorly studied GPCRs that are likely to play roles in the regulation of renal tubule function. Thus, we provide data resources for GPCR expression across the renal tubule, highlighting both well-known GPCRs and understudied receptors to provide guidance for future studies.

Phosphoproteomic identification of vasopressin-regulated protein kinases in collecting duct cells.

BACKGROUND AND PURPOSE: The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a PKA-dependent signalling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins. EXPERIMENTAL APPROACH: We carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct (mpkCCD) cells. Quantification was done using stable isotope labelling (SILAC method). KEY RESULTS: Six hundred forty phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (Cdk18), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (Mapk3) and myosin light chain kinase (Mylk). CONCLUSION AND IMPLICATIONS: In V2 receptor-mediated signalling, PKA is at the head of a complex network that includes at least six downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study are provided as a web-based data resource for future studies of GPCRs.

Phosphoproteomic Identification of Vasopressin/cAMP/Protein Kinase A-Dependent Signaling in Kidney.

Water excretion by the kidney is regulated by the neurohypophyseal peptide hormone vasopressin through actions in renal collecting duct cells to regulate the water channel protein aquaporin-2. Vasopressin signaling is initiated by binding to a G-protein-coupled receptor called V2R, which signals through heterotrimeric G-protein subunit Gs α, adenylyl cyclase 6, and activation of the cAMP-regulated protein kinase (PKA). Signaling events coupling PKA activation and aquaporin-2 regulation were largely unknown until the advent of modern protein mass spectrometry techniques that allow proteome-wide quantification of protein phosphorylation changes (phosphoproteomics). This short review documents phosphoproteomic findings in collecting duct cells describing the response to V2R-selective vasopressin agonists and antagonists, the response to CRISPR-mediated deletion of PKA, results from in vitro phosphorylation studies using recombinant PKA, the response to the broad-spectrum kinase inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide), and the responses underlying lithium-induced nephrogenic diabetes insipidus. These phosphoproteomic data sets have been made available online for modeling vasopressin signaling and signaling downstream from other G-protein-coupled receptors. SIGNIFICANCE STATEMENT: New developments in protein mass spectrometry are facilitating progress in identification of signaling networks. Using mass spectrometry, it is now possible to identify and quantify thousands of phosphorylation sites in a given cell type (phosphoproteomics). The authors describe the use of phosphoproteomics technology to identify signaling mechanisms downstream from a G-protein-coupled receptor, the vasopressin V2 subtype receptor, and its role of the regulation and dysregulation of water excretion in the kidney. Data from multiple phosphoproteomic data sets are provided as web-based resources.

Protein kinase A catalytic-α and catalytic-ß proteins have nonredundant regulatory functions.

Vasopressin regulates osmotic water transport in the renal collecting duct by protein kinase A (PKA)-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic ß (PKA-Cß). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cß was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4,635 phosphopeptides that were quantified, 67 phosphopeptides were significantly altered in abundance with PKA-Cα deletion, whereas 21 phosphopeptides were significantly altered in abundance with PKA-Cß deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, whereas target proteins in PKA-Cß-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cß resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, whereas PKA-Cß deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cß serve substantially different regulatory functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g., mediated by A-kinase anchor proteins, C-kinase anchoring proteins, or PDZ binding.

An integrative proteogenomics approach reveals peptides encoded by annotated lincRNA in the mouse kidney inner medulla.

Long noncoding RNAs (lncRNAs) are intracellular transcripts longer than 200 nucleotides and lack protein-coding information. A subclass of lncRNA known as long intergenic noncoding RNAs (lincRNAs) are transcribed from genomic regions that share no overlap with annotated protein-coding genes. Increasing evidence has shown that some annotated lincRNA transcripts do in fact contain open reading frames (ORFs) encoding functional short peptides in the cell. Few robust methods for lincRNA-encoded peptide identification have been reported, and the tissue-specific expression of these peptides has been largely unexplored. Here we propose an integrative workflow for lincRNA-encoded peptide discovery and test it on the mouse kidney inner medulla (IM). In brief, low molecular weight protein fractions were enriched from homogenate of IMs and trypsinized into shorter peptides, which were sequenced by high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). To curate a hypothetical lincRNA-encoded peptide database for peptide-spectrum matching following LC-MS/MS, we performed RNA-Seq on IMs, computationally removed reads overlapping with annotated protein-coding genes, and remapped the remaining reads to a database of mouse noncoding transcripts to infer lincRNA expression. Expressed lincRNAs were searched for ORFs by an existing rule-based algorithm, and translated ORFs were used for peptide-spectrum matching. Peptides identified by LC-MS/MS were further evaluated by using several quality control criteria and bioinformatics methods. We discovered three novel lincRNA-encoded peptides, which are conserved in mouse, rat, and human. The workflow can be adapted for discovery of small protein-coding genes in any species or tissue where noncoding transcriptome information is available.

PKA-independent vasopressin signaling in renal collecting duct.

Vasopressin regulates renal water excretion by binding to a Gα s-coupled receptor (V2R) in collecting duct cells, resulting in increased water permeability through regulation of the aquaporin-2 (AQP2) water channel. This action is widely accepted to be associated with cAMP-mediated activation of protein kinase A (PKA). Here, we use phosphoproteomics in collecting duct cells in which PKA has been deleted (CRISPR-Cas9) to identify PKA-independent responses to vasopressin. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Upregulated sites in PKA-null cells include Ser256 of AQP2, which is critical to regulation of AQP2 trafficking. In addition, phosphorylation changes in the protein kinases Stk39 (SPAK) and Prkci (an atypical PKC) are consistent with PKA-independent regulation of these protein kinases. Target motif analysis of the phosphopeptides increased in PKA-null cells indicates that vasopressin activates one or more members of the AMPK/SNF1-subfamily of basophilic protein kinases. In vitro phosphorylation assays using recombinant, purified SNF1-subfamily kinases confirmed postulated target specificities. Of interest, measured IBMX-dependent cAMP levels were an order of magnitude higher in PKA-null than in PKA-intact cells, indicative of a PKA-dependent feedback mechanism. Overall, the findings support the conclusion that V2-receptor mediated signaling in collecting duct cells is in part PKA-independent.

CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase.

Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.

Phosphoproteomic identification of vasopressin V2 receptor-dependent signaling in the renal collecting duct.

Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: 1) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position -2 and -3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; 2) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; 3) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and 4) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.