Proteomic profiling of nuclei from native renal inner medullary collecting duct cells using LC-MS/MS

Abstract

Vasopressin is a peptide hormone that regulates renal water excretion in part through its actions on the collecting duct. The regulation occurs in part via control of transcription of genes coding for the water channels aquaporin-2 (Aqp2) and aquaporin-3 (Aqp3). To identify transcription factors expressed in collecting duct cells, we have carried out LC-MS/MS-based proteomic profiling of nuclei isolated from native rat inner medullary collecting ducts (IMCDs). To maximize the number of proteins identified, we matched spectra to rat amino acid sequences using three different search algorithms (SEQUEST, InsPecT, and OMSSA). All searches were coupled to target-decoy methodology to limit false-discovery identifications to 2% of the total for single-peptide identifications. In addition, we developed a computational tool (ProMatch) to identify and eliminate ambiguous identifications. With this approach, we identified >3,500 proteins, including 154 proteins classified as “transcription factor” proteins (Panther Classification System). Among these, are members of CREB, ETS, RXR, NFAT, HOX, GATA, EBOX, EGR, MYT1, KLF, and CP2 families, which were found to have evolutionarily conserved putative binding sites in the 5′-flanking region or first intron of the Aqp2 gene, as well as members of EBOX, NR2, GRE, MAZ, KLF, and SP1 families corresponding to conserved sites in the 5′-flanking region of the Aqp3 gene. In addition, several novel phosphorylation sites in nuclear proteins were identified using the neutral loss-scanning LC-MS³ technique. The newly identified proteins have been incorporated into the IMCD Proteome Database (http://dir.nhlbi.nih.gov/papers/lkem/imcd/).

the inner medullary collecting duct (IMCD) is the final portion of the renal collecting duct system. It is responsible for the controlled reabsorption of water from the tubule lumen into the interstitial space of the kidney for eventual return to the bloodstream. The main controlling factor is the peptide hormone vasopressin. Vasopressin mediates rapid regulation of IMCD water permeability by triggering redistribution of the water channel protein aquaporin-2 (Aqp2) from a largely intracellular location to the apical plasma membrane through vesicular trafficking (30). Addition of water channels to the apical plasma membrane increases its permeability to water, allowing accelerated osmotic water transport. In addition to this “classic” mode of regulation, vasopressin has long term effects on the renal collecting duct to increase the total abundance of the Aqp2 protein (10) as well as that of its basolateral counterpart aquaporin-3 (Aqp3) (13).

Vasopressin has been demonstrated to increase transcription of the Aqp2 gene (22, 28, 52), resulting in increased levels of Aqp2 mRNA (12, 16, 51) and protein (31) in kidney tissue. Water restriction increases, while water loading decreases Aqp2 mRNA levels in rat kidney (9, 29, 40). Administration of an orally acting vasopressin V2 antagonist decreased Aqp2 mRNA (16), a finding subsequently confirmed by Christensen et al. (9) and Murillo-Carretero et al. (29). Christensen et al. (9) showed in addition that treatment of rats with a vasopressin V2 receptor antagonist caused renal Aqp2 mRNA levels to fall within 30 min.

In addition to Aqp2, Aqp3 gene expression is regulated by vasopressin as well, with marked increases in levels of Aqp3 mRNA (12, 29) and Aqp3 protein (13, 45). However, the role of transcriptional mechanisms is largely unexplored for Aqp3.

The transcriptional network that governs long-term responses to vasopressin is largely unknown. Recently, we used mRNA profiling (Affymetrix) of native rat IMCD and mouse mpkCCD collecting duct cells, coupled with computational analysis (Genomatix) to identify conserved transcriptional regulator binding site motifs in the 5′-flanking region of the Aqp2 gene to identify transcriptional regulators (TRs) that potentially regulate Aqp2 gene expression (54). The findings demonstrated SF1, NFAT, FKHD, ETS, RXR, AP2, CREB, GATA, SRF, HOX, and EBOX family TR binding sites as likely components of the transcriptional network responsible for regulation of Aqp2 gene transcription. Although these conserved binding site motifs can predict what TR families may be involved in transcriptional regulation of Aqp2 and other genes, identification of the actual TR proteins expressed in the IMCD is a necessary intermediate step to the design of studies needed to fully resolve transcriptional regulatory networks involved in regulation of Aqp2 gene expression. We have previously carried out extensive transcriptomic profiling of IMCD cells using Affymetrix arrays to learn what transcripts are expressed in the IMCD (46) (see “IMCD Transcriptome Database”: http://dir.nhlbi.nih.gov/papers/lkem/imcdtr/). However, relative transcript levels are not necessarily predictive of the level of the corresponding proteins in cells and direct detection by mass spectrometry is desirable. The low abundance of many TR proteins relative to other categories of proteins has made them difficult to detect by protein mass spectrometry and the current IMCD Proteome Database (33) contains only a few TR proteins such as Stat2 and Pax8, detected in whole cell analysis. Notably, a transcription factor whose role is already established, namely Creb1 (22, 28, 52), was not detected using proteomic methods. This problem can be addressed biochemically by isolating nuclei from IMCD cells to enrich nuclear proteins prior to mass spectrometric identification, making it more likely that TRs and other nuclear proteins will be detected. In this study, we have isolated nuclei from native rat IMCDs and carried out proteomic and phosphoproteomic profiling using 1-D SDS-PAGE followed by LC-MS/MS. To increase the number of proteins identified we utilized a computational protocol that includes a combination of three search algorithms (SEQUEST, InsPecT, and OMSSA) for matching mass spectra to rat protein sequences. To limit false-positive identifications we used target-decoy analysis to set the false discovery rate (FDR) to <2% of the total for single-peptide identifications. In addition we developed a novel computational tool (ProMatch) to identify ambiguous identifications, i.e., tryptic or chymotryptic peptides that are the same in two or more proteins. Finally, since regulation of transcription is in part mediated by regulated phosphorylation of various nuclear proteins, we also carried out MS-based experiments to identify phosphoproteins in nuclei from native IMCD cells.

METHODS

IMCD Sample Preparation and Isolation of Nuclei

Twenty male Sprague-Dawley rats were euthanized (National Heart, Lung, and Blood Institute Animal Care and Use Committee-approved protocol H-0110) and their renal inner medullas (IMs) were resected and pooled. The IMs were minced and digested in a solution containing 250 mM sucrose, 2,000 units/ml hyaluronidase, and 3 mg/ml collagenase B for 75 min at 37°C with continuous stirring. IMCDs were sedimented at 70 g for 20 s, and the supernatant containing non-IMCD elements was discarded. IMCD pellet was resuspended in a homogenization solution [250 ml sucrose solution containing phosphatase inhibitor (Halt phosphatase inhibitor, Thermo Scientific, Waltham, MA) and protease inhibitor (Complete mini, Roche Diagnostics, Indianapolis, IN)]. Previous studies have shown that IMCDs isolated by this technique are at least 85% pure (46), viable, and vasopressin-responsive (35).

Isolated IMCDs were suspended in the homogenization solution and homogenized using a Potter-Elvehjem motor-driven homogenizer on ice for a total of 2 min in 15 s intervals. The homogenized solutions were centrifuged at 1,000 g for 30 min at 4°C. The supernatant from the previous step, which contained the cytoplasmic fraction of lysed IMCD cells, was kept. The cell lysis solutions Cytoplasmic Extraction Reagent I and II (CER I and CERII) from the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) were then added, according to instructions, to the pellet now containing nuclei and unbroken IMCD cells followed by centrifugation for 5 min at 16,000 g. CERI and CERII were again added to the pellet at half the original volumes and the sample was centrifuged as above. All supernatants containing the cytoplasm of lysed cells were pooled (designated “Cyto”). Nuclear extraction solution was then added to the pellet, and the sample was centrifuged according to the kit instructions. The supernatant from the final centrifugation contained soluble nuclear proteins (designated “NE”) and the pellet contained nuclear membrane/ER proteins (designated “NP”).

One-dimensional SDS-PAGE and In-gel Digestion

One-dimensional (1-D) SDS-PAGE was performed on samples from NE, NP, and Cyto fractions (400 μg protein each) using three separate 10% polyacrylamide Ready Gels (Bio-Rad, Hercules, CA). The gels were stained with Imperial Protein Stain (Thermo Scientific). Each gel was sliced into 40 fractions and cut into 1 mm³ blocks. In-gel digestion was performed as described previously (34). Briefly, 25 mM NH₄HCO₃/50% acetonitrile (ACN) was added to each sample three times for 10 min each to dehydrate the gels and samples were dried via a Speed Vac. 10 mM DTT in 25 mM NH₄HCO₃ was added to each sample at 56°C for 1 h followed by the addition of 55 mM iodoacetamide in 25 mM NH₄HCO₃ for 45 min in the dark. The gel blocks were then dried via a Speed Vac. The gel pieces were digested overnight at 37°C with 12.5 ng/μl of Sequencing Grade Modified Trypsin (Promega, Madison, WI). Following the in-gel digestion, peptides were extracted with 50% ACN/0.1% formic acid (FA) then desalted using a ZipTip C18 pipette tip (Millipore, Billerica, MA). In addition, a separate set of 40 NE fractions was in-gel digested overnight at 25°C with 12.5 ng/μl of Sequencing Grade Chymotrypsin (Roche Diagnostics, Indianapolis, IN). In total, 120 samples were analyzed by LC-MS/MS.

Nanospray LC-MS/MS

Each peptide sample was analyzed once via 1-D nanospray LC-MS/MS with a modified ProteomeX 2D LC/MS workstation utilizing a linear ion trap mass spectrometer, LTQ-FT (Thermo, San Jose, CA) (20). In an alternating fashion, two Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) chromatographically separated the peptides. A nanospray ionization source and a reverse-phase PicoFrit column [BioBasic C18, 75 μm × 10 cm, tip = 15 μm (New Objective, Woburn, MA)] were used. The peptides were eluted via an increasing 0–60% gradient of solvent B in solvent A (A = 0.1% FA, B = 100% ACN/0.1% FA) over a 30 min period at a flow rate of ∼200 nl/min.

Post-LC-MS/MS Analysis

Search algorithms.

To maximize the number of peptide identifications, we analyzed the data using three different search algorithms. In addition to utilizing two peak matching algorithms viz. SEQUEST (15) and the Open Mass Spectrometry Search Algorithm (OMSSA) (17), we also employed InsPecT (44), a so-called “hybrid” algorithm that performs partial de novo sequencing generating 3-amino acid tags that are used to narrow down the possible peptide candidates before performing the final peak matching. A fixed +57 Da modification on cysteine and variable +14 Da cysteine and +16 Da methionine modifications were included as part of the search.

The raw data files were searched, using the three search algorithms, against a concatenated forward and reverse database that included the most recent RefSeq rat protein database from the National Center for Biotechnology Information appended with a list of common contaminants (pig and bovine trypsin as well as human isoforms of keratin). Peptide identifications were sorted from best to worst using the following parameters: XCorr (SEQUEST), P value (InsPecT), and E-value (OMSSA). Target-decoy analysis was performed to limit global FDR for each peptide identified using the following formula: FDR = 2R/(F + R), where R is the number of accumulated peptide hits from reversed “decoy” sequences and F is the number of accumulated peptide hits from forward “target” sequences (14). All peptides passing the 2% FDR filter from the three different search algorithms were merged at the spectral level. For a given spectrum, a valid identification was defined if at least 67% of the passed search algorithms agree on the identification (an agreement of 3 out of 3, 2 out of 3, 2 out of 2, or 1 out of 1). If no agreement among the three search algorithms was found, the single best identification (based on FDR) across all algorithms for a given spectrum was selected. Unresolved spectra were discarded.

Peptide to protein matching.

The same peptide may map to more than one protein. However, peak matching programs like SEQUEST often report a single match even when the peptide could match to multiple protein candidates. To detect ambiguous identifications, we have used an in-house program written in Java called ProMatch (executable file available upon request). In this program, each peptide identified is matched against all proteins in the rat RefSeq database, every protein whose amino acid subsequence identically matches to that peptide is extracted.

The following rules were used in reporting the peptide-based protein identifications. A peptide that matched only to a single protein was defined as a “unique peptide” and reported as such. A peptide that matched to multiple proteins was defined as a “nonunique peptide.” If that peptide matched to multiple proteins that are splicing variants or products of alternative transcription start sites in the same gene, it was considered as a unique peptide since it maps to only a single gene. For this determination, we extracted the nucleotide sequences of all the proteins that matched to a particular peptide and then applied the ClustalW2 sequence alignment program (European Bioinformatics Institute) to compare them. Proteins with a similarity score ≥90 were considered to be derived from the same gene and a single RefSeq accession number is reported for all. A protein identification was labeled as “unambiguous” if it was derived from at least one unique peptide. A protein identification was labeled as “ambiguous” if it was derived exclusively from one or more nonunique peptides derived from different genes. Ambiguous protein identifications were not reported.

Phosphopeptide Analysis

Sample preparation.

IMCDs were isolated and pooled from 10 male Sprague-Dawley rats. Nuclear isolation was performed as described above. The NE and NP fractions (250 and 500 μg, respectively) were resuspended with 6 M guanidine solution. Protein samples were reduced with 10 mM DTT solution at 56°C for 1 h then alkylated with 40 mM iodoacetamide solution in the dark at room temperature for 1 h. DTT solution (40 mM) was added after alkylation to quench excess iodoacetamide. Samples were diluted in 25 mM NH₄HCO₃ solution and digested with trypsin (1:30 wt/wt) overnight at 37°C. Peptide samples were desalted with a 1 ml HLB cartridge (Oasis, Milford, MA) and then dried via a Speed Vac.

Enrichment of phosphopeptides.

Dried peptide samples were resuspended in 5% acetic acid and Ga³⁺-IMAC (Phosphopeptide Isolation Kit, Thermo Scientific) was performed as described previously (20). The IMAC flow-through was subjected to an additional phosphopeptide isolation protocol using TopTips with TiO₂ Material (PolyLC, Columbia, MD). Phosphopeptide samples were dried with a Speed Vac, resuspended in 0.5% FA, and desalted with ZipTip C18 pipette tips (Millipore).

LC-MS³ analysis.

The neutral loss scanning LC-MS³ analysis was performed as described (20). Briefly, isolated phosphopeptide samples were analyzed on an Agilent 1100 nanoflow system (Agilent Technologies, Palo Alto, CA) connected to a Finnigan LTQ-FT mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanoelectrospray ion source. The LTQ was used for the full MS scan and subsequent spectra (MS² and MS³). MS³ scans were triggered when the presence of a neutral loss peak (−98, −49, or −32.7 m/z from precursor ion) was detected in MS² scans.

Post LC-MS³ analysis.

SEQUEST, InsPecT, and OMSSA searches were performed against the concatenated forward and reverse database as described above. A fixed +57 Da cysteine modification and variable +16 Da methionine and +80 Da phosphorylation on serine, threonine, and tyrosine modifications were included. In addition, a variable loss of water −18 Da modification was included for serine and threonine when searching MS³ spectra. Identified peptides (both phosphopeptides and nonphosphopeptides) were filtered for a 2% FDR using the target-decoy approach described above. The PhosphoPIC program was used to extract and compile MS² and MS³ spectra of phosphopeptides (21). Phosphorylation site assignment was performed using Ascore (2), Phosphate Localization Score (PLscore) (1), and PhosphoScore (39).

Immunoblotting

Samples were solubilized in 1.5% SDS/Tris, pH 6.8. A BCA assay (Thermo Scientific) using BSA as the standard was used to determine total protein concentrations. Samples were then diluted in Laemmli buffer (10 mM Tris, pH 6.8, 1.5% SDS, 6% glycerol, 0.05% bromphenol blue, and 40 mM DTT). Proteins (5 μg of each sample) were separated by 1-D SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with blocking buffer (LI-COR Biotechnology, Lincoln, NE) then exposed overnight to primary antibodies diluted in the blocking buffer. After a series of washes, membranes were incubated with secondary antibodies as previously described (35) prior to image acquisition on an Odyssey Infrared Imaging System (LI-COR Biotechnology).

Antibodies

Antibodies used are listed as follows: aldose reductase (Alr2) (sc-17735, Santa Cruz Biotechnology, Santa Cruz, CA), BRM/SWI2-related gene 1 (Brg1) (sc-17796, Santa Cruz Biotechnology), and cyclic AMP response element binding protein 1 (Creb1) (9197, Cell Signaling, Danvers, MA). Species-specific secondary antibodies were obtained from Rockland Immunochemicals (Gilbertsville, PA).

Transcription Factor Binding Site Analysis

The 5′-flanking region (1,000 nucleotides) and first intron of Aqp2 and Aqp3 gene of human, rat, and mouse were extracted using UCSC Genome Browser (http://genome.ucsc.edu/). To identify conserved transcription factor binding sites (TFBS), the 5′-flanking region, and first intron sequences were analyzed using the online Genomatix software suite (http://www.genomatix.de/) as previously described (54). Sequence matches to a particular TFBS matrix were scored and filtered based on MatInd algorithm (36).

RESULTS AND DISCUSSION

Confirmation of Nuclear Isolation and Purification from Native Rat IMCD

Common markers for the cytoplasm and nucleus were used to determine the efficacy of the nuclear isolation and enrichment procedure. Figure 1 demonstrates that the nuclear proteins BRM/SWI2-related gene 1 (Brg1) and cyclic-AMP response element binding protein 1 (Creb1) were almost exclusively present in the nuclear fractions. In contrast, the cytosolic marker aldose reductase (Alr2) was far more abundant in the cytoplasmic fractions.

Fig. 1.

Quality control for nuclear isolation. Nuclear marker Brg1 is found only in the nuclear extract (NE) fraction. Another nuclear marker Creb1 is chiefly present in the NE fraction but is also found in the nuclear pellet (NP) fraction. Cytoplasmic marker (Alr2) is predominantly present in the cytoplasmic fractions. (Cytoplasm I = Potter-Elvehjem supernatant, Cytoplasm II = CERI/CERII supernatant.) Note that 5 μg of each sample was loaded. In this study we carried out MS analysis of the pooled Cytoplasm I and II (Cyto) as well as NE and NP fractions.

Proteomic Profiling of Rat IMCD Fractions

LC-MS/MS analysis was performed on three fractions derived from the nuclear isolation protocol: the soluble nuclear fraction (NE), the nuclear membrane pellet (NP), and the pooled cytoplasmic fractions (Cyto). Figure 2 summarizes the proteomic and bioinformatic workflow used for these samples. While the sample preparation protocol is relatively standard, we customized the post-LC-MS/MS data analysis for this study. Specifically, three different search algorithms (SEQUEST, InsPecT, and OMSSA) were used to increase the number of peptides identified from the spectra that were generated. The ProMatch algorithm was then used to check whether identified peptide sequences were unique to a single identified protein to allow ambiguous identifications to be eliminated.

Fig. 2.

Proteomic work flow. Graphical representation of the complete workflow used for this proteomic study. The ovals represent the experimental samples, spectra, or proteomic data. Shaded boxes represent the biochemical and computational methods that were applied to the biological samples and data.

A total of 2,172 proteins were identified in the NE fraction, 1,306 proteins were identified in the NP fraction, and 1,555 proteins were identified in the cytoplasmic fraction. For all three fractions we imposed a single-peptide FDR of 2% or less using a target-decoy algorithm (Fig. 3A). A total of 3,531 proteins were identified in one or more of the three fractions (all protein identifications are listed in Supplementary Table S1).¹ Overall, 45% of protein identifications in this study are based on spectra for 2 or more peptides (Supplementary Table S1). Figure 3B summarizes the number of proteins discovered by each of the three search algorithms (SEQUEST, InsPecT, and OMSSA) in the NE fraction. Also in the NE fraction, 690 proteins were identified from chymotrypsin-digested samples. This yielded 184 proteins that were not found using trypsin and confirmed the presence of 506 proteins found in the NE trypsin dataset (Fig. 3C). It is evident from Fig. 3, B and C, that the use of multiple search algorithms and different proteases are beneficial in increasing the number as well as the confidence of unique peptides and proteins identified. Among the 3,531 protein identifications made in this study, 2,163 (61%) were not identified in the IMCD in previous studies and now have been added to the IMCD Proteome Database (http://dir.nhlbi.nih.gov/papers/lkem/imcd/).

Fig. 3.

Venn diagram of proteomic results. A: the number of proteins identified in the NE, NP, and cytoplasmic fractions (Cyto) with an estimated single-peptide false discovery rate specified as 2% or less. A total of 3,531 proteins were identified in one or more of the 3 fractions. B: the number of proteins identified in the NE fraction by each searching algorithm (SEQUEST, InsPecT, and OMSSA). Intersections between each circle show the number of proteins identified by a combination of search algorithms. C: the number of proteins identified in the NE fraction using 2 different proteases (trypsin and chymotrypsin).

Transcription Factors Identified by LC-MS/MS

To enumerate the transcription factors found in the rat IMCD in this study, proteins identified in all the samples were combined into one dataset (Supplementary Table S1). One hundred fifty-four proteins (4.4% of total) were classified as “transcription factor” proteins (Table 1) using the Panther Classification System (http://www.pantherdb.org/). Figure 4 shows a Venn diagram of the distribution of transcription factors identified in the NE, NP, and Cyto fractions, with the majority of these proteins found in the NE fraction. One hundred twenty-eight of these proteins (83%) were new identifications for the IMCD Proteome Database.

Table 1.

Transcription factors

Accession No.	Gene	Name	Sample
	CREB transcription factors
NP_001002809	Atf6b (Crebl1)	cAMP responsive element binding protein-like 1	NP
NP_604392	Creb1	cAMP responsive element binding protein 1 isoform A	NE
NP_001099749	RGD1565584	similar to tyrosine kinase-associated leucine zipper protein LAZipII	Cyto
	Basic helix-loop-helix transcription factors
NP_036913	Arnt2	aryl hydrocarbon receptor nuclear translocator 2	Cyto
XP_001072661	LOC684826	similar to Basic helix-loop-helix transcription factor scleraxis	NP
NP_001020878	Tfeb	transcription factor EB	NP
NP_113965	Usf1	upstream stimulatory factor 1	NE
NP_112401	Usf2	upstream transcription factor 2	NE
	Homeobox transcription factors
NP_476450	Barhl1	BarH-like homeobox 1	NP
XP_001058888	Chx10	similar to Homeobox protein CHX10 (Ceh-10 homeodomain-containing homolog) isoform 2	NE
XP_347164	Cutl1	similar to Homeobox protein cut-like 1 (CCAAT displacement protein) (CDP) (Homeobox protein Cux)	NE
NP_036713	Hoxa2	homeo box A2	NE
NP_001017480	Hoxb7	homeo box B7	NE
XP_001069122	LOC688999	similar to Homeobox protein Hox-D8 (Hox-4.3) (Hox-5.4)	NE
NP_113925	Nkx6-1	NK6 homeobox 1	Cyto
NP_001099831	Pax2	paired box 2	NE
NP_037133	Pax6	paired box 6	NE
NP_112403	Pax8	paired box 8	NE
NP_001094151	Pbx1	preB-cell leukemia homeobox 1 isoform b	NE
XP_001081290	RGD1562142	similar to Homeobox protein Hox-B6 (Hox-2.2) (MH-22A)	NE
XP_001081293	RGD1562292	similar to Homeobox protein Hox-B5 (Hox-2.1) (MU-1) (H24.1)	Cyto
XP_226533	RGD1562686	similar to genetic suppressor element 1	NE
XP_001073119	RGD1563929	similar to OG2 homeobox	Cyto
XP_234082	RGD1564874	similar to NK1 transcription factor related 2-like,b	Cyto
XP_001058915	Zfhx4	similar to zinc finger homeodomain 4	NE
NP_001040562	Zhx3	zinc fingers and homeoboxes 3	NE, NP
	Nuclear hormone receptors
NP_599227	Dnttip1	deoxynucleotidyltransferase, terminal, interacting protein 1	NE
NP_001008511	Esrra	estrogen related receptor, alpha	NE
NP_976081	Esrrg	estrogen-related receptor gamma	NE
NP_113814	Nr1 h2	nuclear receptor subfamily 1, group H, member 2	NP
NP_542956	Nr2f2	nuclear receptor subfamily 2, group F, member 2	NE
NP_036708	Nr3c1	nuclear receptor subfamily 3, group C, member 1	NP
NP_478117	Sf1	splicing factor 1 isoform 2	NP
	Zinc finger transcription factors
NP_001099559	Ash2l	ash2 (absent, small, or homeotic)-like	NE
NP_446008	Bud31	BUD31 homolog	NE
NP_114012	Ctcf	CCCTC-binding factor (zinc finger protein)	NE, NP
NP_001101986	Dpf2	D4, zinc and double PHD fingers family 2	NE, NP
NP_001099893	Evi1	ecotropic viral integration site 1	NE
NP_001029098	Fhl1	four and a half LIM domains 1 isoform 1	Cyto
NP_001101293	Gfi1b	growth factor independent 1B transcription repressor	NE
XP_218939	Hbxap	similar to remodeling and spacing factor 1	Cyto
NP_001100647	Jarid1b	jumonji, AT rich interactive domain 1B	NP
NP_112397	Klf10	Kruppel-like factor 10	NE
NP_445988	Klf15	Kruppel-like factor 15	Cyto
XP_001072513	LOC684924	similar to zinc finger protein 449	NP
XP_001062820	LOC685209	similar to Zinc finger BED domain containing protein 4	Cyto
XP_001067699	LOC688634	similar to Histone acetyltransferase MYST4 (MYST protein 4) (MOZ, YBF2/SAS3, SAS2 and TIP60 protein 4) (Querkopf protein)	NE
NP_001094368	Mtf2	metal response element binding transcription factor 2	NE
NP_851595	Myst2	MYST histone acetyltransferase 2	NE
NP_001099501	Np	nucleoside phosphorylase	Cyto
NP_001103962	Phf14	PHD finger protein 14	Cyto
NP_001100812	Phf2	PHD finger protein 2	NE
NP_001071116	Prdm2	retinoblastoma protein-binding zinc finger protein	Cyto
NP_001005893	Repin1	replication initiator 1	NE
XP_219346	RGD1305903	similar to mesenchymal stem cell protein DSC43	NE
XP_001061052	RGD1310645	similar to Zinc finger protein 407	NE,NP
XP_001057325	RGD1359201	similar to CG15011-PA	Cyto
XP_001067122	RGD1560834	similar to zinc finger and BTB domain containing 41	Cyto
XP_233885	RGD1561526	similar to zinc finger protein 512	NP
XP_234528	RGD1563945	similar to PHD finger protein 16	NE
XP_001076564	RGD1564807	similar to zinc finger protein, subfamily 1A, 5	NP
NP_001128016	RGD1565545	hypothetical protein LOC308337	Cyto
NP_997714	Ring1	ring finger protein 1	NE
NP_001094197	Rufy1	RUN and FYVE domain containing 1	NE
NP_695222	St18	suppression of tumorigenicity 18	NE
XP_223196	Wdfy3	similar to WD repeat and FYVE domain containing 3	NP
NP_775412	Yy1	YY1 transcription factor	NE, NP
NP_001100567	Zbtb11	zinc finger and BTB domain containing 11	NE
NP_001099350	Zbtb20	zinc finger and BTB domain containing 20	NE, NP
NP_001124009	Zbtb39	zinc finger and BTB domain containing 39	Cyto
NP_446454	Zbtb7a	zinc finger and BTB domain containing 7a	NE
NP_001010963	Zc3 h15	erythropoietin 4 immediate early response	NE
NP_001028873	Zeb2	zinc finger E-box binding homeobox 2	NE, Cyto
NP_001101283	Zfp217	zinc finger protein 217	NE
XP_001059668	Zfp236	similar to Zinc finger protein 236	NE, Cyto
NP_001100701	Zfp278	zinc finger protein 278	Cyto
NP_001100591	Zfp316	zinc finger protein 316	NE
NP_001128057	Zfp426l2	zinc finger protein 426	NP
NP_877975	Zfp472	zinc finger protein 472	NE
NP_714953	Zfp709	zinc finger protein 709	NP
NP_001101279	Znf512b	zinc finger protein 512B	NE
NP_001019429	Znf574	zinc finger protein 574	Cyto
NP_001132956	Znf644	zinc finger protein 644	NP, Cyto
NP_001073676	Znf652	zinc finger protein 652	NP
XP_001080236	Zzef1	similar to zinc finger, ZZ type with EF hand domain 1	Cyto
	Other transcription factors
XP_219832	Aff2	similar to AF4/FMR2 family member 2 (Fragile X mental retardation protein 2 homolog)	NP
XP_341162	Ahctf1	similar to transcription factor ELYS	NE, Cyto
NP_446456	Cand1	cullin-associated and neddylation-dissociated 1	NE, NP, Cyto
NP_001101391	Casp8ap2	caspase 8 associated protein 2	NE
NP_001013209	Cbfb	core-binding factor, beta subunit	NE
NP_445979	Cdc5l	CDC5 cell division cycle 5-like	NE
NP_001028242	Cip29	cytokine induced protein 29 kDa	NE
NP_446118	Dll3	delta-like 3	Cyto
NP_001011914	Dr1	down-regulator of transcription 1	NE
NP_942046	Eif3 h	eukaryotic translation initiation factor 3, subunit H	Cyto
NP_001102912	Erh	enhancer of rudimentary homolog	NE
NP_001100552	Etv5	ets variant 5	Cyto
NP_001014192	Fam48a	transcription factor (p38 interacting protein)	NE
NP_001012137	Fus	fusion, derived from t(12;16) malignant liposarcoma	NE, Cyto
NP_001041351	Ilf2 (Nf45)	interleukin enhancer binding factor 2	NE,NP
XP_001076183	LOC500591	similar to calmodulin-binding transcription activator 1	Cyto
XP_001069951	LOC503338	similar to HBxAg transactivated protein 2	Cyto
XP_001057305	LOC681004	similar to GC-rich sequence DNA-binding factor homolog isoform 2	NE
XP_001081073	LOC688079	similar to TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor	NE
NP_001103001	LOC689296	hypothetical protein LOC689296	Cyto
NP_001101145	Maml3	mastermind like 3	NE
NP_001011999	Morf4l1	mortality factor 4 like 1	NE
NP_001007715	Morf4l2	mortality factor 4 like 2	Cyto
NP_113754	Nfib	nuclear factor I/B	NE
NP_113755	Nfic	nuclear factor I/C	NE
XP_342347	Nfkb1	similar to Nuclear factor NF-kappa-B p105 subunit (DNA-binding factor KBF1) (EBP-1)	NE, NP
NP_036997	Nfya	nuclear transcription factor Y, alpha	NE
NP_113741	Nfyb	nuclear transcription factor-Y beta	NE
NP_001004206	Pa2 g4	ErbB3-binding protein 1	NE, NP
NP_001099311	Ptrf	polymerase I and transcript release factor	NE,NP, Cyto
NP_071786	Pttg1	pituitary tumor-transforming 1	NP
XP_001063244	Pura	similar to Transcriptional activator protein Pur-alpha (Purine-rich single-stranded DNA-binding protein alpha)	NE, NP, Cyto
NP_001017503	Purb	purine rich element binding protein B	NE, Cyto
XP_001071121	Rb1	similar to Retinoblastoma-associated protein (PP105) (RB)	NE, NP
NP_001099414	Rfx1	regulatory factor X, 1 (influences HLA class II expression)	NE
XP_001056829	RGD1308009	similar to CCR4-NOT transcription complex, subunit 1 isoform a	NE, Cyto
XP_001080950	RGD1311017	similar to RIKEN cDNA 4732497O03	NP
XP_001061499	RGD1560020	similar to Myb proto-oncogene protein (C-myb)	NE
NP_001102749	RGD1561926	hypothetical protein LOC500695	NP
XP_220843	RGD1562272	similar to TAF11 RNA polymerase II, TATA box binding protein (TBP)-associated factor	Cyto
XP_228426	RGD1564748	similar to Mortality factor 4-like protein 2 (MORF-related gene X protein)	NP
NP_001012004	Rnf25	ring finger protein 25	NE
NP_001008290	Sbds	Shwachman-Bodian-Diamond syndrome homolog	NE, NP
NP_001007713	Sdpr	serum deprivation response	NP
NP_001100834	Sfmbt2	Scm-like with four mbt domains 2	NE
NP_001102231	Sin3a	SIN3 homolog A, transcription regulator	NE,NP
NP_001108510	Sin3b	SIN3 homolog B, transcription regulator	NE, NP, Cyto
NP_062064	Smad2	SMAD family member 2	NP
NP_037227	Smad3	SMAD family member 3	NE
NP_062148	Smad4	SMAD family member 4	NE
NP_110485	Smad7	SMAD family member 7	NE
NP_112383	Ssrp1	Structure specific recognition protein 1	NE, NP
NP_036879	Stat3	signal transducer and activator of transcription 3	NE, NP
NP_001011979	Tardbp	TAR DNA binding protein	NE, NP, Cyto
NP_001099921	Tbx15	T-box 15	NE
NP_001100504	Tbx4	T-box 4	NE
NP_001124046	Tcf20	transcription factor 20	NP
NP_001128186	Tcfcp2	transcription factor CP2	NE, NP
NP_001100640	Tcfcp2l1	transcription factor CP2-like 1	NP
NP_001032431	Tcfcp2l2	transcription factor CP2-like 2	Cyto
XP_001069278	Tead1	similar to Transcriptional enhancer factor TEF-1 (TEA domain family member 1) isoform 2	NE, NP
NP_001100317	Tfdp2	transcription factor Dp-2 (E2F dimerization partner 2)	Cyto
NP_001009693	Thrap3	thyroid hormone receptor associated protein 3	NE, NP
NP_001020136	Trappc2	trafficking protein particle complex 2	NE
NP_001128309	Trps1	trichorhinophalangeal syndrome I homolog	NP, Cyto
XP_001076644	Ubp1	similar to upstream binding protein 1 (LBP-1a)	NP
NP_001099193	Ubtf	upstream binding transcription factor, RNA polymerase I isoform 1	NE, NP
NP_620809	Xab2	XPA-binding protein 2	NE
NP_113751	Ybx1	Y box binding protein 1	NE, NP, Cyto
XP_001079683	Ybx2	similar to Y-box-binding protein 2 (Germ cell-specific Y-box-binding protein) (FRGY2 homolog)	NE

NE, soluble nuclear fraction; NP, nuclear membrane pellet; Cyto, pooled cytoplasmic fractions.

Fig. 4.

Venn diagram of the distribution of transcription factors identified in the NE, NP, and Cyto fractions.

Computational Analysis of Conserved Transcription Factor Binding Elements in Aqp2 and Aqp3 Genes

To relate the transcription factors found (Table 1) to their possible roles in regulation of Aqp2 and Aqp3 gene transcription, we have mapped conserved binding element motifs present in the 5′-flanking region as well as the first intron of rat Aqp2 and Aqp3 genes (Fig. 5), based on analysis using the Genomatix software suite (methods). In the following, we summarize these findings in the context of existing literature.

Fig. 5.

Transcription factor binding site analysis. The 1,000 bp 5′-flanking region and 1^st intron of the Aqp2 and Aqp3 genes of human, rat, and mouse were analyzed for conserved transcription factor binding sites (TFBS) using Genomatix database and software suite. This figure represents only Rat Aqp2 and Aqp3 genes. Conserved TFBS were found in the 1,000 bp 5′-flanking region of the Aqp2 and Aqp3 genes and the 1st intron of the Aqp2, however, no conserved TFBS were found in the 1st intron of the Aqp3 gene. Transcription factors identified in this study that potentially bind to these TFBS are shown above each TFBS. Abbreviations for Genomatix TFBS family name: V$SF1F = Vertebrate steroidogenic factor; V$NFAT = Nuclear factor of activated T-cells; V$FKHD = Fork head domain factors; V$ETSF = Human and murine ETS1 factors; V$RXRF = RXR heterodimer binding sites; V$AP2F = Activator protein 2; V$CREB = cAMP-responsive element binding proteins; V$GATA = GATA binding factors; V$SRFF = Serum response element binding factor; V$HOXF = Paralog hox genes 1–8 from the four hox clusters A, B, C, D; V$EBOX = E-box binding factors; V$EGRF = EGR/nerve growth factor induced protein C & related factors; V$MYT1 = MYT1 C2HC zinc finger protein; V$KLFS = Krueppel like transcription factors; V$CP2F = CP2-erythrocyte Factor related to drosophila Elf1; V$CEBP = Ccaat/Enhancer Binding Protein; V$PARF = PAR/bZIP family; V$NR2F = Nuclear receptor subfamily 2 factors; V$HAND = Twist subfamily of class B bHLH transcription factors; V$GREF = Glucocorticoid responsive and related elements; V$MAZF = Myc associated zinc fingers; V$ZBPF = Zinc binding protein factors; and V$SP1F = GC-Box factors SP1/GC.

Transcription Factors Potentially Involved in Aqp2 Gene Transcription

Figure 5A shows the transcription factors found in this study (listed in Table 1) that potentially bind conserved transcription factor binding elements in the first 1,000 bp of the 5′-flanking region or first intron of the Aqp2 gene, based on computational analysis using the Genomatix suite. As discussed in the following some of these have been examined experimentally in prior studies.

CREB.

A conserved CREB binding motif (aka CRE) is present at −222 bp from the transcription start site. Originally identified by Uchida and colleagues (48), the importance of this site has been thoroughly documented by the subsequent studies of Hozawa et al. (22), Matsumura et al. (28), Yasui et al. (52), and Cai et al. (5). There are several genes in mammalian genomes that code for proteins that could potentially bind to CRE, including the index protein Creb1, which is activated by phosphorylation at Ser-133 by Ca-calmodulin-dependent kinases, ribosomal S6 kinase, or protein kinase A (41). In this study we identify two of these, Creb1 and Crebl1 (also known as “cAMP responsive element binding protein-like 1”). These contain the so-called “bZIP domain,” comprising a basic region and a leucine zipper region. Crebl1 acts in the unfolded protein response (UPR) pathway by activating UPR target genes induced during endoplasmic reticulum (ER) stress (24). It is a single-pass integral membrane protein that undergoes regulated intramembrane proteolysis in the Golgi during the UPR to yield an ∼400 amino acid cytoplasmic product that translocates into the nucleus.

GATA.

Centered at −214 bp from the transcription start site is a conserved putative GATA sequence, a binding motif for zinc-finger transcription factors of the GATA family. Uchida et al. (47) identified Gata3 mRNA in microdissected collecting ducts and demonstrated that overexpression of Gata3 enhanced Aqp2 promoter-reporter activity. Both Gata2 and Gata3 are expressed in IMCD at many fold above the median signal (46). In the present study, we identified another GATA family member, Trps1 (Tricho-rhino-phalangeal syndrome type I protein), in nuclei from native IMCD cells. This protein is known to bind relatively specifically to GATA sequences and represses GATA-regulated genes (27). Interestingly Rai et al. (37) identified, in the 5′-flanking region of the Aqp2 gene, a region overlapping this GATA site that contained an unidentified cis-element with an apparent negative regulatory role on transcriptional activity. However, in a later study overexpression of the Gata3 transcription factor increased Aqp2 transcription pointing to an enhancer role for this binding element (47), in seeming contradiction to the findings of Rai et al. (37). A possible explanation for this conundrum is that Trps1 normally maintains repression at the GATA site, blocking the enhancer activity of Gata2 and Gata3 that would otherwise occur.

HOX.

In the present study we also identified by mass spectrometry two transcription factors that potentially bind to a conserved HOX binding element centered at −201 bp from the transcription start site, viz., Hoxa2 and Hoxb7. Homeobox or HOX transcription factors are recognized to be involved in renal tubule segmentation as well as collecting duct development (32). The 5′-flanking region of Hoxb7 has been used to target transgene expression specifically to collecting duct cells in mice (38, 42).

NFAT.

Centered at −469 bp upstream from the transcription start site is a potential NFAT binding element. Several NFAT proteins including TonEBP (Nfat5) (19) and the calcineurin-dependent Nfat proteins (Nfatc1–4) (26) have been implicated in Aqp2 transcriptional regulation. Although we did not identify these transcription factors in the present study, we found a protein (Nf45) with weak homology to them that has been previously implicated in regulation of interleukin-2 transcription in lymphocytes (23). Although mRNA levels do not necessarily correlate with protein expression, Affymetrix microarray studies of native IMCD cells demonstrated that Nfat5 (also called TonEBP) is expressed with a signal 12.5-fold above the median signal for all transcripts (46). Interestingly, the same study showed that another Nfat protein is strongly expressed in rat renal IMCD, viz. Nfatc3, which is regulated by intracellular calcium. A rise in calcium activates the Ca-dependent protein phosphatase, calcineurin, which dephosphorylates the Nfat protein allowing it to translocate into the nucleus. A member of this family has recently been implicated in the transcriptional control of the Aqp2 gene (26). The involvement of Ca²⁺-calcineurin-sensitive regulation of Aqp2 gene transcription identifies another mechanism by which vasopressin can regulate Aqp2 expression, since several laboratories have demonstrated that activation of the V2 vasopressin receptor is associated with an elevation of intracellular calcium (6, 8, 11, 35, 43, 53).

ETS.

Centered at −455 bp from the transcriptional start site of the Aqp2 gene is a conserved Ets binding motif that we identified in a previous study (54) as playing an enhancer role in cell-specific expression of Aqp2, and possibly playing a role in the vasopressin response. In the present study, MS analysis identified Etv5 (ets variant gene 5 or PEA3), a member of the ets family. This transcription factor is known to be selectively expressed in ureteric bud (the developmental precursor of the collecting duct system) (7).

RXR.

Centered at −342 bp from the transcription start site is a conserved putative RXR binding motif. RXR binding elements bind dimers of ligand-activated transcription factors, usually RAR/RXR heterodimers. These transcription factors are known to be involved in development of the ureteric bud (4). In addition, RXR can heterodimerize with vitamin D receptors, peroxisome proliferator activator receptors, thyroid receptors, and other ligand-activated nuclear receptors. MS analysis in the present paper found one such ligand-activated transcription factor, viz. Nr1h2 (nuclear receptor subfamily 1 group H member 2, also known as “liver X receptor”). Interestingly, this is one of two transcription factor genes (with Elf1) whose mRNA levels correlated negatively with Aqp2 mRNA levels among subcloned mpkCCD collecting duct cell lines (54). Its endogenous ligand is at present unknown.

EBOX.

A conserved EBOX motif is located at −67 bp relative to the transcription start site of Aqp2. Two transcription factors were identified that potentially bind this site, namely Usf1 and Usf2. These are classical basic helix-loop-helix transcription factors known to be involved in transcriptional regulation in a variety of tissues.

Potential binding elements in the first intron.

We identified four potential transcription factor binding elements in a conserved region of the first intron of the Aqp2 gene (Fig. 5A), viz. binding sites for EGR, MYT1, KLF, and CP2 family transcription factors. The transcription factors identified in IMCD nuclei that correspond to these sites (Zbtb7a, St18, Klf15, Tcfcp2, and Tcfcp2l1) are all of the zinc-finger or the CP2 classes of transcription factors and typically play repressor roles. Klf15 has been previously implicated in regulation of the ClC-K1 chloride channel of the thin ascending limb of Henle (49). Tcfp2l1 is associated with normal duct development in both the salivary gland and kidney (50).

Transcription Factors Potentially Involved in Aqp3 Gene Transcription

Figure 5B shows the transcription factors found in this study (Table 1) that potentially bind conserved transcription factor binding elements in the first 1,000 bp of the 5′-flanking region or first intron of the Aqp3 gene, based on computational analysis using the Genomatix suite. No conserved binding motifs were found in the first intron.

The 5′-flanking region of the Aqp3 gene contains several conserved transcription factor binding element motifs seen in the Aqp2 5′-flanking region or first intron including two EBOX motifs (potentially binding Usf1 and Usf2), an NR2 nuclear receptor site (potentially binding the orphan nuclear receptor Nrf2f), and two KLF sites (potentially binding Klf15). These sites are potentially responsible for the coordinate regulation of Aqp2 and Aqp3 seen in response to vasopressin (45). In addition, the 5′-flanking region of the Aqp3 gene contains some interesting unique sites that may provide some of the explanation for the difference in the tissue distribution of Aqp3 expression vs. Aqp2 expression, as well as differences in regulation. Foremost among these unique sites is a putative GRE (glucocorticoid response element), which can bind either the glucocorticoid receptor (Nr3c1) or the mineralocorticoid receptor (Nr3c2). Although only the former was found by MS analysis of the nuclear fraction in the current study, the latter is also known to be strongly expressed in the rat renal IMCD (46), providing a potential explanation for the large increase in Aqp3 protein abundance seen in the renal collecting duct in response to treatment of rats with the mineralocorticoid, aldosterone (25).

In addition, the 5′-flanking region of the Aqp3 gene contains two highly conserved “MAZ” or “MYC-associated zinc finger” sites at −62 and −79 bp relative to the transcriptional start site. Genomatix analysis predicts that one transcription factor from Table 1 would bind to these sites, viz. Zbtb19 (aka Zfp278 or “protein kinase A RI subunit α-associated protein”). Like other zinc-finger transcription factors, this transcription factor is associated with repressor activity. Finally, the 5′-flanking region of the Aqp3 gene contains a highly conserved SP1 binding element motif at −62 bp, which potentially binds another zinc-finger transcription factor found in this study by mass spectrometry, viz. Klf10 (also known as “TGF-β-inducible early growth response protein 1”), that likely manifests repressor function.

Transcriptional Co-regulators and Nucleic Acid Binding Proteins Identified by Proteomic Analysis of IMCD

Transcriptional co-regulators are listed in Table 2. Proteins classified as “nucleic acid binding” proteins by Panther analysis are listed in Supplementary Table S2. This category includes basal transcription factors, helicases, RNA polymerases, and other ubiquitous proteins. Both lists are made up largely of proteins that would be found in any cell type.

Table 2.

Transcriptional co-regulators

Accession No.	Gene	Name	Sample
NP_062093	Aes	amino-terminal enhancer of split	NP
NP_001101094	Arid5b	AT rich interactive domain 5B (Mrf1 like)	NP
NP_001101923	Cbfa2t3	core-binding factor, runt domain, alpha subunit 2, translocated to, 3	NE
NP_445981	Ciita	class II transactivator	NE
NP_596872	Crebbp	CREB binding protein	NE
NP_445787	Ctbp2	C-terminal binding protein 2	NE
XP_341396	Dcp1a	similar to mRNA decapping enzyme 1A (Smad4-interacting transcriptional co-activator)	NE
NP_001102523	Dtx3l	deltex 3-like	NE
NP_001100027	Edf1	endothelial differentiation-related factor 1	NE
NP_001100774	Ell	elongation factor RNA polymerase II	Cyto
XP_576312	Ep300	similar to E1A binding protein p300	NE
NP_598232	Hdgfrp2	hepatoma-derived growth factor-related protein 2	NE
NP_001009625	Ifi35	interferon-induced protein 35	NE
XP_001061895	LOC360888	similar to lin-9 homolog	NE
XP_001055295	LOC679225	similar to Ladybird homeobox corepressor 1	NP
XP_001054608	LOC679693	similar to Mediator of RNA polymerase II transcription subunit 12 isoform 1	NE
XP_001080941	LOC688035	similar to transformation related protein 53 binding protein 1	NE
XP_001066953	LOC689062	similar to nuclear DNA-binding protein	NE
NP_001019932	Lrrfip2	leucine rich repeat (in FLII) interacting protein 2	NE, NP
NP_001100271	Med17	mediator complex subunit 17	NE
NP_001100035	Med27	mediator complex subunit 27	NP
NP_114010	Ncoa2	nuclear receptor coactivator 2	NP
XP_221724	Nrip1	similar to nuclear receptor interacting protein 1	NE
NP_001101968	Nrip3	nuclear receptor interacting protein 3	NE
NP_001094308	Prkcbp1	protein kinase C binding protein 1	NE
NP_786941	Psip1	PC4 and SFRS1 interacting protein 1	NE, NP
NP_001100644	Rbbp5	retinoblastoma binding protein 5	NP
NP_001101164	Rfx5	regulatory factor X, 5 (influences HLA class II expression)	NE
XP_221241	RGD1304870	similar to transcription elongation factor B (SIII), polypeptide 2	NP
XP_223519	RGD1561605	similar to Ada2b CG9638-PA, isoform A	Cyto
NP_671706	Ruvbl1	RuvB-like 1	NE, NP
NP_001020576	Ruvbl2	RuvB-like 2	NE, NP
NP_071789	Safb	scaffold attachment factor B	NE, Cyto
NP_073185	Snd1	staphylococcal nuclease domain containing 1	NE, Cyto
NP_001009618	Sub1	RNA polymerase II transcriptional coactivator	NE, NP
NP_001100731	Supt16 h	suppressor of Ty 16 homolog	NE, NP
NP_001101374	Tgs1	trimethylguanosine synthase homolog	NE, NP
XP_233081	Thoc2	similar to THO complex subunit 2 (Tho2)	NE
NP_446368	Trim28	tripartite motif-containing 28	NE, NP, Cyto
XP_345267	Trim33	similar to tripartite motif protein 33	NE
NP_001101666	Wtip	WT1-interacting protein	Cyto
NP_976244	Zmynd11	zinc finger, MYND domain containing 11 delta isoform	NE

Phosphoproteomic Analysis

Figure 6 summarizes the workflow utilized for the discovery of phosphoproteins in NE and NP fractions from rat IMCD. Three phosphorylation site verification tools were used [Ascore (2), Phosphate Localization Score (PLscore) (1), and PhosphoScore (39)] to increase our confidence in the identified phosphorylation sites. Table 3 summarizes the 122 phosphorylation sites found. Of those, 63 were previously unidentified phosphorylation sites (not present in Phosphosite, http://www.phosphosite.org). Eight phosphorylation sites in six proteins were identified as a “transcription factor” or “transcription co-regulator” by Panther, namely Bclaf1 (S512), Hbxap (S608 and S1352), Lrrfip2 (S133), Ptrf (S169), Safb (S309), and Trim28 (S52 and S474). The presence of phosphorylated COOH-terminal tails of two aquaporins in the nuclear fractions raises the possibility of a role for regulated intramembrane proteolysis (3) of aquaporin proteins in transcriptional regulation in the IMCD, a possibility that needs further investigation. However, we cannot rule out the presence of these aquaporin phosphopeptides in nuclear fractions owing to contamination with small amounts of ER membranes, which are essentially extensions of the nuclear envelope.

Fig. 6.

Phosphoproteomic work flow. Graphical representation of the sample preparation, LC-MS³, and bioinformatic steps taken in the nuclear phosphoproteomic study. Ovals represent the sample or proteomic data. Shaded boxes represent the biochemical and computational methods that were applied to the sample or proteomic data. The work flow utilized 2 phosphopeptide isolation kits (IMAC and TiO₂ tips) to maximize phosphopeptide recovery and 3 phosphorylation site verification programs (Ascore, PLscore, and PhosphoScore) to ensure phosphosite validity.

Table 3.

Phosphorylation sites identified in nuclear fractions

Accession No.	Gene	Name	Sequence	PhosphoSite
NP_001037859	Ablim1	actin-binding LIM protein 1	S*GLHRPVSTDFAQYNSYGDVSGGVR	S311^
			TLS*PTPSAEGFQDGR	S115^
XP_574618	Ahnak	similar to AHNAK nucleoprotein isoform 1	FKAEAALPS*PK	S2257
XP_216539	Aim1l	similar to absent in melanoma 1	AVRDYCT*PR	T1271^
NP_001029156	Ank3	ankyrin 3, epithelial isoform 2	RQS*FTSLALR	S1458^
NP_036910	Aqp1	aquaporin 1	VWTSGQVEEYDLDADDINS*R	S262^
NP_037041	Aqp2	aquaporin 2 (collecting duct)	QS*VELHS*PQSLPR	S256, S261
			RQS*VELHSPQSLPR	S256
			RQSVELHS*PQSLPR	S261
NP_001029312	Baiap2l1	BAI1-associated protein 2-like 1	SIS*TVDLTEK	S423
NP_001041317	Bclaf1	BCL2-associated transcription factor 1	LKELFDYS*PPLHK	S512^
NP_001102045	Brd3	bromodomain containing 3	SES*PPPLSEPK	S263^
NP_114175	Calm1	calmodulin 1	HVMT*NLGEKLT*DEEVDEMIR	T111^, T118
NP_446072	Cdc42bpb	Cdc42-binding protein kinase beta	HSTPSNSSNPSGPPS*PNSPHR	S1692
NP_001101634	Cgnl1	cingulin-like 1	EGVGEETLS*PR	S251^
			NCFPKPCGS*QPNS*PTPEDLAK	S198, S202
			NCFPKPCGSQPNS*PT*PEDLAK	S202, T204^
NP_001007146	Ctnna1	catenin (cadherin-associated protein), alpha 1, 102 kDa	TPEELDDS*DFETEDFDVRSRTS*VQTEDDQLIAGQSAR	S643S657
NP_001101210	Ctnnd1	catenin (cadherin associated protein), delta 1	GS*LASLDSLRK	S346
			GSLAS*LDSLRK	S349
			VGGS*SVDLHR	S268
XP_225259	Dsp	similar to desmoplakin isoform I isoform 2	NLTIRS*SSLS*DPLEESSPIAAIFDTENLEK	S2613, S2617
NP_114008	Dvl1	dishevelled, dsh homolog 1	HHFLGIS*IVGQS*NDR	S265^, S270^
NP_001013122	Eef1d	eukaryotic translation elongation factor 1 delta	GATPAEDDEDNDIDLFGS*DEEEEDKEAAR	S531
			KGATPAEDDEDNDIDLFGS*DEEEEDKEAAR	S531
NP_067713	Epb4.1l1	erythrocyte protein band 4.1-like 1 isoform L	HQAS*INELKR	S510
XP_217039	eplin	similar to Epithelial protein lost in neoplasm (mEPLIN)	GENEETLGRPAQPPSAGETPHS*PGVEDAPIAKRLS*ENSCSLDDLEIGAGHLSSSAFNSEK	S488S225
			SEAQQPIYTKPLS*PDAR	S360
			SPKPLS*PSLR	S608
			TSSVKS*PKPLS*PSLRK	S603^, S608
			TSSVKS*PKPLSPS*LRK	S603^, S610^
NP_001124037	Fam83 h	family with sequence similarity 83, member H	KGS*PTPAYPER	S860
NP_001128071	Flna	filamin, alpha	RAPS*VANVGSHCDLSLK	S2144
XP_218939	Hbxap	similar to remodeling and spacing factor 1	IES*DEEEDFENVGK	S1352^
			TSVDKDIQRLS*PIPEEVVR	S608^
NP_001020580	Hdac1	histone deacetylase 1	MLPHAPGVQMQAIPEDAIPEES*GDEDEEDPDKR	S393
NP_446159	Hdgf	hepatoma-derived growth factor	AGDMLEDS*PKRPK	S165
			GNAEGS*S*DEEGKLVIDEPAKEK	S132, S133
			RAGDMLEDS*PKRPK	S165
NP_579819	Hist1 h1d	histone cluster 1, H1d	KAS*GPPVSELITK	S36
			RKAS*GPPVSELITK	S36
NP_073177	Hist1 h4b	histone cluster 1, H4b	RIS*GLIYEETR	S48
NP_001101168	Hist2 h3c2	histone cluster 2, H3c2	FQSSAVMALQEASEAY*LVGLFEDTNLCAIHAK	Y100^
NP_058944	Hnrnpa1	heterogeneous nuclear ribonucleoprotein A1	SES*PKEPEQLR	S6
			SES*PKEPEQLRK	S6
NP_476482	Hnrnpk	heterogeneous nuclear ribonucleoprotein K	IIPTLEEGLQLPS*PTATSQLPLESDAVECLNYQHYK	S116^
NP_114176	Hspb1	heat shock protein 1	QLS*S*GVSEIR	S85, S86^
			QLS*SGVSEIR	S85
			SPS*WEPFR	S15
			SPS*WEPFRDWYPAHSR	S15
NP_001101412	Lad1	ladinin	LPS*VEEAEVSKPSPPASKDEGEEFQAILR	S62
NP_001002016	Lmna	lamin A isoform C2	LRLS*PS*PTSQR	S389, S391
			LRLS*PSPTSQR	S389
			LRLSPS*PTSQR	S391
NP_001001515	Lmo7	LIM domain 7	SRS*TTELNDPLIEK	S1476^
XP_001080824	LOC360570	similar to myosin XVIIIa	RFS*FSQR	S140
			SLAPDLS*DDEHDPVDSISRPR	S2020^
XP_341387	LOC361100	similar to topoisomerase (DNA) II beta	KTS*FDQDSDVDIFPSDFTSEPPALPR	S367^
			KTSFDQDS*DVDIFPSDFTSEPPALPR	S372
			KVVEPANS*DS*DSELGNIPK	S312^, S314^
			VKAS*PITNDGEDEFVPS*DGIDKDEYAFSPGK	S189, S202^
			VKAS*PITNDGEDEFVPSDGIDKDEYAFSPGK	S189
XP_573444	LOC498222	similar to specifically androgen-regulated protein	APAQPAS*PALVR	S396^
XP_001056725	LOC681423	similar to delangin isoform A	AITSLLGGGS*PK	S2431^
XP_001061576	LOC682450	similar to CG31613-PA	FQS*S*AVIALQEASEAYLVGLFEDTNLCAIHAK	S87^, S88^
XP_001068152	LOC684233	similar to Putative RNA-binding protein 15 (RNA-binding motif protein 15) (One-twenty two protein)	DRT*PPLLYRLLLLERPS*PVRDR	T567S701^
XP_001071565	LOC684681	similar to Histone H1.2 (H1 VAR.1) (H1c)	S*ET*APAAPAAAPPAEKAPAK	S108^, T110^
XP_001055673	LOC685179	similar to SWI/SNF-related matrix-associated actin-dependent regulator of chromatin c2 isoform b isoform 2	DMDEPS*PVPNVEEVTLPK	S347^
XP_001078152	LOC686579	similar to AHNAK nucleoprotein isoform 2 isoform 1	KGDRS*PEPGQTWAHEVFSSR	S94
NP_001019932	Lrrfip2	leucine rich repeat (in FLII) interacting protein 2	RGS*GDTSSLIDPDTSLSELR	S133^
NP_037198	Map2	microtubule-associated protein 2	ARVDHGAEIITQS*PSR	S1780
NP_113708	Myh10	myosin, heavy chain 10, nonmuscle	QLHIEGAS*LELS*DDDTESKQLHIEGASLELS*DDDTESK	S1952^, S1956S1956
			RQLHIEGASLELS*DDDTESK	S1956
NP_037326	Myh9	myosin, heavy chain 9, nonmuscle	GTGDCS*DEEVDGKADGADAK	S1944
NP_631977	Nexn	nexilin (F actin binding protein) isoform b	AEIKDMLAS*DEEEEPSKVEK	S16
NP_037124	Npm1	nucleophosmin (nucleolar phosphoprotein B23, numatrin)	CGSGPVHISGQHLVAVEEDAES*EDEDEEDVK	S125
NP_073636	Nucks1	nuclear casein kinase and cyclin-dependent kinase substrate 1	TSAS*PPLEK	S223^
XP_218972	Numa1	similar to nuclear mitotic apparatus protein 1	AAQLQGS*PAPEKGEVLGDALQLDTLKQEAAK	S416^
NP_001106799	Pde4d	phosphodiesterase 4D isoform 1	KSRMS*WPSSFQGLR	S137^
NP_071796	Plec1	plectin 1 isoform 1	KQIT*VEELVR	T4033^
			SSS*VGSSSSYPISSAVPR	S4389
NP_001101576	Plxnb2	plexin B2	ADPAY*RCVWCRGQNR	Y779^
XP_343923	Polr2a	similar to DNA-directed RNA polymerase II largest subunit (RPB1)	GHGGCGRY*QPRIR	Y187^
NP_001099311	Ptrf	polymerase I and transcript release factor	LPAKLS*VSK	S169^
NP_001013225	Rbm39	RNA binding motif protein 39	DKS*PVREPIDNLTPEER	S136
XP_578547	RGD1562028	similar to Mitogen-activated protein kinase kinase kinase kinase 5 (MAPK/ERK kinase kinase kinase 5) (MEK kinase kinase 5) (MEKKK 5)	TAS*EINFDKLQFEPPLRK	S316^
XP_001075668	RGD1564067	hypothetical protein	T*KVEEEPPPPPPPPPPPPPPPPPR	T104^
NP_071789	Safb	scaffold attachment factor B	APTAAPS*PEPR	S309
XP_217021	Sbf1	similar to SET binding factor 1 isoform a	LGLGTLSS*SLS*R	S1142^, S1145^
NP_476489	Sept2	septin 2	IYHLPDAES*DEDEDFKEQTR	S218
			IYHLPDAESDEDEDFKEQT*R	T228^
NP_789826	Sept9	septin 9 isoform 2	LVDTLSQRS*PKPSLR	S67^
NP_001009255	Sfrs9	splicing factor, arginine/serine rich 9	GS*PHYFSPFRPY	S211
NP_690921	Slc4a9	solute carrier family 4, sodium bicarbonate cotransporter, member 9	WS*APHVPTLALPSLQK	S89^
NP_037165	Slc5a1	solute carrier family 5 (sodium/glucose cotransporter), member 1	MT*KEEEEAMKLK	T622^
NP_062251	Snip	SNAP25-interacting protein	RCRVVT*DTLAQIRR	T853^
NP_001011981	Snx17	sorting nexin 17	S*QEYKIVLRK	S194^
NP_741984	Spna2	alpha-spectrin 2	WRS*LQQLAEER	S1217
NP_001101456	Srrm1	serine/arginine repetitive matrix 1	EKS*PELPEPSVR	S220^
			RYS*PPIQR	S601
NP_001099736	Tjp1	tight junction protein 1	VQIPVSHPDPDPVS*DNEDDSYDEDVHDPR	S113^
			VQIPVSHPDPDPVSDNEDDS*YDEDVHDPR	S119^
NP_446225	Tjp2	tight junction protein 2	KVQVAPLQGS*PPLSHDDR	S107
NP_037019	Tmpo	thymopoietin	GPPDFSSDEEREPT*PVLGSGASVGR	T74
NP_446368	Trim28	tripartite motif-containing 28	RPAASSAAAASASASS*PAGGGGEAQELLEHCGVCR	S52^
			SRS*GEGEVSGLMR	S474^
NP_001026829	Trip12	thyroid hormone receptor interactor 12	S*ESPPAELPSLR	S310^
NP_037202	Utrn	utrophin	AAQAS*LSALNDPSAVEQALQEK	S933^
NP_973718	Xirp2	xin actin-binding repeat containing 2	CFET*QPLY*VIR	T507^, Y511^
NP_001101423	Zcchc11	zinc finger, CCHC domain containing 11	FILTS*GKPPTIVCS*ICK	S768^, S777^

^{^}Previously unidentified.

Kinases in Nuclear Fractions

Table 4 categorizes the kinases found in this study, which include 58 kinases present in the nuclear extract fraction. While all are candidates for regulatory roles in transcription in IMCD, some already have well documented roles in other tissues. For example, all three of the kinases known to phosphorylate Creb1 at Ser133 are present in either the NE or NP fraction, viz. protein kinase A catalytic subunit α (Prkaca), calcium/calmodulin-dependent protein kinase II δ and γ (Camk2d and Camk2g), and ribosomal protein S6 kinase (Rps6ka5 and Rps6kc1) (41). In addition several MAP kinases were detected, including Erk1 (Mapk3), Erk2 (Mapk1), Erk3 (Mapk6), and p38α (Mapk14), which are known to phosphorylate transcription factors of the ETS, AP1, and GATA families (Phosphosite: http://www.phosphosite.org/). Finally, the Hipk2 protein phosphorylates a number of transcription factors including p53, Pax6, and Zbtb4 (Phosphosite).

Table 4.

Protein kinases in nuclear fractions

Accession No.	Gene	Name	Sample
	AGC Ser/Thr protein kinase family
NP_001101987	Ccdc88b	coiled-coil domain containing 88B	NP
NP_446072	Cdc42bpb	Cdc42-binding protein kinase beta	NE, NP, Cyto
NP_001025082	Cit	citron (rho-interacting, serine/threonine kinase 21)	NE
NP_112358	Grk1	G protein-coupled receptor kinase 1	NE, NP, Cyto
NP_001100737	Lats2	large tumor suppressor 2	NE
NP_851603	Mast1	microtubule associated serine/threonine kinase 1	NE
NP_001099225	Pkn2	protein kinase N2	NE
NP_001094392	Prkaca	cAMP-dependent protein kinase catalytic subunit alpha	NP, Cyto
NP_112360	Rock1	Rho-associated coiled-coil containing protein kinase 1	NE, Cyto
NP_037154	Rock2	Rho-associated coiled-coil containing protein kinase 2	NE, Cyto
NP_001101518	Rps6ka5	ribosomal protein S6 kinase, polypeptide 5	NP
NP_001099454	Rps6kc1	ribosomal protein S6 kinase, 52 kDa, polypeptide 1	NP
NP_001076805	Stk38l	serine/threonine kinase 38 like	NE
	Atypical: PDK/BCKDK protein kinase family
NP_110499	Pdk2	pyruvate dehydrogenase kinase, isozyme 2	NE
	Atypical: PI3/PI4-kinase family
NP_001100291	Atm	ataxia telangiectasia mutated homolog	NE
NP_063971	Mtor	FK506 binding protein 12-rapamycin associated protein 1	NE, Cyto
NP_001101797	Prkdc	protein kinase, DNA activated, catalytic polypeptide	NE
	CAMK Ser/Thr protein kinase family
NP_036651	Camk2d	calcium/calmodulin-dependent protein kinase II delta	NE, NP, Cyto
NP_598289	Camk2 g	calcium/calmodulin-dependent protein kinase II gamma	NE
NP_071991	Dapk3	Death-associated protein kinase 3	NE
NP_067731	Mark2	MAP/microtubule affinity-regulating kinase 2	NE
NP_570105	Mark3	MAP/microtubule affinity-regulating kinase 3	NE
NP_001007618	Nuak2	NUAK family, SNF1-like kinase, 2	NP, Cyto
NP_001009362	Pask	PAS domain containing serine/threonine kinase	NP
NP_062015	Prkaa1	protein kinase, AMP-activated, alpha 1 catalytic subunit	NP
XP_230713	RGD1565143	Sperm motility kinase X	NE
NP_001011900	Tssk1	testis-specific serine kinase 1	NE
	CK1 Ser/Thr protein kinase family
NP_446067	Csnk1a1	casein kinase 1, alpha 1	NE, NP
NP_001012194	Vrk1	vaccinia related kinase 1	NE, NP
	CMGC Ser/Thr protein kinase family
NP_543161	Cdk5	cyclin-dependent kinase 5	NP
NP_059040	Gsk3a	glycogen synthase kinase 3 alpha	NE
NP_114469	Gsk3b	glycogen synthase kinase 3 beta	NE
NP_001102092	Hipk2	homeodomain interacting protein kinase 2	NE
NP_446294	Mapk1	mitogen activated protein kinase 1	NE, NP
NP_112282	Mapk14	mitogen-activated protein kinase 14	NP
NP_059043	Mapk3	mitogen activated protein kinase 3	NE
NP_113810	Mapk6	mitogen-activated protein kinase 6	NE
NP_001093976	Pctk3	PCTAIRE protein kinase 3	NE, NP, Cyto
NP_001011923	Prpf4b	PRP4 premRNA processing factor 4 homolog B	NE, NP
NP_001020897	Srpk1	SFRS protein kinase 1	NE
	NEK Ser/Thr protein kinase family
XP_224971	Nek3	similar to Serine/threonine-protein kinase Nek3 (NimA-related protein kinase 3)	NP
NP_001099274	Nek8	NIMA-related kinase 8	NE
	STE Ser/Thr protein kinase family
NP_596900	Ilk	integrin linked kinase	NE, NP
XP_001079680	LOC687705	similar to misshapen-like kinase 1 isoform 1 isoform 1	NE
NP_113831	Map2k1	mitogen-activated protein kinase kinase 1	NP
NP_579817	Map2k2	mitogen activated protein kinase kinase 2	NE
NP_001094144	Map2k3	mitogen activated protein kinase kinase 3	NP
NP_001100374	Map4k4	mitogen-activated protein kinase kinase kinase kinase 4	NE
NP_001101664	Oxsr1	oxidative-stress responsive 1	NP
NP_445758	Pak2	p21-activated kinase 2	NP
NP_001099708	Pak4	p21 (CDKN1A)-activated kinase 4	NE
XP_001070192	RGD1563568	similar to serine/threonine protein kinase MASK	NE, NP
NP_062222	Slk	serine/threonine kinase 2	NE, NP, Cyto
XP_577078	Stk10	similar to Serine/threonine-protein kinase 10 (Lymphocyte-oriented kinase)	NE
NP_001120966	Stk24	serine/threonine kinase 24	NE
NP_113923	Stk3	serine/threonine kinase 3	NP
NP_062235	Stk39	Ste-20 related kinase (SPAK)	NP
NP_775449	Taok1	TAO kinase 1	NE
	Tyr protein kinase family
XP_001079831	Abl1	similar to Proto-oncogene tyrosine-protein kinase ABL1 (p150)	NE
NP_001025210	Csk	c-src tyrosine kinase	NE,NP
NP_001102447	Epha2	ephrin receptor EphA2	NE, NP, Cyto
NP_599158	Epha7	ephrin receptor EphA7	NE
NP_001120791	Ephb2	Eph receptor B2	NE, Cyto
NP_077059	Fgr	proto-oncogene tyrosine-protein kinase Fgr	NE
NP_077344	Frk	fyn-related kinase	NP, Cyto
NP_036887	Fyn	FYN oncogene related to SRC, FGR, YES	NE
NP_058767	Insr	insulin receptor	NP
NP_445918	Jak1	Janus kinase 1	NP
NP_110484	Lyn	Yamaguchi sarcoma viral (v-yes-1) oncogene homolog isoform A	NE,NP
NP_001100785	Pragmin	pragmin	NE, Cyto
NP_114183	Src	v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog	NP, Cyto
NP_001099207	Tek	TEK tyrosine kinase, endothelial	NE
NP_001100482	Tnk1	tyrosine kinase, nonreceptor, 1	NP
NP_150640	Yes1	Yamaguchi sarcoma viral oncogene homolog 1	NE, NP, Cyto
	Other protein kinases
NP_112628	Camkk2	calcium/calmodulin-dependent protein kinase 2 beta	NP
NP_446276	Csnk2a1	casein kinase 2, alpha 1 polypeptide	NE, Cyto
NP_001100879	Csnk2a2	casein kinase 2, alpha prime polypeptide	NE, Cyto
NP_062208	Eif2ak2	eukaryotic translation initiation factor 2-alpha kinase 2	NP
XP_226572	RGD1306091	similar to cDNA sequence BC021891	NP
XP_577704	RGD1560972	similar to serine/threonine kinase	NE
XP_001076790	RGD1562028	similar to Mitogen-activated protein kinase kinase kinase kinase 5 (MEK kinase kinase 5) (MEKKK 5)	NE
XP_001080090	RGD1562674	similar to kinase suppressor of ras 2	NE

In general, of the 82 kinases listed in Table 4, 36 of them are classified by Gene Ontology (component) as located in “nucleus.” Among these are kinases that have housekeeping roles in the nucleus, such as DNA repair (Atm and Prkdc), mRNA splicing (Stk3), and cell cycle regulation (Cit, Cdk5, Gsk3β, and Mtor).

Phosphatases

Table 5 categorizes the 17 phosphatase proteins found in nuclear fractions in this study. These proteins include receptor and nonreceptor tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases. All could potentially play roles in transcriptional regulation in the collecting duct.

Table 5.

Protein phosphatases in nuclear fractions

Accession No	Gene	Name	Sample
	Dual-specificity phosphatases
NP_001012352	Dusp26	Dual-specificity phosphatase 26	NP
XP_001071732	Dusp27	PREDICTED: similar to Dual specificity protein phosphatase 13 (Testis- and skeletal-muscle-specific DSP)	NE
NP_001013065	Mtm1	X-linked myotubular myopathy gene 1	NP
NP_001101593	Mtmr2	myotubularin-related protein 2	NE
	Receptor tyrosine phosphatases
NP_062122	Ptprf	protein tyrosine phosphatase, receptor type, F	NE, NP
NP_599183	Ptprg	protein tyrosine phosphatase, receptor type, G	NE
NP_058965	Ptprj	protein tyrosine phosphatase, receptor type, J	NP
XP_001063025	Ptpru	PREDICTED: similar to protein tyrosine phosphatase, receptor type, L	NE
	Nonreceptor tyrosine phosphatases
NP_446360	Ptpn6	protein tyrosine phosphatase, nonreceptor type 6	NE, NP
NP_037220	Ptpn11	protein tyrosine phosphatase, nonreceptor type 11	NE, NP
XP_213997	Ptpn13	PREDICTED: similar to Tyrosine-protein phosphatase nonreceptor type 13	NE
NP_001100670	Ptpn14	protein tyrosine phosphatase, nonreceptor type 14	NP
	Serine/threonine phosphatases
NP_037197	Ppp1cb	protein phosphatase 1, catalytic subunit, beta	NE, Cyto
NP_071943	Ppp1 ml	protein phosphatase 1, catalytic subunit, gamma isoform	NE
NP_058735	Ppp2ca	protein phosphatase 2a, catalytic subunit, alpha isoform	NE
NP_476481	Ppp2r1a	alpha isoform of regulatory subunit A, protein phosphatase 2	NE, NP, Cyto
NP_852044	Ppp2r5b	protein phosphatase 2, regulatory subunit B', beta isoform	NE

General Observations

In addition to investigating the regulation of the water channels Aqp2 and Aqp3, a general goal of our studies is the development of tools, including proteomics databases, that can be useful to the kidney research community. In so doing, we have developed several proteomics and transcriptomic databases profiling genes expressed in the IMCD, thick ascending limb and proximal tubule (https:http://dirweb.nhlbi.nih.gov/Centers/CBPC/LKEM_G/LKEM/Pages/--TranscriptomicandProteomicDatabases.aspx). Our nuclear proteomics data have been added to an upgraded IMCD Proteome Database website (http://dir.nhlbi.nih.gov/papers/lkem/imcd/).

An additional goal has been to develop improved computational approaches to increase the yield of peptides that can be identified from high-quality spectra without a high rate of false positive identifications. In this study, the use of multiple search algorithms [SEQUEST (15), InsPecT (44), and OMSSA (17)] gave substantial numbers of identifications unique to each search algorithm. The largest increment in identifications was found with the InsPecT algorithm, a hybrid approach that uses de novo sequencing of a portion of each peptide to narrow the search space for pattern matching. InsPecT allowed the identification of a subset of peptides often missed by strictly peak-matching software such as SEQUEST or OMSSA. Further steps, such as target-decoy analysis as well as performing ambiguity checks on identified peptides using an in-house algorithm ProMatch, limited the number of false positive identifications and eliminated ambiguous identifications. We emphasize that target-decoy analysis has been demonstrated to be superior in limiting false positive identifications compared with use of the “two-peptide rule,” requiring protein identifications to those established by two or more peptides (18).

Overall, with the new data presented in this study, we have laid the groundwork for future studies of the role of vasopressin and other hormones in transcriptional regulation in renal collecting duct cells. The data provide a guide to proteins present in the nucleus of the “cells of interest” for such studies, which become candidates for genetic manipulation in cell culture and mouse models. The ultimate goal is to identify the relevant transcriptional regulatory networks involved in the control of Aqp2 and Aqp3 gene transcription. By sharing our data in a publicly accessible database, we encourage other investigators to partake in these investigations.

DISCLOSURES

No conflicts of interest are declared by the authors.