Quantitative mass spectrometry was used to identify hormonedependent signaling pathways in renal medullary thick ascending limb (mTAL) cells via phosphoproteomic analysis. Active transport of NaCl across the mTAL epithelium is accelerated by hormones that increase cAMP levels (vasopressin, glucagon, parathyroid hormone, and calcitonin). mTAL suspensions from rat kidneys were exposed (15 min) to a mixture of these four hormones. Tryptic phosphopeptides (immobilized metal affinity chromatography-enriched) were identified and quantified by mass spectrometry (LTQ-Orbitrap) using label-free methodology. We quantified a total of 654 phosphopeptides, of which 414 were quantified in three experimental pairs (hormone vs. vehicle). Of these phosphopeptides, 82% were statistically unchanged in abundance in response to the hormone mixture. In contrast, 48 phosphopeptides were significantly increased, whereas 28 were significantly decreased. The population of up-regulated phosphopeptides was highly enriched in basophilic kinase substrate motifs (AGC or calmodulin-sensitive kinase families), whereas the down-regulated sites were dominated by "proline-directed" motifs (cyclin-dependent or MAP kinase families). Bioinformatic classification uncovered overrepresentation of transmembrane transporters, protein phosphatase regulators, and cytoskeletal binding proteins among the regulated proteins. Immunoblotting with phospho-specific antibodies confirmed cAMP/vasopressin-dependent phosphorylation at Thr96, Ser126, and Ser874 of the Na + :K + :2Cl − cotransporter NKCC2, at Ser552 of the Na + :H + exchanger NHE3, and at Ser552 of β-catenin. Vasopressin also increased phosphorylation of NKCC2 at both Ser126 (more than fivefold) and Ser874 (more than threefold) in rats in vivo. Both sites were phosphorylated by purified protein kinase A during in vitro assays. These results support the view that, although protein kinase A plays a central role in mTAL signaling, additional kinases, including those that target proline-directed motifs, may be involved.protein phosphatase | glucose transporters | mass spectrometry | ion transporters | protein kinase T he thick ascending limb (TAL) of Henle's loop is a nephron segment that plays a critical role in the control of mammalian water excretion. Active NaCl transport by the medullary TAL (mTAL) drives the countercurrent multiplication process that concentrates the urine (1). Hormones that increase the concentration of the intracellular second messenger, cAMP, have been shown to enhance the rate of NaCl transport in mTAL cells (2). These hormones include parathyroid hormone (PTH), calcitonin, glucagon, and vasopressin (2). Among these, only vasopressin plays a selective role in regulation of water balance. The molecular targets for cAMP-mediated regulation in the mTAL include the apical Na + : K + :2Cl − cotransporter NKCC2 (gene symbol: Slc12a1) and the apical Na + :H + exchanger NHE3 (Slc9a3) (3).The signaling network that accounts for cAMP-dependent regulation of these transporters is largely unk...

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called "PhosphoLogo," uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions -2 and -3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4.

The transcription factor TonEBP/OREBP promotes cell survival during osmotic stress. High NaCl-induced phosphorylation of TonEBP/OREBP at tyrosine-143 was known to be an important factor in increasing its activity in cell culture. We now find that TonEBP/OREBP also is phosphorylated at tyrosine-143 in rat renal inner medulla, dependent on the interstitial osmolality. c-Abl seemed likely to be the kinase that phosphorylates TonEBP/OREBP because Y143 is in a consensus c-Abl phosphorylation site. We now confirm that, as follows. High NaCl increases c-Abl activity. Specific inhibition of c-Abl by imatinib, siRNA, or c-Abl kinase dead drastically reduces high NaCl-induced TonEBP/OREBP activity by reducing its nuclear location and transactivating activity. c-Abl associates with TonEBP/OREBP (coimmunoprecipitation) and phosphorylates TonEBP/OREBP-Y143 both in cell and in vitro. High NaCl-induced activation of ataxia telangiectasia mutated, previously known to contribute to activation of TonEBP/OREBP, depends on c-Abl activity. Thus, c-Abl is the kinase responsible for high NaCl-induced phosphorylation of TonEBP/OREBP-Y143, which contributes to its increased activity.