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Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells.

ABSTRACT: Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K(+) ((86)Rb(+)) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37 °C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl(-)] media to reduce cell [Cl(-)] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na(+)-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates.

SUBMITTER: Hannemann A

PROVIDER: S-EPMC3064583 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells.

Hannemann Anke A Flatman Peter W PW

PloS one 20110325 3

Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K(+) ...[more]

PMID: 21464992

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Project description:Precise regulation of the intracellular concentration of chloride [Cl-]i is necessary for proper cell volume regulation, transepithelial transport, and GABA neurotransmission. The Na-K-2Cl (NKCCs) and K-Cl (KCCs) cotransporters, related SLC12A transporters mediating cellular chloride influx and efflux, respectively, are key determinants of [Cl-]i in numerous cell types, including red blood cells, epithelial cells, and neurons. A common "chloride/volume-sensitive kinase", or related system of kinases, has long been hypothesized to mediate the reciprocal but coordinated phosphoregulation of the NKCCs and the KCCs, but the identity of these kinase(s) has remained unknown. Recent evidence suggests that the WNK (with no lysine = K) serine-threonine kinases directly or indirectly via the downstream Ste20-type kinases SPAK/OSR1, are critical components of this signaling pathway. Hypertonic stress (cell shrinkage), and possibly decreased [Cl-]i, triggers the phosphorylation and activation of specific WNKs, promoting NKCC activation and KCC inhibition via net transporter phosphorylation. Silencing WNK kinase activity can promote NKCC inhibition and KCC activation via net transporter dephosphorylation, revealing a dynamic ability of the WNKs to modulate [Cl-]. This pathway is essential for the defense of cell volume during osmotic perturbation, coordination of epithelial transport, and gating of sensory information in the peripheral system. Commiserate with their importance in serving these critical roles in humans, mutations in WNKs underlie two different Mendelian diseases, pseudohypoaldosteronism type II (an inherited form of salt-sensitive hypertension), and hereditary sensory and autonomic neuropathy type 2. WNKs also regulate ion transport in lower multicellular organisms, including Caenorhabditis elegans, suggesting that their functions are evolutionarily-conserved. An increased understanding of how the WNKs regulate the Na-K-2Cl and K-Cl cotransporters may provide novel opportunities for the selective modulation of these transporters, with ramifications for common human diseases like hypertension, sickle cell disease, neuropathic pain, and epilepsy.

Project description:BackgroundPituitary tumor transforming gene (PTTG), also known as securin, is highly expressed in various tumors including pituitary, thyroid, colon, ovary, testis, lung, and breast. An overexpression of PTTG enhances cell proliferation, induces cellular transformation in vitro, and promotes tumor development in nude mice. PTTG also inhibits separation of sister chromatids leading to aneuploidy and genetic instability. A great amount of work has been undertaken to understand the biology of PTTG and its expression in various tumors. However, mechanisms by which PTTG mediates its tumorigenic function are not fully understood. To utilize this gene for cancer therapy, identification of the downstream signaling genes regulated by PTTG in mediation of its tumorigenic function is necessary. For this purpose, we expressed PTTG in human embryonic kidney (HEK293) cells that do not express PTTG and analyzed the downstream genes using microarray analysis.ResultsA total of 22,277 genes printed on an Affymetrix HG-U133A 2.0 GeneChip array were screened with labeled cRNA prepared from HEK293 cells infected with adenovirus vector expressing PTTG cDNA (AdPTTG cDNA) and compared with labeled cRNA prepared from HEK293 cells infected with control adenovirus (control Ad) or adenovirus vector expressing GFP (AdGFP). Out of 22,277 genes, 71 genes were down-regulated and 35 genes were up-regulated with an FDR corrected p-value of < or = 0.05 and a fold change of > or =2. Most of the altered genes identified are involved in the cell cycle and cell apoptosis; a few are involved in mRNA processing and nitrogen metabolism. Most of the up-regulated genes belong to the histone protein family.ConclusionPTTG is a well-studied oncogene for its role in tumorigenesis. In addition to its importance in regulation of the cell cycle, this gene has also been recently shown to play a role in the induction of cell apoptosis. The microarray analysis in the present study demonstrated that PTTG may induce apoptosis by down-regulation of oncogenes such as v-Jun and v-maf and up-regulation of the histone family of genes.

Dataset Information

Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells.

Publications

Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells.

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