Na-K-Cl协同转运蛋白研究进展

Na-K-Cl协同转运蛋白是一类膜蛋白,负责转运Na、K、Cl离子进出上皮细胞与非上皮细胞。Na-K-Cl介导的转运过程是电中性的,多数情况下是1Na：1K：2C1（乌贼轴突中是2Na：1K：3C1）,其活性被布美他尼（bumetanide）和呋塞米（furosemide）所抑制。迄今为止,Na-K-Cl协同转运蛋白被鉴定出来两个同源异构体：NKCCl和NKCC2。NKCCl存在于多个组织中,合有NKCCl的上皮大多数属于分泌上皮,而且会有Na-K-Cl协同转运蛋白位于基底膜外侧;NKCC2只存在于肾脏,位于上皮细胞致密斑的顶膜上。Na-K-Cl协同转运蛋白的调控在不同的细胞和组织中是不同的。Na-K-Cl协同转运蛋白的活性会受激素刺激和细胞体积变化的影响;有些组织中,这种调控作用（尤其是NKCCl亚基）是通过特定的激酶使该转运蛋白自身发生氧化／硝化、磷酸化／去磷酸化来实现的;蛋白过表达在Na-K-Cl协同转运蛋白的激活中也起重要作用。     

The Na-K-Cl Cotransporters

The Na-K-Cl cotransporters are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. The transport process mediated by Na-K-Cl cotransporters is characterized by electroneutrality (almost always with stoichiometry of 1Na:1K:2Cl) and inhibition by the loop diuretics bumetanide, benzmetanide, and furosemide. Presently, two distinct Na-K-Cl cotransporter isoforms have been identified by cDNA cloning and expression; genes encoding these two isoforms are located on different chromosomes and their gene products share approximately 60% amino acid sequence identity. The NKCC1 (CCC1, BSC2) isoform is present in a wide variety of tissues; most epithelia containing NKCC1 are secretory epithelia with the Na-K-Cl cotransporter localized to the basolateral membrane. By contrast, NKCC2 (CCC2, BSC1) is found only in the kidney, localized to the apical membrane of the epithelial cells of the thick ascending limb of Henle's loop and of the macula densa. Mutations in the NKCC2 gene result in Bartter's syndrome, an inherited disease characterized by hypokalemic metabolic alkalosis, hypercalciuria, salt wasting, and volume depletion. The two Na-K-Cl cotransporter isoforms are also part of a superfamily of cation-chloride cotransporters, which includes electroneutral K-Cl and Na-Cl cotransporters. Na-K-Cl cotransporter activity is affected by a large variety of hormonal stimuli as well as by changes in cell volume; in many tissues this regulation (particularly of the NKCCl isoform) occurs through direct phosphorylation/dephosphorylation of the cotransport protein itself though the specific protein kinases involved remain unknown. An important regulator of cotransporter activity in secretory epithelia and other cells as well is intracellular [Cl] ([Cl]_i), with a reduction in [Cl]_i being the apparent means by which basolateral Na-K-Cl cotransport activity is increased and thus coordinated with that of stimulated apical Cl channels in actively secreting epithelia.     

Towards genomic and proteomic studies of protein phosphorylation in plant-pathogen interactions

Phosphorylation is an effective method of post-translational protein modification but understanding its significance is hindered by its biological complexity. Many protein kinases and phosphatases have been identified that connect signal perception mechanisms to plant defence responses. Recent studies of mitogen-activated protein kinases, calcium-dependent protein kinases and other kinases and phosphatases have revealed some important mechanisms, but have also raised new questions. The regulation of any phosphorylation pathway is complex and dynamic. There are many protein kinases and phosphatases in the plant genome, which makes it hard to delineate the phosphorylation machinery fully. Genomics and proteomics have already identified new components and will continue to influence the study of phosphorylation profoundly in plant-pathogen interactions.     

The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway

Volume-sensitive K-Cl cotransport occurs in red blood cells of many species. In intact cells, activation of K-Cl cotransport by swelling requires dephosphorylation of some cell protein, but maximal activity requires the presence of intracellular ATP. We have examined the relation between K-Cl cotransport activity and ATP in ghosts prepared from human red blood cells. K-Cl cotransport activity in swollen ghosts increased by ATP, and the increase requires Mg so that it almost certainly results from the phosphorylation of some membrane component. However, even in ATP-free ghosts residual volume-sensitive K-Cl cotransport can be demonstrated. This residual cotransport in ATP-free ghosts is greater in the presence of vanadate, a tyrosyl phosphatase inhibitor, and in ghosts that contain ATP cotransport is reduced by genistein, a tyrosyl kinase inhibitor. Okadaic acid, an inhibitor of serine and threonine phosphatases, inhibits K-Cl cotransport in ghosts as it does in intact cells. Experiments in which ghosts were preexposed to okadaic acid showed that the protein dephosphorylation that permits K-Cl cotransport can proceed to completion before the ghosts are swollen and K transport measured and therefore dephosphorylation is not a response to ghost swelling. In experiments with ATP-free ghosts we found that phosphorylation is not necessary to increase the cotransport rate when shrunken ghosts are swollen, nor is rephosphorylation necessary to decrease the cotransport rate when swollen ghosts are shrunken. Cotransport is greater in swollen than in shrunken ghosts even when the swollen and shrunken ghosts have the same concentration of cytoplasmic solutes. We conclude that, although phosphorylation and dephosphorylation modify the activity of the cotransporter in swollen and in shrunken ghosts, neither of these processes nor any other known messenger is involved in signal transduction between the cell volume sensor and the cotransporter as originally proposed by Jennings and Al- Rohil (Jennings, M. L., and N. Al-Rohil. 1990. Journal of General Physiology. 95: 1021-1040).     

Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells

The trabecular meshwork (TM) of the eye plays a central role inmodulating intraocular pressure by regulating aqueous humor outflow,although the mechanisms are largely unknown. We and others have shownpreviously that aqueous humor outflow facility is modulated byconditions that alter TM cell volume. We have also shown that theNa-K-Cl cotransport system is a primary regulator of TM cell volume andthat its activity appears to be coordinated with net efflux pathways tomaintain steady-state volume. However, the cellular mechanisms thatregulate cotransport activity and cell volume in TM cells have yet tobe elucidated. The present study was conducted to investigate thehypothesis that intracellular Cl concentration([Cl]_i) acts toregulate TM cell Na-K-Cl cotransport activity, as has been shownpreviously for some other cell types. We demonstrate here that thehuman TM cell Na-K-Cl cotransporter is highly sensitive to changes in[Cl]_i. Our findingsreveal a marked stimulation of Na-K-Cl cotransport activity, assessedas ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means ofreducing [Cl]_i. Incontrast, preincubation of cells with media containing elevated Kconcentrations as a means of increasing [Cl]_i results ininhibition of Na-K-Cl cotransport activity. The effects of reducing[Cl]_i, as well aselevating [Cl]_i, onNa-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced[Cl]_i is additive withcell-shrinkage-induced stimulation of the cotransporter. Our studiesalso show that TM cell Na-K-Cl cotransport activity is altered by avariety of Cl channel modulators, presumably through changes in[Cl]_i. These findingssupport the hypothesis that regulation of Na-K-Cl cotransport activity,and thus cell volume, by[Cl]_i may participatein modulating outflow facility across the TM.

     

On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases in intracellular signaling

Intracellular signaling proteins are very often regulated by site-specific phosphorylation. For example, growth factor receptors in eukaryotic cells contain intrinsic tyrosine kinase activity and use inter- and intra-molecular interactions to recruit and orient potential protein substrates for phosphorylation. Equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalysed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, SHP-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and we show how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.     

3H]bumetanide binding to avian erythrocyte membranes. Correlation with activation and deactivation of Na/K/2Cl cotransport

The relationship between Na/K/2Cl cotransport activation in duck erythrocytes and binding of the diuretic [3H]bumetanide to isolated membranes from stimulated cells has been assessed. Cotransport was activated by either cAMP-dependent (norepinephrine) or -independent (fluoride, hypertonicity) pathways. Membranes isolated from unstimulated cells possessed no specific bumetanide binding. In the presence of norepinephrine, cotransport and saturable binding rose in parallel, reaching a maximum after 5-7 min. In membranes from maximally stimulated cells the K1/2 and Bmax for bumetanide binding were 100 nM and 1.7 pmol/mg protein, respectively. The diuretic binding properties of these membranes were characteristic of interactions of ligands with the Na/K/2Cl cotransporter: specific binding required the presence of all three cotransported ions (Na, K, and Cl), and the rank order of potency for diuretic competition with bumetanide for binding sites was benzmetanide greater than bumetanide greater than furosemide. The appearance of specific bumetanide binding was also seen in membranes from erythrocytes activated by non-cAMP-dependent stimuli, with an excellent temporal correlation between cotransport activation and diuretic binding. On removal of all stimuli both cotransport and bumetanide binding declined in parallel. Duck erythrocytes treated with norepinephrine in a solution containing 15 mM K+ swell to a new stable cell volume after 60 min, during which time cotransport becomes inoperative. Bumetanide binding to both whole cells and isolated membranes paralleled the decline in cotransport activity. It is concluded that bumetanide binding to isolated membranes faithfully reflects the state of activation of the Na/K/2Cl cotransporter in intact cells under a variety of conditions.     

The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid

The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of     

Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

Regulation of cell adhesion by protein-tyrosine phosphatases: II. Cell-cell adhesion

Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. The stability of many adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly. The level of tyrosine phosphorylation reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. Many protein-tyrosine phosphatases associate with the cadherin-catenin complex, directly regulating the phosphorylation of these proteins, thereby affecting their interactions and the integrity of cell-cell junctions. Tyrosine phosphatases can also affect cell-cell adhesions indirectly by regulating the signaling pathways that control the activities of Rho family G proteins. In addition, receptor-type tyrosine phosphatases can mediate outside-in signaling through both ligand binding and dimerization of their extracellular domains. This review will discuss the role of protein-tyrosine phosphatases in cell-cell interactions, with an emphasis on cadherin-mediated adhesions.     

The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.     

Phosphoregulation of K+‐Cl− cotransporters during cell swelling: Novel insights

The K⁺‐Cl^? cotransporters (KCCs) belong to the cation‐Cl^? cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K⁺ and Cl^? ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl^?] (Cl_i), but the mechanisms at play are still ill‐defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine‐deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1‐related proline/alanine‐rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C‐terminus of full‐length KCC4 led to higher levels of heterologous K⁺‐Cl^? cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric‐sensitive kinase‐dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.     

Protein tyrosine phosphatases as negative regulators of mitogenic signaling

The regulation of tyrosine phosphorylation represents a key mechanism governing cell proliferation. In fibroblasts, inputs from both growth factor and extracellular matrix receptors are required for cell division. Triggering such receptors induces a wave of tyrosine phosphorylation on key signaling molecules, culminating in the activation of cyclin-dependent kinases and cell cycle progression. In general, protein tyrosine kinases stimulate, while protein tyrosine phosphatases inhibit, such cell proliferation pathways. The role of protein tyrosine kinases in mitogenesis has been extensively studied, but the identity and targets of the protein tyrosine phosphatases that regulate cell growth are not well described. In this review, I will survey recent advances in the identification and regulation of protein tyrosine phosphatases that downregulate cell proliferation. J. Cell. Physiol. 180:173–181, 1999. © 1999 Wiley-Liss, Inc.     

The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation.

We determined the relationship between the activation state and phosphorylation state of the Na-K-Cl cotransport protein in tubules isolated from the shark rectal gland, a prototypic chloride-secreting epithelium. In response to cAMP-dependent secretagogues (e.g. vasoactive intestinal peptide, adenosine, and forskolin) or osmotically induced changes in cell volume, the activation state of the cotransport protein (assessed from measurements of loop diuretic binding) increased 5-10 fold. The response was temporally associated with a comparable increase (3-9 fold) in cotransport protein phosphorylation. Graded changes in cotransporter activation evoked proportional changes in cotransporter phosphorylation. Under the conditions of our experiments, the 195-kDa cotransporter was the only membrane protein whose phosphorylation state increased conspicuously in response to both cAMP and cell shrinkage. Both stimuli promoted phosphorylation of the cotransport protein at serine and threonine residues. One of the cAMP-sensitive phosphoacceptors was found within a segment of the cotransport protein comprised of a sequence (Phe-Gly-His-Asn-Thr*-Ile-Asp-Ala-Val-Pro) that corresponds to a segment of the Na-K-Cl cotransport protein predicted by cDNA analysis, where the phosphoacceptor (Thr*) is threonine 189. Incubation of rectal gland tubules with K-252a or H-8, structurally different protein kinase inhibitors, rendered the cotransporter insensitive to both cAMP and cell shrinkage. We conclude that the rectal gland Na-K-Cl cotransport protein is regulated by direct reversible phosphorylation at serine and threonine sites.     

The sulfhydryl reagent N-ethylmaleimide induces hyperphosphorylation on tyrosine residues in the Jurkat T-cell line.

Tyrosine protein kinases have been shown to be functionally involved in regulation of cellular signalling, proliferation and transformation. The activity of tyrosine protein kinases is counterbalanced by phospho tyrosine phosphatases that maintain constitutively low levels of protein phosphotyrosine in most cells. In this study the effect of N-ethylmaleimide on the protein tyrosine phosphorylation was tested in Jurkat T-cells. Treatment of intact cells for 5-10 mins with 50-100 microM N-ethylmaleimide resulted in a dramatic increase in phosphorylation on tyrosine residues. Phosphoaminoacid analysis revealed an up to ten-fold increase in the content of phosphotyrosine. N-ethylmaleimide blocked the phospho tyrosine phosphatases activity of immunoprecipitated CD45 while in a kinase assay N-ethylmaleimide did not affect the 32P-gamma-ATP phosphorylation of substrates. The N-ethylmaleimide-induced hyperphosphorylation was reversed by treatment with 2 mM dithiotreitol. It is concluded that N-ethylmaleimide offers a novel useful tool for identification of substrates for tyrosine protein kinases and for studies on phosphotyrosine-dependent protein interactions.     

蛋白酪氨酸磷酸酶-PEST在生理调节及相关疾病中的作用

蛋白质分子中酪氨酸残基可逆性的磷酸化是细胞内信号分子传导的基本方式。两类作用相反的酶参与磷酸化的调节:蛋白酪氨酸激酶(protein tyrosinekinase,PTK)和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)。含脯氨酸-谷氨酸-丝氨酸-苏氨酸(P-E-S-T)结构域的蛋白酪氨酸磷酸酶(PTP-PEST)属于非受体型酪氨酸磷酸酶类,其本身能与多种蛋白质相互作用,并在细胞迁移、免疫细胞活化和胚胎发育等生理过程中发挥重要作用。本文对PTP-PEST的结构特点、生理功效、介导的信号传导途径和近年来PTP-PEST在疾病中的作用作一综述。     

Protein tyrosine phosphatases in cell transformation

The role of tyrosine phosphorylation in cell transformation has been well established. It has been proposed that protein tyrosine phosphatases (PTPases) may be capable of dephosphorylating critical substrates involved in the transformation process, suggesting that they represent a tumor suppressor family of enzymes. Indeed, recent work showed that overexpression of some PTPases in malignant cells counteracted the action of oncogenic tyrosine kinases although overexpression of other forms of these enzymes increased tumorigenicity. The work described herein has provided some insight into the action, both antagonistic and synergistic, of the kinases and phosphatases on cell growth and transformation.     

Docking interactions induce exposure of activation loop in the MAP kinase ERK2

MAP kinases bind activating kinases, phosphatases, and substrates through docking interactions. Here, we report a 1.9 A crystallographic analysis of inactive ERK2 bound to a "D motif" docking peptide (pepHePTP) derived from hematopoietic tyrosine phosphatase, a negative regulator of ERK2. In this complex, the complete D motif interaction defined by mutagenic analysis is observed, including extensive electrostatic interactions with the "CD" site of the kinase. Large conformational changes occur in the activation loop where the dual phosphorylation sites, which are buried in the inactive form of ERK2, become exposed to solvent in the complex. Similar conformational changes occur in a complex between ERK2 and a MEK2 (MAP/ERK kinase-2)-derived D motif peptide (pepMEK2). D motif peptides are known to bind homologous loci in the MAP kinases p38alpha and JNK1, also inducing conformational changes in these enzymes. However, the binding interactions and conformational changes are unique to each, thus contributing to specificity among MAP kinases.