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1.
Regulation of the cyclic activity of asparaginase (obtained as a purified protein complex) by a reversible auto-phosphorylation process has been previously reported in the fungus Leptosphaeria michotii (West) Sacc. In the present study, the protein complex was purified in the presence of either a mixture of 3 protein phosphatase inhibitors (fluoride, vanadate and molybdate) or EGTA, during the cycle of asparaginase activity, and the protein kinase and protein phosphatase activities characterized. (I) At the phase of increasing asparaginase activity, a Ca2+/calmodulin-dependent kinase activity was identified by (a) its inhibition by calmidazolium, reversed by calmodulin, and its inhibition by EGTA, but not by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole or polylysine (b) an increasing level of calmodulin bound to the complex, as estimated by enzyme-linked immunosorbent assay (ELISA). (2) At the phase of decreasing asparaginase activity, the Ca2+-calmodulin-dependent kinase activity disappeared and a little calmodulin remained associated with the complex: phosphorylation of the complex was increased several-fold by 1 nM okadaic acid and 25 nM inhibitor-2, and was not affected by EGTA, indicating a protein phosphatase-2A-like activity. (3) When asparaginase activity was low, a little calmodulin was bound to the complex. The kinase could phosphorylate casein and phosvitin. was inhibited by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole and heparin, stimulated by polylysine and not affected by calmidazolium or EGTA, just as a casein kinase 2. A Ca2+-dependent but calmodulin-independent protein phosphatase activity, not affected by okadaic acid and inhibitor-2. was then identified. We postulate the presence in the complex, of (a) only one protein kinase and one protein phosphatase, whose properties could change during the cycle of asparaginase activity: (b) two Ca2+/-binding proteins: first calmodulin, which could bind to Ca2+ and the casein kinase-2 form to give a Ca2+/calmodulin-dependent kinase, which could become Ca2+/calmodulin-independent following an auto-phosphorylation process: second a protein homologous to calmodulin, able to bind to the protein phosphatase-2A catalytic subunit to give a protein phosphatase-2B catalytic subunit. 相似文献
2.
Involvement of protein kinase C in the phosphorylation of an insulin-granule membrane protein. 总被引:2,自引:2,他引:0
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The involvement of protein kinase C in the Ca2+-dependent phosphorylation of a 29 000-Mr insulin-granule membrane protein prepared from a rat insulinoma was investigated. Protein kinase C activity towards exogenous lysine-rich histone was detected in a cytosolic fraction prepared from an insulinoma homogenate in the presence of EGTA. This activity bound reversibly to insulin granules in a Ca2+-dependent manner. Phosphatidylserine liposomes removed both protein kinase C activity and the 29 000-Mr protein-phosphorylating activity from the cytosolic fraction in a Ca2+-dependent fashion. Protein kinase C activity and the enzymic activity responsible for the phosphorylation of the 29 000-Mr granule protein behaved identically on sucrose-density-gradient centrifugation, ion-exchange chromatography, (NH4)2SO4 fractionation and gel filtration of the cytosolic fraction. These results are consistent with protein kinase C being the enzyme responsible for the phosphorylation of the 29 000-Mr insulin-granule membrane protein. 相似文献
3.
4.
Regulation of the dual specificity protein phosphatase, DsPTP1, through interactions with calmodulin
Yoo JH Cheong MS Park CY Moon BC Kim MC Kang YH Park HC Choi MS Lee JH Jung WY Yoon HW Chung WS Lim CO Lee SY Cho MJ 《The Journal of biological chemistry》2004,279(2):848-858
Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation. 相似文献
5.
Involvement of calcium-dependent protein kinase in rice (Oryza sativa L.) lamina inclination caused by brassinolide 总被引:12,自引:0,他引:12
Promotive effect of brassinolide (BL) on green lamina inclination was concentration-dependent when excised rice (Oryza sativa L.) lamina was floated on BL solution under continuous light conditions. Protein kinase inhibitor staurosporine and Ca2+ channel blocker LaCl3 could completely, while Ca2+ chelator EGTA could partially inhibit the lamina inclination caused by BL. Two protein kinases with apparent molecular masses of 45 and 54 kDa were detected using an in-gel kinase assay with histone III-S as a substrate. In particular, the changes in 45 kDa protein kinase activity correlated with lamina inclination caused by BL. The 45 kDa kinase activity was inhibited by Ca2+ chelator EGTA, protein kinase inhibitor, staurosporine and calmodulin antagonist W-7. Therefore, this 45 kDa protein kinase was identified as a Ca2+ -dependent protein kinase (CDPK). Patterns of 2-dimensional PAGE after in vitro phosphorylation of crude extracts showed that the phosphorylation of 56 and 41 kDa proteins, which was Ca2+ -dependent, was strongly increased by BL treatment. These results suggested that CDPK and Ca2+ -dependent protein phosphorylation are involved in BL-induced rice lamina inclination. 相似文献
6.
Purified Ca2+-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S6 when incubated with 40S ribosomal subunits from rat liver or from hamster fibroblasts. The phosphorylation was dependent on Ca2+ and phospholipid, and occurred under ionic conditions similar to those which support protein biosynthesis in vitro. Protein kinase C phosphorylated at least three sites on ribosomal protein S6 when incubated with unphosphorylated ribosomes, and increased the extent of phosphorylation of ribosomes previously phosphorylated predominantly on two sites by cyclic-AMP-dependent protein kinase, converting some molecules to the tetraphosphorylated or pentaphosphorylated form. This indicates that protein kinase C can phosphorylate sites on ribosomal protein S6 other than those phosphorylated by the cyclic-AMP-dependent protein kinase, and this conclusion was confirmed by analysis of tryptic phosphopeptides. These results strengthen the possibility that protein kinase C might be involved in catalysing the multisite phosphorylation of ribosomal protein S6 in certain circumstances in vivo. 相似文献
7.
The properties of L-asparaginase (EC 3.5.1.1) in Leptosphaeria michotii (West) Sacc., which has previously been shown to have an activity rhythm, were analyzed. Two forms of L-asparaginase were isolated from acetic acid and ammonium sulfate fractionations followed by DEAE-Sephacel chromatography. The activity of L-asparaginase changed rhythmically with the same period as that of crude extracts, but the rhythms of the two enzyme forms were out of phase. The two asparaginase forms differed in their isoelectric points and the substrate concentrations for attaining half-maximal velocity; non-Michaelis-Menten kinetics for hydrolysis of L-asparagine were observed. Analyses of asparaginase form II by polyacrylamide gel electrophoresis showed that four proteins, irrespective of the phase of the activity rhythm at which the enzyme was extracted, could be detected: asparaginase oligomer (Mr 130 000 to 140 000), its dimer, an aggregate (Mr 500 000 to 600 000) having a low asparaginase activity, and a protein (Mr 60 000) without asparaginase activity; the same proteins were found in asparaginase form I. These results indicate that L. michotii asparaginase could be implicated in a protein complex. 相似文献
8.
The ciliated protozoan Paramecium tetraurelia contained two protein kinase activities that were dependent on Ca2+. We purified one of the enzymes to homogeneity by Ca2+-dependent affinity chromatography on phenyl-Sepharose and ion exchange chromatography. The purified enzyme contained polypeptides of 50 and 55 kDa, with the 50-kDa species predominant. From its Stokes radius (32 A) and sedimentation coefficient (3.9 S), we calculated a native molecular weight of 51,000, suggesting that the active form is a monomer. Its specific activity was 65-130 nmol X min-1 X mg-1 and the Km for ATP was 17-35 microM, depending on the exogenous substrate used. Kinase activity was completely dependent upon Ca2+; half-maximal activation occurred at approximately 1 microM free Ca2+ at pH 7.2. Phosphatidylserine and diacylglycerol did not stimulate activity, nor did the addition of purified Paramecium calmodulin. The enzyme phosphorylated casein and histones, forming primarily phosphoserine and phosphothreonine, respectively. It also catalyzed its own phosphorylation in a Ca2+-dependent reaction; the half-maximal rate of autophosphorylation occurred at approximately 1-1.5 microM free Ca2+, and both the 50- and 55-kDa species were autophosphorylated. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation in situ, the 50-kDa protein retained its Ca2+-dependent ability to phosphorylate casein, suggesting that Ca2+ interacts directly with this polypeptide. This was confirmed by direct binding studies; when the enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis transferred to nitrocellulose, and renatured, there was 45Ca2+-binding in situ to both the 50- and 55-kDa polypeptides. The Paramecium enzyme appears to be a new and unique type of Ca2+-dependent protein kinase. 相似文献
9.
Specificity of protein phosphatases in the dephosphorylation of protein kinase C. 总被引:2,自引:2,他引:0
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Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed. 相似文献
10.
The phosphorylation of the whole troponin complex and of the cardiac and skeletal troponin components by Ca2+-phospholipid-dependent protein kinase was studied. The activity of enzyme isolated from rat brain by ion-exchange chromatography on DEAE-Sephadex and by affinity chromatography on phosphatidylserine immobilized on polyacrylamide gel was shown to be completely dependent on Ca2+ and phospholipids and was equal to 0.4-0.6 mumol of phosphate/min.mg protein with histone H1 as substrate. The resulting preparation of Ca2+-phospholipid-dependent protein kinase was able to phosphorylate the isolated troponin I; the amount of phosphate transferred per mol of cardiac and skeletal troponin I was equal to 1.1 and 0.4, respectively. The maximal degree of phosphorylation of isolated troponin T by Ca2+-phospholipid-dependent protein kinase was 0.6 mol of phosphate per mol of troponin T both for skeletal and cardiac proteins. The rate and degree of phosphorylation were independent of the initial level of troponin T phosphorylation. Ca2+-phospholipid-dependent protein kinase did not phosphorylate the first serine residue of troponin T, i.e., the site which was phosphorylated in the highest degree after isolation of troponin T from skeletal muscles. The data obtained and the fact that the rate and degree of phosphorylation of troponins I and T within the whole troponin complex are 10-20 times less than those for isolated components provide little evidence for the participation of protein kinase C in troponin phosphorylation in vivo. 相似文献
11.
T Nishizaki J H Walent J A Kowalchyk T F Martin 《The Journal of biological chemistry》1992,267(33):23972-23981
Cytoplasmic Ca2+ is a major regulator of exocytosis in secretory cells; however, the Ca(2+)-dependent mechanisms that trigger secretion have not been elucidated. Protein kinase C (PKC) has been proposed to be an important Ca(2+)-dependent component of this regulation; however, the effects of this enzyme on the exocytotic apparatus have not been identified. We developed a PKC-deficient, semi-intact PC12 cell system in which direct stimulatory effects of purified PKC on Ca(2+)-dependent norepinephrine secretion were studied. The reconstitution of optimal Ca(2+)-activated norepinephrine secretion by semi-intact PC12 cells required the addition of MgATP and cytosolic proteins. PKC-deficient cytosol exhibited reduced reconstituting activity that was fully restored by the addition of purified PKC. The restoration of Ca(2+)-dependent norepinephrine secretion by PKC required the presence of other proteins in the cytosol, in particular, a high molecular weight protein. The high molecular weight protein was identified as p145, a recently characterized 145-kDa brain protein. The addition of PKC enhanced phosphorylation of p145 under conditions of fully reconstituted Ca(2+)-activated norepinephrine secretion. The results indicate that 1) PKC is neither necessary nor sufficient for Ca(2+)-activated secretion, whereas other cytosolic proteins are required; and 2) the stimulation of Ca(2+)-activated secretion by PKC is dependent upon cytosolic proteins such as p145 and may be largely mediated through the phosphorylation of p145. 相似文献
12.
Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase
Reinert RB Oberle LM Wek SA Bunpo P Wang XP Mileva I Goodwin LO Aldrich CJ Durden DL McNurlan MA Wek RC Anthony TG 《The Journal of biological chemistry》2006,281(42):31222-31233
L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal. 相似文献
13.
Activation of protein kinase in the bovine corpus luteum by phospholipid and Ca2+. 总被引:4,自引:1,他引:3
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A new species of protein kinase has been identified in cytosol preparations from bovine corpora lutea. Enzyme activity required the simultaneous presence of Ca2+ and phospholipid, and was also enhanced by glyceryl dioleate. Phosphatidylserine was the most effective phospholipid for stimulating histone phosphorylation. Other phospholipids capable of supporting enzymic activity were, in order of decreasing activity, phosphatidylinositol, phosphatidic acid, cardiolipin and phosphatidylglycerol. Several other phospholipids tested were ineffective. A cyclic AMP-dependent protein kinase was also present in the luteal cytosol. This enzyme activity was eliminated by protein kinase inhibitor without affecting the Ca2+- and phospholipid-stimulated activity. Lysine-rich histone (IIIS) was a much better substrate than type-IIA histone for Ca2+- and phospholipid-dependent phosphorylation. Ca2+ and phospholipid also enhanced phosphorylation of endogenous luteal cytosol protein. Calmodulin, alone or in the presence of Ca2+, was unable to increase phosphorylation. Trifluoperazine inhibited protein kinase activity stimulated by Ca2+ and phospholipid. These data suggest that a phospholipid-sensitive, Ca2+-dependent protein kinase may provide an important link between hormonally-induced changes in phospholipid metabolism and corpus-luteum function. 相似文献
14.
Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis 总被引:1,自引:0,他引:1
Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis. Gossypol inhibited Ca2+-dependent activity of the enzyme without affecting its basal activity. The IC50 value (concentration causing 50% inhibition) was 31 microM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (Ki = 31 microM) or lysine-rich histone (Ki = 30 microM), and competitively with respect to phosphatidylserine (Ki = 2.1 microM). With Ca2+, irrespective of the presence or absence of 1,3-diolein, the compound lowered Vmax and increased the apparent Ka for Ca2+. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrates in the testis for the enzyme located in nucleosome), with an IC50 value of 88 microM. These results suggested that a phospholipid-sensitive Ca2+-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis. 相似文献
15.
Hormone-induced protein phosphorylation. III. regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMP in vitro 总被引:2,自引:1,他引:1
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In the preceding papers, we demonstrated that the endogenous phosphorylation of a 29,000-dalton protein is stimulated in response to secretagogue application to intact cells from the rat exocrine pancreas and parotid and dephosphorylated upon termination of secretagogue action. One- and two-dimensional gel analysis of 32Pi-labeled pancreatic and parotid lobules as well as their respective subcellular fractions revealed that the same protein was covalently modified in both tissues and was localized to the ribosomal fraction. To identify the intracellular second messengers which may mediate or modulate the phosphorylation of the 29,000-dalton protein in intact cells, the effects of Ca2+, cAMP, and cGMP on the endogenous phosphorylation of this protein were assessed in subcellular fractions from the rat pancreas and parotid. Our results demonstrate that the phosphorylation of the 29,000-dalton polypeptide may be regulated by both Ca2+ and cAMP in the pancreas and in the parotid. No cGMP-dependent protein phosphorylation was found in either tissue. As in the in situ phosphorylation studies, the Ca2+- and cAMP-dependent phosphorylation of this same protein was localized to the ribosomal fraction. The cAMP-dependent protein kinase activity was found primarily in the postmicrosomal supernatant in contrast to the Ca2+-dependent protein kinase that appeared to be tightly associated with the substrate in addition to being present in the postmicrosomal supernatant. The data suggest that, in cells from the exocrine pancreas and parotid, secretagogues may regulate the phosphorylation of the 29,000-dalton protein through Ca2+ and/or cAMP. 相似文献
16.
Franz-Wachtel M Eisler SA Krug K Wahl S Carpy A Nordheim A Pfizenmaier K Hausser A Macek B 《Molecular & cellular proteomics : MCP》2012,11(5):160-170
Protein kinase D (PKD) is a cytosolic serine/threonine kinase implicated in regulation of several cellular processes such as response to oxidative stress, directed cell migration, invasion, differentiation, and fission of the vesicles at the trans-Golgi network. Its variety of functions must be mediated by numerous substrates; however, only a couple of PKD substrates have been identified so far. Here we perform stable isotope labeling of amino acids in cell culture-based quantitative phosphoproteomic analysis to detect phosphorylation events dependent on PKD1 activity in human cells. We compare relative phosphorylation levels between constitutively active and kinase dead PKD1 strains of HEK293 cells, both treated with nocodazole, a microtubule-depolymerizing reagent that disrupts the Golgi complex and activates PKD1. We identify 124 phosphorylation sites that are significantly down-regulated upon decrease of PKD1 activity and show that the PKD target motif is significantly enriched among down-regulated phosphorylation events, pointing to the presence of direct PKD1 substrates. We further perform PKD1 target motif analysis, showing that a proline residue at position +1 relative to the phosphorylation site serves as an inhibitory cue for PKD1 activity. Among PKD1-dependent phosphorylation events, we detect predominantly proteins with localization at Golgi membranes and function in protein sorting, among them several sorting nexins and members of the insulin-like growth factor 2 receptor pathway. This study presents the first global detection of PKD1-dependent phosphorylation events and provides a wealth of information for functional follow-up of PKD1 activity upon disruption of the Golgi network in human cells. 相似文献
17.
Tyrosine phosphorylation of a 55- and 60-kDa protein was observed when EDTA-treated P2 membrane fraction from monkey basal ganglia was incubated with [gamma-32P]-ATP in the presence of Zn2+. Other metal ions were less effective in this phosphorylation. The effect of Zn2+ did not appear to be due to its inhibition of a tyrosine phosphatase. In the presence of Mg2+/Triton X-100 instead of Zn2+, phosphorylation on tyrosine residues of a 17-kDa protein and the external substrate poly(Glu, Tyr) 4:1 copolymer was observed. Both Mg2+ and Triton X-100 were essential for this and Zn2+ inhibited both of these phosphorylations. Convincing evidence for the existence of Zn2+-dependent and Mg2+/Triton X-100-dependent tyrosine protein kinases was obtained when the two kinases could be separated by extraction of the membranes by Triton X-100. The Zn2+-dependent phosphorylation was present exclusively in the Triton-solubilized supernatant whereas the Mg2+/Triton X-100-dependent phosphorylation was found associated with the Triton-insoluble membrane fractions. Externally added histone could also be phosphorylated on tyrosine residues in a Zn2+- or Mg2+/Triton X-100-dependent manner by the supernatant or membrane fraction, respectively. 相似文献
18.
Activation of tyrosine hydroxylase by micromolar concentrations of calcium in digitonin-permeabilized adrenal medullary cells 总被引:1,自引:0,他引:1
N Yanagihara Y Uezono Y Koda A Wada F Izumi 《Biochemical and biophysical research communications》1987,146(2):530-536
In digitonin-permeabilized bovine adrenal medullary cells, Ca2+ (0.1-1.0 microM) caused an activation of tyrosine hydroxylase which was dependent on the presence of ATP. This Ca2+-induced activation of the enzyme was observed even in the presence of optimal concentration of either cyclic AMP or 12-O-tetradecanoylphorbol-13-acetate (TPA) which by itself increased the enzyme activity. Calmodulin inhibitors, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W-7), had little effect on the Ca2+-evoked activation of enzyme. These results suggest that micromolar concentrations of Ca2+ activate the activity of tyrosine hydroxylase probably through a Ca2+-dependent phosphorylation in digitonin-permeabilized adrenal medullary cells although the protein kinase(s) responsible for it still remains to be determined. 相似文献
19.
Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition. 相似文献
20.
Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction. 相似文献