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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia
Submitted 19 July 2007 ; accepted in final form 13 October 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Src kinase; SH2; SH3; peroxynitrite; tyrosine; calcium channel
-subunit with c-Src (9) and tyrosine phosphorylation has also been demonstrated for the smooth muscle calcium channel (7, 11). The regulation of the calcium channel by c-Src kinase appears to be altered during inflammation. In colonic inflammation, Ca2+ currents and contractile force in smooth muscle are significantly attenuated. Our laboratory (2) and others (13, 15) have previously demonstrated that Ca2+ currents are reduced by almost 70% in experimental colitis. Interestingly, in inflamed cells, the ability of Src to modulate calcium channels is also reduced, thereby affecting calcium-mediated contractions (12, 18). Recently, our laboratory showed that this may arise due to nitration of tyrosine residues of the calcium channel during colonic inflammation, which decreases the ability of Src-dependent regulation of the calcium channel (18). Nitrosylation of tyrosine involves a covalent modification, resulting in the addition of a nitro group onto the ortho carbon of the tyrosine residue (17). Nitrotyrosine formation has been established in several pathophysiological states, including inflammatory bowel disease (20). Peroxynitrite (ONOO–), formed from the reaction of nitric oxide with superoxide anion radical, is thought to be the major nitrating agent in in vivo and in vitro chemical studies and can disable the tyrosine phosphorylating regulatory mechanisms by nitrating the tyrosine residues of the target proteins (14).
The carboxyl terminus of the calcium channel appears to be the major site for Src regulation. The mechanism by which Src enhances calcium currents is not entirely clear, but may involve conversion of the channel into a second open state, resulting in increased availability (16). The protein-protein interaction between c-Src and the calcium channels may involve both binding through the SH3 and SH2 domains of Src, as well as direct phosphorylation of the COOH terminus. Consensus sequences within the COOH-terminal region of the calcium channel for the SH3 domain include a proline-rich domain (PRD), whereas binding to SH2 domain requires phosphorylated tyrosine residues. Bence-Hanulec et al. (3) and Gui et al. (7) demonstrated Src-mediated IGF-I-induced potentiation and integrin-mediated enhancement of the rat neuronal 1c, respectively, which required the phosphorylation of the tyrosine 2122 within the carboxyl terminus. However, the sequence alignment of the human smooth muscle calcium channel (GenBank accession no. AAM70049) COOH-terminal region against the rat neuronal 1c (GenBank accession no. AAA18905) shows that this particular tyrosine is replaced by arginine within the consensus region EDESCVY2122 (see 
Fig. 5A). This raises the question of whether similar regulation may occur in human smooth muscle and whether alteration of the tyrosine residues through nitrosylation can affect calcium channel function of the human smooth muscle isoform.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1c-subunit of human jejunal Ca2+ channel and mutants using the Genejemmer transfection reagent (Invitrogen). Forty-eight hours posttransfection, cells were washed in phosphate-buffered saline (PBS) and solubilized in RIPA buffer (Santa Cruz), supplemented with protease inhibitors containing 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml Calpain I, 10 µg/ml Calpain II, and 0.1 mM sodium orthovanadate. After incubation for 30 min on ice, cell debris was pelleted by centrifugation (10,000 g, 10 min, 4°C). The supernatant was aliquoted and stored at –80°C. Membrane proteins were prepared by Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce), according to the manufacturer's protocols. Protein concentration was determined by the BCA Protein Assay Kit (Pierce) before use in the experiments. The protein samples were incubated with anti-voltage-gated calcium channel (Cav) 1.2 antibody at 4°C overnight. The immunoprecipitates were washed with wash buffer (20 mM Na2HPO4, pH 7.4, 150 mM NaCl, and 0.1% Triton X-100) and analyzed using SDS-PAGE gel and immunoblots with anti-pY20 antibody (Santa Cruz) or anti-nitrotyrosine antibody (Santa Cruz) at concentration of 1:500 to 1:1,000.
Glutathione S-transferase fusion protein construction and preparation.
The COOH terminus fragments of human 1c-subunit, SH2 and SH3 binding domains of mouse c-Src, were subcloned into the pGEX-6P-3 (Pharmacia Amersham) to generate the fusion proteins glutathione S-transferase (GST)-CT (amino acids 1809-2138), GST-SH2 (150–247), and GST-SH3 (83–144). The sense primer was designed to contain a BamHI site in the 5' end, and the antisense primer was designed to contain an EcoRI site at the 5' end. The resultant PCR product was subcloned in frame with the GST open frame of the bacterial expression plasmid pGEX-6P-3 between BamHI and EcoRI sites. GST-
PRD fusion proteins were generated from GST-CT as a template DNA using the site-directed mutagenesis kit (Stratagene). Mutations of the GST fusion proteins and wild type of human 1c (hCav12.b) were also constructed by PCR using the site-directed mutagenesis kit. All constructs were confirmed by DNA sequencing. All primers in this study were designed using Vector NTI software (InforMax). The sequence of the primers used are shown in Table 1.
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GST pull-down and immunoblots.
GST pull-down experiments were performed by incubating 50-µg GST fusion proteins with 3 µl of purified c-Src (3 U/µl) in 1-ml cell lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM EDTA, pH 8.0, 1% Triton X-100). Twenty microliters of 50% slurry of glutathione sepharose beads were added, and the mixture was incubated for 3 h at 4°C, and then beads were centrifuged at 10,000 g for 2 min. The pellet was washed three times with cell lysis buffer, and the beads were pelleted after repeating the steps of centrifugation and washing. Twenty microliters of SDS loading buffer were added, and the mixture was heated 5 min at 100°C before loading on SDS-PAGE gel. Standard protocols for Western blots were performed using site-specific antibodies. Protein samples were run on 5% and 10% SDS-PAGE and transferred on either nitrocellulose or polyvinylidene difluoride membrane (Bio-Rad). Membranes were blocked with 3% nonfat milk blocking buffer with 0.1% Tween 20. Anti-1c antibodies (BD Biosciences Pharmingen), anti-c-Src antibodies (Santa Cruz), anti-pY20 antibodies (Santa Cruz), anti-GST antibodies (Santa Cruz), and anti-nitrotyrosine antibodies (Calbiochem) were used for blotting at concentration of 1:200 to 1:500. The membranes were then incubated with secondary antibodies at concentration of 1:1,000 to 1:5,000 and visualized using either the enhanced chemiluminescence plus Western blot detection system (Amersham) or the Odyssey Infrared Imaging System (LI-COR Biosciences).
In vitro phosphorylation.
GST-CT and GST-PRD were phosphorylated with purified c-Src kinase for in vitro phosphorylation. Twenty micrograms of GST fusion proteins were incubated with 3 µl of purified c-Src (3 U/µl) in 1x Src kinase buffer (Upstate Biotechnology) at room temperature for 1 h. SDS-polyacrylamide gel electrophoresis and immunoblots with anti-pY20 antibody after GST pull-down were then performed using standard methods.
Overlay binding. Fifty-microgram protein samples were loaded onto SDS-PAGE gels and were transferred onto nitrocellulose membrane. After blocking with 5% (wt/vol) nonfat milk blocking buffer, filter strips were cut and incubated with GST-SH3 or GST-SH2 fusion protein (0.1 µg/ml) in blocking buffer for 1 h or overnight at 4°C. Filter strips were then washed twice with PBS and once with PBS buffer containing 0.1% (vol/vol) Tween 20. GST-SH3 and GST-SH2 protein bound to filters were probed using anti-GST antibody.
In vitro nitrosylation by ONOO–. Nitrosylation by ONOO– was achieved as described previously (18). GST-fusion proteins or the cleaved fusion proteins were incubated in 3-morpholinosydnonimine (SIN-1) (500 µM, Tocris) for 1 h followed by ONOO– (150 µM, Cayman Chemical) three times at 4-min intervals and washed with calcium-free high-K+ physiological saline solution.
Pretreated sample proteins were incubated in 0.5-ml GST binding buffer and incubated at room temperature for 1 h. Fifty-microliter glutathione separose beads were added and incubated for 30 min. The pellet was centrifuged at 500 g for 5 min and washed three times with PBS buffer. SDS loading buffer was added and then subjected to SDS-PAGE.
Electrophysiological recording.
Electrophysiological studies were performed on HEK-293 cells transfected with human jejunal smooth muscle 1c-subunit (hCav1.2b) wild type or mutated at specific tyrosine residues together with β2-subunit. Ca2+ currents were recorded at room temperature using EPC 10 patch-clamp amplifier (HEKA). Patch micropipettes with resistances of 3–5 M
were pulled from borosilicate glass capillaries on a Flaming-Brown P97 (Sutter Instruments) electrode puller. The cells were continuously perfused with HEPES-buffered physiological salt solution (135 mM NaCl, 5.4 mM KCl, 0.33 mM NaH2PO4, 5 mM HEPES, 1 mM MgCl2, 20 mM BaCl2, and 5.5 mM glucose: pH 7.4 with NaOH) and the internal pipette solution containing 100 mM cesium aspartate, 30 mM CsCl, 2 mM MgCl2, 5 mM HEPES, 5 mM EGTA, and 5 mM adenosine triphosphate (disodium salt) (pH 7.2 with CsOH). Pulse generation and data acquisition were performed using Patchmaster version 2.15 software (HEKA). The cells were voltage clamped at –60 mV, and membrane currents were recorded at a voltage step to +20 mV over a period of 500 ms. After basal recording, the cells were perfused with SIN-1 (10 µM; 15 min) and sodium ONOO– (150 µM) for 15 min and washed with HEPES-buffered physiological salt solution before the Ca2+ currents were recorded again. The inhibition of Ca2+ currents was calculated as a percentage of the initial peak current.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To determine the effect of nitration on the regulation of calcium currents by c-Src kinase, hCav1.2b transfected cells were subjected to whole cell voltage clamp. As demonstrated previously (11, 12, 24), calcium currents were significantly reduced by the Src kinase inhibitor PP2 (10 µM) by 58 ± 5% (Fig. 2). When cells were pretreated with ONOO–, the currents were inhibited by 64 ± 6%, and additional perfusion with PP2 did not further reduce the calcium currents (73 ± 6%) (Fig. 2, B and C).
The data above suggest that hCav1.2b is regulated by c-Src kinase in a nitrosylation-sensitive manner.
Phosphorylation by c-Src kinase is required for the interaction between the COOH terminus of Ca2+ channel 1c-subunit and Src-kinase SH2 domain.
To determine the binding sites and the protein-protein interactions, GST fusion proteins were constructed for the COOH terminal region of hCav1.2b. c-Src kinase has two potential protein interacting domains, SH2 and SH3. The SH2 domain recognizes phosphotyrosine, and SH3 binds to proline-rich regions. Consensus sites from both domains are present in the COOH-terminus region of the smooth muscle L-type Ca2+ channels. GST fusion proteins were constructed by PCR of the distal portion of the COOH terminus (amino acids 1809-2138) and with deletion of PRD (PRD) within this region (Fig. 3A). c-Src SH2 and SH3 binding domain were also constructed from mouse c-Src kinase. The GST fusion proteins were incubated with purified c-Src kinase and subjected to immunoblots after pull-down with glutathione sepharose beads. Immunoblot with anti-c-Src antibody demonstrated pull-down of c-Src with GST-CT but was consistently less with PRD (n = 3) (Fig. 3B). We next examined whether the PRD is required for the interaction with c-Src kinase. Overlay binding assay of CT and PRD cleaved from the GST tag were performed with GST-SH2 and GST-SH3 fusion proteins. Immunoblots with anti-GST antibodies demonstrated that CT bound to the SH3 domain of c-Src but not to SH2 domain (Fig. 3C, top). Neither SH2 nor SH3 fusion proteins bound to PRD. To determine whether phosphorylation by Src kinase is necessary for the interaction of COOH terminus of 1c-subunit with SH2 domain, cleaved CT and PRD were phosphorylated by c-Src kinase in the presence of kinase buffer and subjected to overlay binding assays with GST-SH2 and GST-SH3 fusion protein. Immunoblots with anti-GST antibody showed that, following phosphorylation, GST-SH2 binding was detected for both CT and PRD (Fig. 3C, bottom). Phosphorylation of tyrosine residues by purified c-Src kinase was confirmed by immunoblot with anti-pY20 antibody (Fig. 3D). These data suggest that phosphorylation of tyrosine residues is necessary for the interaction with the SH2 domain.
Effect of ONOO– on the interaction between COOH terminus of Ca2+ channels and SH2 binding. To test whether nitrosylation of the COOH terminus affects SH2 binding, overlay binding and pull-down assays were performed. Cleaved CT fragment were treated with c-Src kinase (in kinase buffer) in the absence and presence of ONOO–, incubated with GST-SH2, and subjected to pull-down assay. Figure 4A shows the native SDS-PAGE gel whereby the CT fragment could be pulled down by GST-SH2 following Src kinase phosphorylation, but not when pretreated with ONOO–. Figure 4B shows that ONOO– prevented phosphorylation of the CT fragments by c-Src kinase. GST-CT fusion proteins were incubated with c-Src kinase (in kinase buffer) and subjected to pull-down. This was followed by immunoblot with anti-phosphotyrosine and ant-nitrotyrosine antibodies. Tyrosine phosphorylation due to c-Src kinase was downregulated by ONOO– and resulted in nitrotyrosine formation.
Within the distal 330-amino acid COOH terminus of human jejunal 1c, three tyrosine residues (Y1837, Y1861, and Y2134) that are potential sites for phosphorylation were mutated individually to phenylalanine (F) by site-directed mutagenesis (Fig. 5). Full-length mutants were transfected into HEK-293 cells and subjected to pull-down with GST-SH2 to determine which phosphorylated tyrosine residue is critical for SH2 binding. Sequence alignment of the rat neuronal and the human jejunum 1c shows that the potential SH2 binding previously shown for the rat neuron, Y2122, is replaced by an arginine in the human isoform (Fig. 5A). Figure 6A shows that, of the three mutants, only Y2134F did not pull down with GST-SH2, suggesting that this residue was important for SH2 binding. When treated with ONOO–, mutants Y1837F and Y1861F failed to bind SH2, suggesting that nitrosylation of Y2134 prevents interaction with SH2 domain. Interestingly, immunoblot with anti-nitrotyrosine antibody showed that all three mutants were equally nitrosylated (Fig. 6B), suggesting that, while Y2134 may be important for SH2 binding, other tyrosine residues can be nitrosylated. Decreased tyrosine phosphorylation of Y2134 was further confirmed in cells cotransfected with c-Src. The calcium channel was immunoprecipitated with anti-Cav1.2 antibody and immunoblotted with anti-phosphotyrosine antibody (Fig. 6C). The Y2134 mutation reduced tyrosine phosphorylation that was prevented by ONOO– treatment. That tyrosine phosphorylation was not completely abolished by the Y2134F mutation suggests that other tyrosine residues may also be phosphorylated. To test this, we constructed a combination of double and triple mutants in the CT fragment. Of the various double-mutant combinations, Y1837-2134F prevented tyrosine phosphorylation, as did the triple mutations (Fig. 7).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1c was mediated by c-Src kinase-dependent phosphorylation of the tyrosine Y2122. This finding was confirmed by Gui et al. (7), who showed integrin-mediated enhancement required phosphorylation of Y2122. As shown in Fig. 1, sequence alignment demonstrates that this particular residue is absent in human jejenum smooth muscle isoform and, therefore, raised the question of whether similar regulation as that seen in mouse models of inflammation may occur in the human. This is of particular relevance, as disruption of Src kinase regulation due to nitration of tyrosine residues results in decreased calcium influx affecting contraction. Nitrotyrosylation has been shown to occur in inflammatory bowel disease (20) and may underlie the decreased motility during colonic inflammation (18). Evidence that smooth muscle calcium channels from several tissue types are regulated by Src kinase under basal unstimulated conditions come from 1) inhibition of calcium currents and depolarization-induced calcium influx by the Src kinase inhibitor PP2; 2) enhancement of the currents by the tyrosine phosphatase inhibitor sodium orthovanadate; 3) cotransfection with v-Src enhances calcium currents; and 4) coimmunoprecipitation of the calcium channels with Src kinase (8, 9, 11, 22). Similar to these previous studies, hCav1.2b was also found to be under basal regulation of Src kinase. This was determined by inhibition of the calcium currents by the Src kinase inhibitor PP2. Treatment with ONOO– also resulted in a decrease in the calcium currents and prevented the effects of PP2, thus defining the modulatory role of Src kinase.
Src kinase contains two protein binding domains, SH2 and SH3, that recognize specific consensus sequences within the target protein. The SH3 domain binds to proline-rich regions, which, in the case of hCav1.2b, consists of PPQP (amino acids 1965-1968). Deletion of this region resulted in decreased Src and GST-SH3 binding. However, the binding to SH3 domain was not prerequisite for the SH2 interaction with phosphorylated tyrosine. The PRD is functionally important in membrane tethering the COOH terminus of the calcium channel (6). It has been known since the early work on the cardiac L-type calcium channel (21) that truncation of the distal COOH terminus results in enhanced currents, suggesting an autoinhibitory function residing within the most terminal end [
150 amino acids (5)]. Proteolytic processing of the COOH terminus has been demonstrated in both Cav1.2 and Cav1.1 (5, 10) at sites more proximal to the proline-rich region, resulting in the removal of the inhibitory domain and thus enhancing calcium currents. Under in vivo conditions, the COOH terminal fragment remains tethered to the main body of the channel, which appears to require the presence of the PRD. Gerhardstein et al. (6) showed that deletion of the PRD resulted in the dissociation of the COOH terminal fragment from the main body and induced enhanced currents. Our finding that deletion of the PRD precluded SH3 binding but not tyrosine phosphorylation suggests that the two protein-protein interacting domains may be mutually exclusive in the regulation of the hCav1.2b. However, further experiments will be necessary to define this thesis.
The SH2 domain of c-Src requires consensus sequences containing phosphotyrosine for binding (19). Phosphorylation of the GST-CT fragments by c-Src in the presence of a Src kinase buffer resulted in GST-SH2 binding, which was prevented by nitrotyrosylation. In the full-length hCav1.2b, mutation of Y2134 prevented SH2 binding and reduced tyrosine phosphorylation of the channel. Interestingly, tyrosine phosphorylation was not completely prevented by a single mutation, but required mutations in both Y2134 and Y1837. Utilizing the effect of PP2, a Src kinase inhibitor, as a means to determine the effect of Src kinase on calcium currents, the double mutation significantly prevented the regulation of the calcium currents by the tyrosine kinase, as did the triple mutant.
Colonic inflammation results in decreased smooth muscle contraction. Ross et al. (18) showed that, in a murine 2,4,6-trinitrobenzene sulfonic acid-induced colitis model, depolarization induced contractions were attenuated compared with controls and were less sensitive to the Src kinase inhibitor. The calcium channels from inflamed colon were nitrotyrosylated, and, under basal unstimulated conditions, nitration resulted in decreased calcium currents. Similar results are obtained from nitrotyrosylation of the hCav1.2b. Interestingly, all three tyrosine residues appear to have the propensity for nitrosylation; however, only mutations affecting Y2134 significantly affect calcium currents.
In summary, these data show that the COOH terminus of the hCav1.2b is importantly involved in calcium channel regulation by c-Src kinase and that nitrosylation of Src kinase regulatory sites, as occurs during inflammation, results in marked downregulation.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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5. Gao T, Cuadra AE, Ma H, Bunemann M, Gerhardstein BL, Cheng T, Eick RT, Hosey MM. C-terminal fragments of the alpha 1C (CaV1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated alpha 1C subunits. J Biol Chem 276: 21089–21097, 2001.
6. Gerhardstein BL, Gao T, Bunemann M, Puri TS, Adair A, Ma H, Hosey MM. Proteolytic processing of the C terminus of the alpha(1C) subunit of L-type calcium channels and the role of a proline-rich domain in membrane tethering of proteolytic fragments. J Biol Chem 275: 8556–8563, 2000.
7. Gui P, Wu X, Ling S, Stotz SC, Winkfein RJ, Wilson E, Davis GE, Braun AP, Zamponi GW, Davis MJ. Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways. J Biol Chem 281: 14015–14025, 2006.
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9. Hu XQ, Singh N, Mukhopadhyay D, Akbarali HI. Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase. J Biol Chem 273: 5337–5342, 1998.
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