|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/1998//1637 $5.00
The Journal of Cell Biology, Volume 141, Number 7,
, 1998 1637-1646
Articles |
Src Family Tyrosine Kinase Regulates Intracellular pH in Cardiomyocytes
Centre National de Recherche Scientifique, Faculté de Pharmacie, 34295 Montpellier Cedex, France
The Anion Cl–/HCO3– Exchangers AE1, AE2, and AE3 are membrane pH regulatory ion transporters ubiquitously expressed in vertebrate tissues. Besides relieving intracellular alkaline and CO2 loads, the AEs have an important function during development and cell death and play a central role in such cellular properties as cell shape, metabolism, and contractility. The activity of AE(s) are regulated by neurohormones. However, little is known as to the intracellular signal transduction pathways that underlie this modulation. We show here that, in cardiomyocytes that express both AE1 and AE3, the purinergic agonist, ATP, triggers activation of anion exchange. The AE activation is observed in cells in which AE3 expression was blocked but not in cells microinjected with neutralizing anti-AE1 antibodies. ATP induces tyrosine phosphorylation of AE1, activation of the tyrosine kinase Fyn, and association of both Fyn and FAK with AE1. Inhibition of Src family kinases in vivo by genistein, herbimycin A, or ST638 prevents purinergic activation of AE1. Microinjection of either anti-Cst.1 antibody or recombinant CSK, both of which prevent activation of Src family kinase, significantly decreases ATP-induced activation of AE. Microinjection of an anti-FAK antibody as well as expression in cardiomyocytes of Phe397 FAK dominant negative mutant, also prevents purinergic activation of AE. Therefore, tyrosine kinases play a key role in acute regulation of intracellular pH and thus in cell function including excitation–contraction coupling of the myocardium.
Abbreviations used in this paper: AA, amino acids; AE, anion Cl–/ HCO3– exchangers; FAK, focal adhesion kinase; GFP, green fluorescent protein; ODNAS, oligodeoxynucleotide antisense; PI3K, phosphatidylinositol-3-kinase; pHi, intracellular pH; PKC, protein kinase C.
Address correspondence to Michel Pucéat, INSERM U-390, Laboratoire de Physiopathologie Cardiovasculaire, C.H.U. Arnaud de Villeneuve, 34295 Montpellier Cedex, France. Tel.: 33-4-6741-5240. Fax: 33-4-741-5242. E-mail: puceat{at}u390.montp.inserm.fr
INTRACELLULAR pH (pHi)1 regulates various cellular functions including Ca2+ homeostasis, ionic channels (Busa and Nuccitelli, 1984), gene expression (Isfort et al., 1993), and cell death (Reynolds et al., 1996). In cardiac cells, pHi is a major regulator of the excitation–contraction coupling. pHi modulates excitability (e.g., ionic conductances), contractility (e.g., Ca-sensitivity of myofilaments), and conduction (e.g., gap–junction conductance; Orchard and Kentish, 1990). Besides proton pumps and channels, most cells possess three membrane ionic transporters that closely regulate their pHi. Under acid loads, both a Na+/H+ antiport and a Na+-dependent HCO3– transporter are switched on to restore basal pHi. In contrast, a Na-independent Cl–/HCO3– exchanger that operates as a HCO3– extruder alleviates intracellular alkaline and CO2 loads (Madshus, 1988).
The anion Cl–/HCO3– exchangers (AE) belong to a multigenic family that comprises AE1, AE2, and AE3. The AEs are ubiquitously expressed in vertebrate tissues (Alper, 1991; Kay et al., 1995). AE1 is expressed in erythrocytes and encodes the Band3 protein, a major membrane protein that has been extensively studied (Wang, 1994). Truncated forms of AE1 are also present in kidney (Brosius et al., 1989) and heart (Puceat et al., 1995). AE2 is an housekeeping gene that is expressed in most tissues at least at the level of mRNA (Kudrycki et al., 1990). AE3 is expressed in brain (bAE3) as a protein of 160 kD (Kopito et al., 1989) and as a shorter polypeptide (cAE3, 120 kD) in heart (Linn et al., 1992).
Besides cytosolic pH regulation, the presence of AEs in nuclear, golgi, and mitochondrial membranes (Kay et al., 1990) suggests that they may play a role in regulation of pH of these intracellular compartments. The AEs also feature non-pH regulatory functions. They have critical functions in embryogenesis and morphogenesis during development (Zhao et al., 1995). AE1 participates in cell death; it generates a surface senescent cell antigen, a protein that appears on old cells and marks them for removal by the immune system (Kay, 1981). The AE proteins also play a central role in such cellular properties as cell shape by anchoring the cytoskeleton to the plasma membrane (Jay, 1996), metabolism by binding glycolytic enzymes (Low et al., 1993), and cell contractility (Puceat et al., 1995).
The anion exchange performed by the AEs proteins is modulated by pHi and by hormones or neurotransmitters in various tissues (Ganz et al., 1990; Muallem and Loessberg, 1990; Puceat et al., 1991; Desilets et al., 1994). However, no insight into the mechanism of this neuromodulation has been yet provided in any cell type.
Thus, our goal was to identify the particular AE protein that is activated by a neurohormonal stimulus and to uncover the signal transduction pathway that mediates this effect. To this end, we took advantage of ATP, a purinergic agonist that strongly activates the Cl–/HCO3– exchanger in cardiac cells (Puceat et al., 1991a), in osteoclasts (Yu and Ferrier, 1995) and likely in neurons (de Souza et al., 1995) to investigate both the AE isoform involved and the signal transduction pathway that links the purinergic receptor to activation of AE. It was found that the purinergic agonist, ATP, triggered concomitant and specific tyrosine phosphorylation of AE1 and activation of anion exchange. ATP induced activation of the tyrosine kinases Src and Fyn and association of both Fyn and FAK with AE1. Specific inhibition of Src family kinases in vivo prevented purinergic activation of AE1. Thus, these results suggest that tyrosine kinases play a major role in acute regulation of intracellular pH and exert an important function in excitation–contraction coupling of the myocardium, a concept that could also be extended to other excitable or nonexcitable cells in which AE functions have been shown to be modulated by neurohormones.
 |
Materials and Methods |
|---|
 TopAbstract Materials and Methods ResultsDiscussionReferences |
|---|
Transfection and Transferrinfection of Neonatal Rat Cardiomyocytes
Neonatal rat cardiomyocytes maintained in culture for 2–3 d were transfected with 250 nM AE3 oligodeoxynucleotide sense (AGGGGAATGACAAG) or antisense (CTTGTCATTCCCCT) corresponding to nucleotides –6 to +7 of rat AE3 cDNA (Linn et al., 1992) using 3 µg/ml DOSPER (Boehringer, Meylan, France) for 6 h in serum- and antibiotic-free medium. This protocol allows for 95% of transfection efficiency as assessed by FACS® analysis of cells transfected with fluorescein-conjugated ODN. Confocal microscopy of the transfected cells further showed the intracellular distribution of the ODN. The high yield of transfected cells favored the use of neonatal rat cardiac cells in this set of experiments. After washing, the cells were kept in medium added with 5% heat-inactivated fetal calf serum for 36 h and pHi was monitored as described above.
To transfect plasmids encoding wild-type FAKY397 or the dominant negative mutant FAKF397 in neonatal rat cardiomyocytes, a transferrinfection protocol using the replication-deficient Adl5dl312 adenovirus was used (Curiel et al., 1991; Wagner et al., 1992). The virus was amplified in HEK293 cells. In brief, HEK393 cells were infected with 1 PFU/cell of Adl5dl312 virus. Cells were scraped 24 or 48 h later and lysed by three cycles of freezing and thawing in phosphate-buffered solution. The virus was then purified by density centrifugation on a two step CsCl gradient. The band containing the viral particles was collected and dialyzed at 4°C for 2 h against 10 mM Tris, 1 mM MgCl2, and 10% glycerol.The virus preparation was then pooled and frozen in aliquot at –80°C. 4 x 1010 viral particles were incubated with 1.2 µg poly-L-lysine in 400 µl serum-free medium for 30 min at 20°C. Then 0.5 µg of FAK plasmid and 0.5 µg of pGREEN LANTERN-1 plasmid (GIBCO BRL, Eragny, France) containing the reporter gene green fluorescent protein (GFP) were added and the mixture was further incubated for 30 min. 0.3 µg of poly-L-lysine was added and the complex adenovirus-poly-L-lysine-DNA was added to the cells in a final volume of 1 ml. Cells were infected for 1 h at 37°C and then extensively washed. The experiments were carried out 24 h after transferrinfection. The fluorescence of GFP allowed us to recognize the transferrinfected cells to record pHi. To estimate the level of expression of FAK Y397 or F397, cells were immunostained with the antibody directed against the NH2-terminal domain of FAK and observed using confocal microscopy as previously described (Pucéat et al., 1995).
Immunoprecipitation of Proteins
Whole cell lysates were prepared by incubation of adult rat cardiac cells for 20 min at 4°C in NET-N buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, 50 mM NaF, 1 mM Na3VO4, 1% NP40, 1 mM PMSF, and 10 µg/ml leupeptin. The cell sample was spun at 12,000 g for 15 min and precleared for 1 h at 4°C with protein A–Sepharose. The lysates were spun and the supernatant was used for immunoprecipitation. The primary antibody was added for 2 h at 4°C. Protein antibody complex bound to protein A–Sepharose was pelleted by spinning down the lysates. The pellets were washed twice with NET-N buffer, once with NET buffer and boiled in the presence of Laemmli electrophoresis buffer. The samples were run in 7.5% SDS PAGE and electrophoretically transferred to nitrocellulose. The blots were treated as previously described (Puceat et al., 1995) and probed with the antibody recognizing the protein of interest and a secondary peroxidase-conjugated antibody. The proteins were revealed using ECL reagent. The bands on the films were quantified by an imaging system (SCION IMAGE software; NIH, Bethesda, MD). The Swiss protein database revealed that the amino acid sequence of the peptides (RKFKNSTYFPGK and FKPKYHPDVPF amino acids (AA) 607–618 and 814– 826, respectively (second and fifth intracellular loop of mouse AE1), used to raise the anti-rat AE1 antibodies do not share homology with any other known protein sequence (accession number of mouse AE1 P04919). The antibody did not cross-react with AE2 or AE3 (data not shown). The anti-AE3 antibodies were raised against the cardiac-specific NH2-terminal domain (AA 18–30) and the COOH-terminal domain (AA 999–1010) that is shared by both brain and cardiac rat AE3 (Linn et al., 1992; Puceat et al., 1995). The preimmune AE1 serum and/or the antiserum incubated in the presence of the peptide used to raise it was used as a control in the immunoprecipitation and coimmunoprecipitation experiments.
Measurement of Tyrosine Kinase Activity
Cell lysates were subjected to immunoprecipitation using an anti-Cst.1, a specific anti-Src (clone 2:17) or anti-Fyn (anti-Fyn.2) antibody. The immune complex was washed with NET-N buffer three times, once with TBS, and once with kinase buffer (10 mM Pipes and 10 mM MnCl2, pH7). The kinase reaction was run for 10 min at 30°C in the presence of 5 µCi 
-[32P]ATP and 6 µg acid-denatured enolase or no substrate for autophosphorylation. The reaction was stopped by adding Laemmli buffer. The complex was run in SDS-PAGE. After staining and destaining, the gel was dried and exposed to autoradiography films. In the autophosphorylation experiments, the gels were treated with KOH (1 N) for 1 h at 60°C before autoradiography. Phosphorylation of enolase was quantified using SCION IMAGE software. In some experiments, after the in vitro kinase assay, the anti-Src or anti-Fyn immunocomplex was electrophoresed and transferred to a nitrocellulose membrane. The blot was cut, the upper part was probed with the anti-Src or -Fyn antibody, and the lower part was subjected to autoradiography to reveal enolase phosphorylation.
Microinjection of Cardiomyocytes
Microinjection of neonatal rat cardiac cells was performed according to Shubeita et al. (1994). The pipette concentration of antibodies was 500 µg/ml (unless otherwise mentioned) in 150 mM KCl, 0.025 mM EGTA, and 1 mM Pipes, pH 7.2. Antibodies were affinity-purified and concentrated as described in Roche et al. (1995a). Polyclonal anti-Yes.6 antibody was raised against the AA 15–35 of mouse Yes. CSK purified from Sf-9 insect cells expressing human CSK (Koegl et al., 1994) was used at 100 µg/ml and boiled CSK (3 min, 100°C) served as a control. AE activity was recorded on cells 1 h after microinjection. Neonatal rat cells were chosen in this set of experiments because diffusion of antibodies (microinjected close to the nucleus) was facilitated by the small volume of the cytosol and by the shape of the myocytes.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
|
ATP Induces AE1 Association with Fyn and FAK
We then tested whether physical interactions between tyrosine kinases and AE1 occur in vivo. AE1 and Fyn coimmunoprecipitated from both unstimulated and ATP-stimulated cells. More immunoreactive Fyn and AE1 was detected in the AE1 and Fyn immunocomplex, respectively, from ATP-stimulated cells. (Fig. 5, A and B). To assess whether Fyn was activated in the AE1 immunocomplex, we investigated whether AE1 immunocomplex displayed a tyrosine kinase activity using enolase as a substrate. After AE1 immunoprecipitation, we observed a basal kinase activity that was further enhanced by purinergic stimulation of cells as early as one minute. Kinase activity was not detectable when the immunoprecipitation was done with the anti-AE1 antibody preincubated with the peptide used to raise this antibody (Fig. 5 C). In line with AE1 and Fyn association, we also observed a coimmunoprecipitation of Fyn and FAK in ATP-activated cells. We failed to detect any coimmunoprecipitation of Src with AE1 or FAK (not shown), although we cannot exclude that it was due to the low level of this kinase in cardiac cells. Moreover, the 60-kD tyrosine phosphorylated protein coimmunoprecipitated with AE1 (Fig. 2 A) was recognized by the anti Fyn antibody (data not shown). AE1 was found associated with FAK in unstimulated cells and the association was increased upon ATP-stimulation (Fig. 5 D).
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Cardiac cells express two isoforms of the AE family, namely AE1 and AE3 (Korichneva et al., 1995; Puceat et al., 1995). Although AE2 mRNA could be detected by RT-PCR or Northern blot by us and others (Kudrycki et al., 1990; Wang et al., 1996), we could never detect the AE2 protein. Furthermore, the poor DIDS sensitivity of AE2 protein (EC50,150 µM; Lee et al., 1991) makes it an unlikely candidate for the cardiac Cl–/HCO3– exchange that is fully inhibited by low concentrations of DIDS (EC50, 3 µM; Korichneva et al., 1995; Puceat et al., 1995). The involvement of AE1 and AE3 was tested in experiments designed to block their expression or function. The knock out of expression of cardiac AE3 by the antisense oligonucleotide targeted to the translation initiation codon did not affect the purinergic stimulation of the anion exchange. The lack of effect on AE1 expression demonstrated that the cAE3 oligonucleotide was AE3 specific although not fully specific of cAE3. Indeed, it did also decrease bAE3 expression. The latter effect could be attributed to the binding of the ODNAS to the sequence nt 306–332 and nt 1320–1326 of bAE3 cDNA (Kudrycki et al., 1990) that share 66% homology with the initiation translation site of cAE3 and to the low level of bAE3 protein in isolated rat ventricular cardiomyocytes. Although the antisense strategy resulted in a 70–80% decrease in AE3 expression, it could be argued that the 20–30% of cAE3 left after treatment of cells with antisense could be sufficient to mediate the purinergic stimulation of the anionic transport. Thus, we also used an alternative approach in which we microinjected anti-AE1 antibodies into neonatal cardiomyocytes. The antibodies dramatically decreased the anionic exchange activity as previously observed with an anti-whole band3 protein antibody AE3 (Puceat et al., 1995); they further abolished the purinergic stimulation of the anion exchange. Altogether, our data demonstrate that AE1 is the particular isoform that mediates the purinergic stimulation of the anion exchange. We also recorded ATP-induced activation of the DIDS-sensitive Cl–/HCO3– exchange activity in MDCK cells that possess endogenous AE1 and AE2 but not AE3 proteins which suggests that purinergic activation of AE1 is not a mechanism restricted to cardiac cells.
The main question we addressed in this study was to uncover the signal transduction pathway that underlies the purinergic activation of the Cl–/HCO3– exchanger. We found that ATP strongly and rapidly activates Src and Fyn and induces tyrosine phosphorylation of FAK. That AE1 is a target for these kinases can be seen both from the concomitant phosphorylation on tyrosine residues of AE1 and from Fyn and FAK associations with this anion exchanger. Using enolase as a substrate, we could also demonstrate a tyrosine kinase activity that was further activated in ATP-stimulated cells in AE1 immunocomplex. Since FAK was phosphorylated and found associated with AE1 in ATP-stimulated cells, this kinase may also be involved in the process. Microinjection of an anti-FAK antibody directed against the NH2-terminal domain of the kinase or expression of a dominant negative FAKF397 mutant that are likely to prevent or displace endogenous Fyn/FAK or FAK/AE1 interaction, respectively, significantly inhibited ATP-induced activation of AE1.We could not detect FAK activation in ATP-stimulated cells. Dissociation of tyrosine phosphorylation from activation of PYK2, a tyrosine kinase that belongs to the same family as FAK, has previously been observed in PC12 overexpressing activated Src (Dikic et al., 1996). FAK was also recently suggested to play a role of adaptor protein rather than the one of a kinase (Burgaya et al., 1997). Thus, we rather suggest that Fyn is responsible for AE1 phosphorylation and that FAK serves as a docking protein for Fyn.
We also attempted to understand how Fyn was activated after purinergic stimulation of cardiac cells. Wortmannin, used at a concentration high enough to inhibit PI3K including the G protein regulated P 110 PI3K isotype highly expressed in the heart (Stephens et al., 1994; Stoyanov et al., 1995), did not significantly prevent Src/Fyn or AE activation. Pertussis toxin treatment also did not affect kinase or AE activity. Thus, our findings rule out the participation of a Gi protein and of PI3K in the purinergic signaling pathway.
Three mechanisms for Src/Fyn activation have been proposed: first, dephosphorylation of the inhibitory tyrosine 527, second, displacement of the C-tail from the SH2 domain, and third, displacement of the SH2-SH1 linker from the SH3 domain (Shalloway and Taylor, 1997). Microinjection of CSK prevented purinergic activation of the AE, strongly suggesting that a dephosphorylation of tyrosine 527 of Fyn is necessary for its activation and in turn stimulation of AE. Thus, we predict that the earliest event in the purinergic signal transduction pathway is activation of a tyrosine phosphatase.
To test whether activation of Src family kinases is mandatory for ATP-induced activation of AE, we used three pharmacological agents (e.g., genistein, herbimycin, ST638) that each displays different inhibitory mechanisms to prevent ATP-induced activation of tyrosine kinases in cardiac cells. Whatever their mechanism of action (e.g., blocking of ATP binding site, catalytic site, or reduction of sulfhydryl groups), they all blocked ATP-mediated stimulation of anion exchange activity. To further characterize the tyrosine kinase involved in the phosphorylation of AE1, we used a more specific approach. We microinjected blocking antibodies into cardiac cells. The anti-Cst.1 antibody that inhibits Src, Fyn, and Yes but not IgG or anti-Yes antibody prevented ATP-induced stimulation of anion exchange showing the specificity of the anti-Cst.1 antibody inhibition. The anti-Src antibody did not significantly affect the purinergic effect suggesting a role of Fyn in AE1 activation; this is in agreement with the association of Fyn but not Src with AE1 and with the similar time courses of Fyn autophosphorylation and AE1 phosphorylation. Intracellular injection of a purified fraction of CSK that phosphorylates the inhibitory tyrosine residue 527 of Src family kinases also strongly prevented ATP-triggered anion exchange activation. Purinergic stimulation of cardiomyocytes induces the tyrosine phosphorylation of many proteins (Puceat and Vassort, 1996) in addition to AE1. Whether direct AE1 phosphorylation is required for activation of the anion exchange is likely but cannot be ascertained from our data. Phosphorylation on tyrosine residue of a regulatory protein or of cytoskeleton proteins may also result in AE1 activation. When cardiac truncated AE1 will be cloned, mutation of the tyrosine phosphorylation, and/ or Fyn-binding sites will be helpful to bring further information as to the role of AE1 phosphorylation in the neurohormonal activation of the anion exchange. Nevertheless, we can conclude that activation of Src family tyrosine kinase is a critical required step for ATP-induced stimulation of anion exchange.
How does AE1 get activated after ATP stimulation? We postulate that ATP-induced phosphorylation of FAK provides a docking site for Fyn through its SH2 and SH3 domains (Cobb et al., 1994; Thomas et al., 1997). FAK then recruits Fyn to the costameres where it phosphorylates AE1. Interestingly, the role of tyrosine kinases in cardiac cell homeostasis is not restricted to AE. Tyrosine phosphorylation of PLC accounts for ATP-induced IP3 generation (Puceat and Vassort, 1996) and this phenomenon is likely to modulate Ca release from intracellular stores. The later process is facilitated by AE-mediated acidosis that contributes to a large extent to purinergic arrhythmogenesis (Puceat et al., 1991b). Thus, rapid activation of tyrosine kinases of the Src family by neuromediators allows for an acute regulation of both H+ and Ca2+ homeostasis in cardiac cells. Tyrosine kinases are indeed expected in this fashion to play a major role in excitation–contraction coupling of the myocardium and to be involved in cardiovascular diseases such as arrhythmia. In other excitable or nonexcitable cells that all express AE family proteins, tyrosine kinases activated by purinergic or other G-protein– coupled receptor agonists may play an important regulatory role in H+ homeostasis, a major modulator of cell function.
|
Acknowledgments |
|---|
This work was supported by INSERM and in part by a grant from the American Heart Association (Grant in Aid to M. Pucéat, visiting Scientist at Mayo Clinic, Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic, Mayo Foundation, Rochester, MN) and the Action Concertée, ACC SVN°9-1A021A from The Ministére de l'Education Nationale, de la Recherche et de la Technologie.
Submitted: 4 November 1997
Revised: 13 May 1998
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M & Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases, J Biol Chem, 1987, 262, 5592–5595.
Alper SL. The band 3-related anion exchanger (AE) gene family, Annu Rev Physiol, 1991, 53, 549–564.[Medline]
Brosius FCD, Alper SL, Garcia AM & Lodish HF. The major kidney band 3 gene transcript predicts an amino-terminal truncated band 3 polypeptide, J Biol Chem, 1989, 264, 7784–7787.
Burgaya F, Toutan M, Studler JM, Costa A, Le Bert M, Gelman M & Girault J-A. Alternatively spliced focal adhesion kinase in rat brain with increased autophosphorylation activity, J Biol Chem, 1997, 272, 28720–28725.
Busa WB & Nuccitelli R. Metabolic regulation via intracellular pH, Am J Physiol, 1984, 246, R409–R438.[Medline]
Cobb BS, Schaller MD, Leu TH & Parsons JT. Stable association of pp60src and pp59fyn with the focal adhesion- associated protein tyrosine kinase, pp125FAK, Mol Cell Biol, 1994, 14, 147–155.
Cooper JA, Gould KL, Cartwright CA & Hunter T. Tyr527 is phosphorylated in pp60c-src: implications for regulation, Science, 1986, 231, 1431–1434.
Curiel DT, Agarwal S, Wagner E & Cotten M. Adenovirus enhancement of transferrin-polylysine-mediated gene delivery, Proc Natl Acad Sci USA, 1991, 88, 8850–8854.
De Souza LR, Moore H, Raha S & Reed JK. Purine and pyrimidine nucleotides activate distinct signalling pathways in PC12 cells, J Neurosci Res, 1995, 41, 753–763.[Medline]
Desilets M, Puceat M & Vassort G. Chloride dependence of pH modulation by beta-adrenergic agonist in rat cardiomyocytes, Circ Res, 1994, 75, 862–869.
Dikic I, Tokiwa G, Lev S, Courtneidge SA & Schlessinger J. A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation, Nature, 1996, 383, 547–550.[Medline]
Fukazawa H, Li PM, Yamamoto C, Murakami Y, Mizuno S & Uehara Y. Specific inhibition of cytoplasmic protein tyrosine kinases by herbimycin A in vitro, Biochem Pharmacol, 1991, 42, 1661–1671.[Medline]
Ganz MB, Perfetto MC & Boron WF. Effects of mitogens and other agents on rat mesangial cell proliferation, pH, and Ca2+, Am J Physiol, 1990, 259, F269–F278.[Medline]
Harrison ML, Isaacson CC, Burg DL, Geahlen RL & Low PS. Phosphorylation of human erythrocyte band 3 by endogenous p72syk, J Biol Chem, 1994, 269, 955–959.
Isfort RJ, Cody DB, Asquith TN, Ridder GM, Stuard SB & LeBoeuf RA. Induction of protein phosphorylation, protein synthesis, immediate- early-gene expression and cellular proliferation by intracellular pH modulation. Implications for the role of hydrogen ions in signal transduction, Eur J Biochem, 1993, 213, 349–357.[Medline]
Jay DG. Role of band 3 in homeostasis and cell shape, Cell, 1996, 86, 853–854.[Medline]
Kay MM. Isolation of the phagocytosis-inducing IgG-binding antigen on senescent somatic cells, Nature, 1981, 289, 491–494.[Medline]
Kay MM, Marchalonis JJ, Hughes J, Watanabe K & Schluter SF. Definition of a physiologic aging autoantigen by using synthetic peptides of membrane protein band 3: localization of the active antigenic sites, Proc Natl Acad Sci USA, 1990, 87, 5734–5738.
Kay MM, Cover C, Schluter SF, Bernstein RM & Marchalonis JJ. Band 3, the anion transporter, is conserved during evolution: implications for aging and vertebrate evolution, Cell Mol Biol, 1995, 41, 833–842.[Medline]
Koegl M, Kypta RM, Bergman M, Alitalo K & Courtneidge SA. Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85, Biochem J, 1994, 302, 737–744.[Medline]
Kopito RR, Lee BS, Simmons DM, Lindsey AE, Morgans CW & Schneider K. Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger, Cell, 1989, 59, 927–937.[Medline]
Korichneva I, Puceat M, Cassoly R & Vassort G. Cl–/HCO3–exchange in developing neonatal rat cardiac cells. Biochemical identification and immunolocalization of band 3-like proteins, Circ Res, 1995, 77, 556–564.
Kudrycki KE, Newman PR & Shull GE. cDNA cloning and tissue distribution of mRnas for two proteins that are related to the band 3 Cl–/ HCO3–exchanger, J Biol Chem, 1990, 265, 462–471.
Lee BS, Gunn RB & Kopito RR. Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line, J Biol Chem, 1991, 266, 11448–11454.
Linn SC, Kudrycki KE & Shull GE. The predicted translation product of a cardiac AE3 mRNA contains an N terminus distinct from that of the brain AE3 Cl–/HCO3–exchanger. Cloning of a cardiac AE3 cDNA, organization of the AE3 gene, and identification of an alternative transcription initiation site, J Biol Chem, 1992, 267, 7927–7935.
Lopez-Ilasaca M, Li W, Uren A, Yu JC, Kazlauskas A, Gutkind JS & Heidaran MA. Requirement of phosphatidylinositol-3 kinase for activation of JNK/SAPKs by PDGF, Biochem Biophys Res Commun, 1997, 232, 273–277.[Medline]
Low PS, Rathinavelu P & Harrison ML. Regulation of glycolysis via reversible enzyme binding to the membrane protein, band 3, J Biol Chem, 1993, 268, 14627–14631.
Luttrell LM, Hawes BE, van Biesen T, Luttrell DK, Lansing TJ & Lefkowitz RJ. Role of c-Src tyrosine kinase in G protein-coupled receptor- and G subunit-mediated activation of mitogen-activated protein kinases, J Biol Chem, 1996, 271, 19443–19450.
Madshus IH. Regulation of intracellular pH in eukaryotic cells, Biochem J, 1988, 250, 1–8.[Medline]
Muallem S & Loessberg PA. Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H+ and HCO3– transporters by Ca2+-mobilizing agonists, J Biol Chem, 1990, 265, 12813–12819.
Muller-Berger S, Karbach D, Kang D, Aranibar N, Wood PG, Ruterjans H & Passow H. Roles of histidine 752 and glutamate 699 in the pH dependence of mouse band 3 protein-mediated anion transport, Biochemistry, 1995, 34, 9325–9332.[Medline]
Orchard CH & Kentish JC. Effects of changes of pH on the contractile function of cardiac muscle, Am J Physiol, 1990, 258, C967–C981.[Medline]
Puceat M, Clement O & Vassort G. Extracellular MgATP activates the Cl–/HCO3–exchanger in single rat cardiac cells, J Physiol, 1991a, 444, 241–256.
Puceat M, Clement O, Scamps F & Vassort G. Extracellular ATP-induced acidification leads to cytosolic calcium transient rise in single rat cardiac myocytes, Biochem J, 1991b, 274, 55–62.[Medline]
Puceat M, Hilal-Dandan R, Strulovici B, Brunton LL & Brown JH. Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes, J Biol Chem, 1994, 269, 16938–16944.
Puceat M, Korichneva I, Cassoly R & Vassort G. Identification of band 3-like proteins and Cl–/HCO3–exchange in isolated cardiomyocytes, J Biol Chem, 1995, 270, 1315–1322.
Puceat M & Vassort G. Purinergic stimulation of rat cardiomyocytes induces tyrosine phosphorylation and membrane association of phospholipase C: a major mechanism for InsP3generation, Biochem J, 1996, 318, 23–28.
Reynolds JE, Li J & Eastman A. Detection of apoptosis by flow cytometry of cells simultaneously stained for intracellular pH (carboxy SNARF-1) and membrane permeability (Hoechst 33342), Cytometry, 1996, 25, 349–357.[Medline]
Roche S, Koegl M, Barone MV, Roussel MF & Courtneidge SA. DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases, Mol Cell Biol, 1995a, 15, 1102–1109.[Abstract]
Roche S, Fumagalli S & Courtneidge SA. Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division, Science, 1995b, 269, 1567–1569.
Shalloway D & Taylor SJ. Src: more than the sum of its part, Trends Cell Biol, 1997, 7, 215–217.[Medline]
Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB & Parsons JT. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions, Proc Natl Acad Sci USA, 1992, 89, 5192–5196.
Schlaepfer DD & Hunter T. Focal adhesion kinase overexpression enhances ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src, J Biol Chem, 1997, 272, 13189–13195.
Shiraishi T, Owada MK, Tatsuka M, Yamashita T, Watanabe K & Kakunaga T. Specific inhibitors of tyrosine-specific protein kinases: properties of 4-hydroxycinnamamide derivatives in vitro, Cancer Res, 1989, 49, 2374–2378.
Stephens L, Smrcka A, Cooke FT, Jackson TR, Sternweis PC & Hawkins PT. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits, Cell, 1994, 77, 83–93.[Medline]
Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nurnberg B et al.. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase, Science, 1995, 269, 690–693.
Thomas JW, Ellis B, Boerner RJ, Knight WB, White GC II & Schaller MD. SH2- and SH3-mediated interactions between focal adhesion kinase and Src, J Biol Chem, 1997, 273, 577–583.[Medline]
Wagner E, Zatloukal K, Cotten M, Kirlappos H, Mechtler K, Curiel DT & Birnstiel ML. Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes , Proc Natl Acad Sci USA, 1992, 89, 6099–6103.
Wang DN. Band 3 protein: structure, flexibility and function, FEBS Lett, 1994, 346, 26–31.[Medline]
Wang Z, Schultheis PJ & Shull GE. Three N-terminal variants of the AE2 Cl–/HCO3–exchanger are encoded by mRNAs transcribed from alternative promoters, J Biol Chem, 1996, 271, 7835–7843.
Watts JD, Brabb T, Bures EJ, Wange RL, Samelson LE & Aebersold R. Identification and characterization of a substrate specific for the T cell protein tyrosine kinase ZAP-70, FEBS Lett, 1996, 398, 217–222.[Medline]
Yannoukakos D, Vasseur C, Piau JP, Wajcman H & Bursaux E. Phosphorylation sites in human erythrocyte band 3 protein, Biochim Biophys Acta, 1991, 1061, 253–266.[Medline]
Yu H & Ferrier J. Osteoclast ATP receptor activation leads to a transient decrease in intracellular pH, J Cell Sci, 1995, 108, 3051–3058.[Abstract]
Zhao Y, Chauvet PJ, Alper SL & Baltz JM. Expression and function of bicarbonate/chloride exchangers in the preimplantation mouse embryo, J Biol Chem, 1995, 270, 24428–24434.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
-FAK N-term) domain of FAK was microinjected into cardiomyocytes and AE activity was measured 1 h later. The antibody preincubated with the hexahistidine fusion protein bound to Ni-agarose was microinjected as a control (control ab). Results are expressed as means ± SEM (n, numbers of microinjected cells investigated from at least three different cell cultures are indicated below the bars). (B) Neonatal rat cardiac cells were transferrinfected with plasmids encoding wild-type FAK (FAKY397) or the dominant negative FAK mutant (FAKF397). AE activity was measured 24 h after transferrinfection in 14 and 13 infected cells (GFP positive), respectively. Results are expressed as means ± SEM.