Abstract
The product of the viral src gene (v-src) is the protein tyrosine kinase pp60v-src. Among the known consequences of pp60v-src activity is the reduction in permeability of gap junctions, an effect that is counteracted by the calcium antagonist TMB-8 (8-N,N-[diethylamino]octyl- 3,4,5-trimethoxybenzoate). We show here that a decrease in intracellular pH (pHi) also counteracts the v-src effect: junctional permeability of cells containing active v-src kinase rose with decreasing pHi in the range 7.15 to 6.75, whereas junctional permeability of cells containing inactive v-src kinase or no v-src at all was insensitive to pH in that range. Low pH also counteracted the known action of diacylglycerol on junction, but only when pp60v-src kinase was inactive. Immunoblots of whole-cell lysates using an antibody against phosphotyrosine show that phosphorylation on tyrosine of at least one cellular protein, specific for pp60v-src kinase activity, was reduced by low pH but not by TMB-8. These results suggest that TMB-8 does not inhibit v-src action on junctional permeability by interfering with tyrosine phosphorylation of a protein crucial for closure of gap junction channels, but that the inhibition by low pH may be via this mechanism.
Full Text
The Full Text of this article is available as a PDF (1.4 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Atkinson M. M., Menko A. S., Johnson R. G., Sheppard J. R., Sheridan J. D. Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus. J Cell Biol. 1981 Nov;91(2 Pt 1):573–578. doi: 10.1083/jcb.91.2.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Azarnia R., Loewenstein W. R. Intercellular communication and the control of growth: X. Alteration of junctional permeability by the src gene. A study with temperature-sensitive mutant Rous sarcoma virus. J Membr Biol. 1984;82(3):191–205. doi: 10.1007/BF01871629. [DOI] [PubMed] [Google Scholar]
- Azarnia R., Reddy S., Kmiecik T. E., Shalloway D., Loewenstein W. R. The cellular src gene product regulates junctional cell-to-cell communication. Science. 1988 Jan 22;239(4838):398–401. doi: 10.1126/science.2447651. [DOI] [PubMed] [Google Scholar]
- Berridge M. J., Heslop J. P., Irvine R. F., Brown K. D. Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem J. 1984 Aug 15;222(1):195–201. doi: 10.1042/bj2220195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
- Beyer E. C., Paul D. L., Goodenough D. A. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol. 1987 Dec;105(6 Pt 1):2621–2629. doi: 10.1083/jcb.105.6.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brugge J. S., Darrow D. Rous sarcoma virus-induced phosphorylation of a 50,000-molecular weight cellular protein. Nature. 1982 Jan 21;295(5846):250–253. doi: 10.1038/295250a0. [DOI] [PubMed] [Google Scholar]
- Chiou C. Y., Malagodi M. H. Studies on the mechanism of action of a new Ca-2+ antagonist, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride in smooth and skeletal muscles. Br J Pharmacol. 1975 Feb;53(2):279–285. doi: 10.1111/j.1476-5381.1975.tb07359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cooper J. A., Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. doi: 10.1128/mcb.1.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cooper J. A., Hunter T. Identification and characterization of cellular targets for tyrosine protein kinases. J Biol Chem. 1983 Jan 25;258(2):1108–1115. [PubMed] [Google Scholar]
- Cooper J. A., Sefton B. M., Hunter T. Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells. Mol Cell Biol. 1984 Jan;4(1):30–37. doi: 10.1128/mcb.4.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dahl G., Isenberg G. Decoupling of heart muscle cells: correlation with increased cytoplasmic calcium activity and with changes of nexus ultrastructure. J Membr Biol. 1980 Mar 31;53(1):63–75. doi: 10.1007/BF01871173. [DOI] [PubMed] [Google Scholar]
- Diringer H., Friis R. R. Changes in phosphatidylinositol metabolism correlated to growth state of normal and Rous sarcoma virus-transformed Japanese quail cells. Cancer Res. 1977 Sep;37(9):2979–2984. [PubMed] [Google Scholar]
- Enomoto T., Sasaki Y., Shiba Y., Kanno Y., Yamasaki H. Tumor promoters cause a rapid and reversible inhibition of the formation and maintenance of electrical cell coupling in culture. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5628–5632. doi: 10.1073/pnas.78.9.5628. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Enomoto T., Yamasaki H. Rapid inhibition of intercellular communication between BALB/c 3T3 cells by diacylglycerol, a possible endogenous functional analogue of phorbol esters. Cancer Res. 1985 Aug;45(8):3706–3710. [PubMed] [Google Scholar]
- Gainer H. S., Murray A. W. Diacylglycerol inhibits gap junctional communication in cultured epidermal cells: evidence for a role of protein kinase C. Biochem Biophys Res Commun. 1985 Feb 15;126(3):1109–1113. doi: 10.1016/0006-291x(85)90300-6. [DOI] [PubMed] [Google Scholar]
- Gilmore T., Martin G. S. Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine. Nature. 1983 Dec 1;306(5942):487–490. doi: 10.1038/306487a0. [DOI] [PubMed] [Google Scholar]
- Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
- Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kojima I., Kojima K., Rasmussen H. Mechanism of inhibitory action of TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] on aldosterone secretion in adrenal glomerulosa cells. Biochem J. 1985 Nov 15;232(1):87–92. doi: 10.1042/bj2320087. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kojima I., Shibata H., Ogata E. Action of TMB-8 (8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate) on cytoplasmic free calcium in adrenal glomerulosa cell. Biochim Biophys Acta. 1986 Aug 29;888(1):25–29. doi: 10.1016/0167-4889(86)90066-2. [DOI] [PubMed] [Google Scholar]
- Kumar N. M., Gilula N. B. Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J Cell Biol. 1986 Sep;103(3):767–776. doi: 10.1083/jcb.103.3.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leis J. F., Kaplan N. O. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6507–6511. doi: 10.1073/pnas.79.21.6507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loewenstein W. R., Nakas M., Socolar S. J. Junctional membrane uncoupling. Permeability transformations at a cell membrane junction. J Gen Physiol. 1967 Aug;50(7):1865–1891. doi: 10.1085/jgp.50.7.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Macara I. G., Marinetti G. V., Balduzzi P. C. Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis. Proc Natl Acad Sci U S A. 1984 May;81(9):2728–2732. doi: 10.1073/pnas.81.9.2728. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Macara I. G. Oncogenes, ions, and phospholipids. Am J Physiol. 1985 Jan;248(1 Pt 1):C3–11. doi: 10.1152/ajpcell.1985.248.1.C3. [DOI] [PubMed] [Google Scholar]
- Mix L. L., Dinerstein R. J., Villereal M. L. Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts. Biochem Biophys Res Commun. 1984 Feb 29;119(1):69–75. doi: 10.1016/0006-291x(84)91619-x. [DOI] [PubMed] [Google Scholar]
- Morris A. P., Gallacher D. V., Irvine R. F., Petersen O. H. Synergism of inositol trisphosphate and tetrakisphosphate in activating Ca2+-dependent K+ channels. Nature. 1987 Dec 17;330(6149):653–655. doi: 10.1038/330653a0. [DOI] [PubMed] [Google Scholar]
- Murray A. W., Fitzgerald D. J. Tumor promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells. Biochem Biophys Res Commun. 1979 Nov 28;91(2):395–401. doi: 10.1016/0006-291x(79)91535-3. [DOI] [PubMed] [Google Scholar]
- Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
- Noma A., Tsuboi N. Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea-pig. J Physiol. 1987 Jan;382:193–211. doi: 10.1113/jphysiol.1987.sp016363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paradiso A. M., Tsien R. Y., Machen T. E. Digital image processing of intracellular pH in gastric oxyntic and chief cells. 1987 Jan 29-Feb 4Nature. 325(6103):447–450. doi: 10.1038/325447a0. [DOI] [PubMed] [Google Scholar]
- Paradiso A. M., Tsien R. Y., Machen T. E. Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7436–7440. doi: 10.1073/pnas.81.23.7436. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paul D. L. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol. 1986 Jul;103(1):123–134. doi: 10.1083/jcb.103.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richert N. D., Blithe D. L., Pastan I. Properties of the src kinase purified from Rous sarcoma virus-induced rat tumors. J Biol Chem. 1982 Jun 25;257(12):7143–7150. [PubMed] [Google Scholar]
- Rink T. J., Tsien R. Y., Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 1982 Oct;95(1):189–196. doi: 10.1083/jcb.95.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rose B., Loewenstein W. R. Permeability of a cell junction and the local cytoplasmic free ionized calcium concentration: a study with aequorin. J Membr Biol. 1976 Aug 27;28(1):87–119. doi: 10.1007/BF01869692. [DOI] [PubMed] [Google Scholar]
- Rose B., Rick R. Intracellular pH, intracellular free Ca, and junctional cell-cell coupling. J Membr Biol. 1978 Dec 29;44(3-4):377–415. doi: 10.1007/BF01944230. [DOI] [PubMed] [Google Scholar]
- Rose B., Yada T., Loewenstein W. R. Downregulation of cell-to-cell communication by the viral src gene is blocked by TMB-8 and recovery of communication is blocked by vanadate. J Membr Biol. 1986;94(2):129–142. doi: 10.1007/BF01871193. [DOI] [PubMed] [Google Scholar]
- Sawamura M. Inhibition of protein kinase C activation by 8-(N,N-diethylamino)-octyl-3, 4, 5-trimethoxybenzoate (TMB-8), an intracellular Ca2+ antagonist. Kobe J Med Sci. 1985 Dec;31(6):221–232. [PubMed] [Google Scholar]
- Somogyi R., Batzer A., Kolb H. A. Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C. J Membr Biol. 1989 Jun;108(3):273–282. doi: 10.1007/BF01871742. [DOI] [PubMed] [Google Scholar]
- Spray D. C., Bennett M. V. Physiology and pharmacology of gap junctions. Annu Rev Physiol. 1985;47:281–303. doi: 10.1146/annurev.ph.47.030185.001433. [DOI] [PubMed] [Google Scholar]
- Streb H., Irvine R. F., Berridge M. J., Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature. 1983 Nov 3;306(5938):67–69. doi: 10.1038/306067a0. [DOI] [PubMed] [Google Scholar]
- Sugimoto Y., Whitman M., Cantley L. C., Erikson R. L. Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2117–2121. doi: 10.1073/pnas.81.7.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takeda A., Hashimoto E., Yamamura H., Shimazu T. Phosphorylation of liver gap junction protein by protein kinase C. FEBS Lett. 1987 Jan 5;210(2):169–172. doi: 10.1016/0014-5793(87)81330-3. [DOI] [PubMed] [Google Scholar]
- Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
- Willingham M. C., Jay G., Pastan I. Localization of the ASV src gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1979 Sep;18(1):125–134. doi: 10.1016/0092-8674(79)90361-1. [DOI] [PubMed] [Google Scholar]
- Yada T., Rose B., Loewenstein W. R. Diacylglycerol downregulates junctional membrane permeability. TMB-8 blocks this effect. J Membr Biol. 1985;88(3):217–232. doi: 10.1007/BF01871087. [DOI] [PubMed] [Google Scholar]
- Yancey S. B., John S. A., Lal R., Austin B. J., Revel J. P. The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains. J Cell Biol. 1989 Jun;108(6):2241–2254. doi: 10.1083/jcb.108.6.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yotti L. P., Chang C. C., Trosko J. E. Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science. 1979 Nov 30;206(4422):1089–1091. doi: 10.1126/science.493994. [DOI] [PubMed] [Google Scholar]