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. 1997 Apr 15;99(8):1831–1841. doi: 10.1172/JCI119350

1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation.

S Khare 1, M J Bolt 1, R K Wali 1, S F Skarosi 1, H K Roy 1, S Niedziela 1, B Scaglione-Sewell 1, B Aquino 1, C Abraham 1, M D Sitrin 1, T A Brasitus 1, M Bissonnette 1
PMCID: PMC508007  PMID: 9109427

Abstract

Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-gamma as well as c-Src in rat colonocytes, and indicate that PLC-gamma is a direct substrate of secosteroid-activated c-Src in these cells.

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Selected References

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  1. Bell L., Luthringer D. J., Madri J. A., Warren S. L. Autocrine angiotensin system regulation of bovine aortic endothelial cell migration and plasminogen activator involves modulation of proto-oncogene pp60c-src expression. J Clin Invest. 1992 Jan;89(1):315–320. doi: 10.1172/JCI115578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Belleli A., Shany S., Levy J., Guberman R., Lamprecht S. A. A protective role of 1,25-dihydroxyvitamin D3 in chemically induced rat colon carcinogenesis. Carcinogenesis. 1992 Dec;13(12):2293–2298. doi: 10.1093/carcin/13.12.2293. [DOI] [PubMed] [Google Scholar]
  3. Berry D. M., Antochi R., Bhatia M., Meckling-Gill K. A. 1,25-Dihydroxyvitamin D3 stimulates expression and translocation of protein kinase Calpha and Cdelta via a nongenomic mechanism and rapidly induces phosphorylation of a 33-kDa protein in acute promyelocytic NB4 cells. J Biol Chem. 1996 Jul 5;271(27):16090–16096. doi: 10.1074/jbc.271.27.16090. [DOI] [PubMed] [Google Scholar]
  4. Bissonnette M., Wali R. K., Hartmann S. C., Niedziela S. M., Roy H. K., Tien X. Y., Sitrin M. D., Brasitus T. A. 1,25-Dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol 13-acetate cause differential activation of Ca(2+)-dependent and Ca(2+)-independent isoforms of protein kinase C in rat colonocytes. J Clin Invest. 1995 May;95(5):2215–2221. doi: 10.1172/JCI117911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bolt M. J., Bissonnette B. M., Wali R. K., Hartmann S. C., Brasitus T. A., Sitrin M. D. Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes. Biochem J. 1993 May 15;292(Pt 1):271–276. doi: 10.1042/bj2920271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brasitus T. A., Keresztes R. S. Protein-lipid interactions in antipodal plasma membranes of rat colonocytes. Biochim Biophys Acta. 1984 Jun 27;773(2):290–300. doi: 10.1016/0005-2736(84)90093-2. [DOI] [PubMed] [Google Scholar]
  7. Brugge J., Cotton P., Lustig A., Yonemoto W., Lipsich L., Coussens P., Barrett J. N., Nonner D., Keane R. W. Characterization of the altered form of the c-src gene product in neuronal cells. Genes Dev. 1987 May;1(3):287–296. doi: 10.1101/gad.1.3.287. [DOI] [PubMed] [Google Scholar]
  8. Calalb M. B., Polte T. R., Hanks S. K. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995 Feb;15(2):954–963. doi: 10.1128/mcb.15.2.954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carpenter G., Hernández-Sotomayor T., Jones G. Tyrosine phosphorylation of phospholipase C-gamma 1. Adv Second Messenger Phosphoprotein Res. 1993;28:179–185. [PubMed] [Google Scholar]
  10. Cheng H. C., Nishio H., Hatase O., Ralph S., Wang J. H. A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases. J Biol Chem. 1992 May 5;267(13):9248–9256. [PubMed] [Google Scholar]
  11. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  12. Cooper J. A., Gould K. L., Cartwright C. A., Hunter T. Tyr527 is phosphorylated in pp60c-src: implications for regulation. Science. 1986 Mar 21;231(4744):1431–1434. doi: 10.1126/science.2420005. [DOI] [PubMed] [Google Scholar]
  13. Cooper J. A., King C. S. Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src. Mol Cell Biol. 1986 Dec;6(12):4467–4477. doi: 10.1128/mcb.6.12.4467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Courtneidge S. A. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. doi: 10.1002/j.1460-2075.1985.tb03805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dhar A., Shukla S. D. Electrotransjection of pp60v-src monoclonal antibody inhibits activation of phospholipase C in platelets. A new mechanism for platelet-activating factor responses. J Biol Chem. 1994 Mar 25;269(12):9123–9127. [PubMed] [Google Scholar]
  16. Garland C., Shekelle R. B., Barrett-Connor E., Criqui M. H., Rossof A. H., Paul O. Dietary vitamin D and calcium and risk of colorectal cancer: a 19-year prospective study in men. Lancet. 1985 Feb 9;1(8424):307–309. doi: 10.1016/s0140-6736(85)91082-7. [DOI] [PubMed] [Google Scholar]
  17. Golden A., Nemeth S. P., Brugge J. S. Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity. Proc Natl Acad Sci U S A. 1986 Feb;83(4):852–856. doi: 10.1073/pnas.83.4.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldschmidt-Clermont P. J., Kim J. W., Machesky L. M., Rhee S. G., Pollard T. D. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation. Science. 1991 Mar 8;251(4998):1231–1233. doi: 10.1126/science.1848725. [DOI] [PubMed] [Google Scholar]
  19. Hanke J. H., Gardner J. P., Dow R. L., Changelian P. S., Brissette W. H., Weringer E. J., Pollok B. A., Connelly P. A. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem. 1996 Jan 12;271(2):695–701. doi: 10.1074/jbc.271.2.695. [DOI] [PubMed] [Google Scholar]
  20. Ilić D., Furuta Y., Kanazawa S., Takeda N., Sobue K., Nakatsuji N., Nomura S., Fujimoto J., Okada M., Yamamoto T. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 1995 Oct 12;377(6549):539–544. doi: 10.1038/377539a0. [DOI] [PubMed] [Google Scholar]
  21. Janmey P. A. Polyproline affinity method for purification of platelet profilin and modification with pyrene-maleimide. Methods Enzymol. 1991;196:92–99. doi: 10.1016/0076-6879(91)96011-f. [DOI] [PubMed] [Google Scholar]
  22. Kremer N. E., D'Arcangelo G., Thomas S. M., DeMarco M., Brugge J. S., Halegoua S. Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of src and ras actions. J Cell Biol. 1991 Nov;115(3):809–819. doi: 10.1083/jcb.115.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liao F., Shin H. S., Rhee S. G. In vitro tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2 by src-family protein tyrosine kinases. Biochem Biophys Res Commun. 1993 Mar 31;191(3):1028–1033. doi: 10.1006/bbrc.1993.1320. [DOI] [PubMed] [Google Scholar]
  24. Lydon N. B., Gay B., Mett H., Murray B., Liebetanz J., Gutzwiller A., Piwnica-Worms H., Roberts T. M., McGlynn E. Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase. Biochem J. 1992 Nov 1;287(Pt 3):985–993. doi: 10.1042/bj2870985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marrero M. B., Schieffer B., Paxton W. G., Schieffer E., Bernstein K. E. Electroporation of pp60c-src antibodies inhibits the angiotensin II activation of phospholipase C-gamma 1 in rat aortic smooth muscle cells. J Biol Chem. 1995 Jun 30;270(26):15734–15738. doi: 10.1074/jbc.270.26.15734. [DOI] [PubMed] [Google Scholar]
  26. Nakanishi O., Shibasaki F., Hidaka M., Homma Y., Takenawa T. Phospholipase C-gamma 1 associates with viral and cellular src kinases. J Biol Chem. 1993 May 25;268(15):10754–10759. [PubMed] [Google Scholar]
  27. Nemere I., Dormanen M. C., Hammond M. W., Okamura W. H., Norman A. W. Identification of a specific binding protein for 1 alpha,25-dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium and relationship to transcaltachia. J Biol Chem. 1994 Sep 23;269(38):23750–23756. [PubMed] [Google Scholar]
  28. Pillai S., Bikle D. D., Su M. J., Ratnam A., Abe J. 1,25-Dihydroxyvitamin D3 upregulates the phosphatidylinositol signaling pathway in human keratinocytes by increasing phospholipase C levels. J Clin Invest. 1995 Jul;96(1):602–609. doi: 10.1172/JCI118075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Piwnica-Worms H., Saunders K. B., Roberts T. M., Smith A. E., Cheng S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell. 1987 Apr 10;49(1):75–82. doi: 10.1016/0092-8674(87)90757-4. [DOI] [PubMed] [Google Scholar]
  30. Roche S., Koegl M., Barone M. V., Roussel M. F., Courtneidge S. A. DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases. Mol Cell Biol. 1995 Feb;15(2):1102–1109. doi: 10.1128/mcb.15.2.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  32. Schoenmakers T. J., Visser G. J., Flik G., Theuvenet A. P. CHELATOR: an improved method for computing metal ion concentrations in physiological solutions. Biotechniques. 1992 Jun;12(6):870-4, 876-9. [PubMed] [Google Scholar]
  33. Sitrin M. D., Halline A. G., Abrahams C., Brasitus T. A. Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat. Cancer Res. 1991 Oct 15;51(20):5608–5613. [PubMed] [Google Scholar]
  34. Slater S. J., Kelly M. B., Taddeo F. J., Larkin J. D., Yeager M. D., McLane J. A., Ho C., Stubbs C. D. Direct activation of protein kinase C by 1 alpha,25-dihydroxyvitamin D3. J Biol Chem. 1995 Mar 24;270(12):6639–6643. doi: 10.1074/jbc.270.12.6639. [DOI] [PubMed] [Google Scholar]
  35. Stover D. R., Becker M., Liebetanz J., Lydon N. B. Src phosphorylation of the epidermal growth factor receptor at novel sites mediates receptor interaction with Src and P85 alpha. J Biol Chem. 1995 Jun 30;270(26):15591–15597. doi: 10.1074/jbc.270.26.15591. [DOI] [PubMed] [Google Scholar]
  36. Twamley-Stein G. M., Pepperkok R., Ansorge W., Courtneidge S. A. The Src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH 3T3 cells. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7696–7700. doi: 10.1073/pnas.90.16.7696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wali R. K., Baum C. L., Sitrin M. D., Brasitus T. A. 1,25(OH)2 vitamin D3 stimulates membrane phosphoinositide turnover, activates protein kinase C, and increases cytosolic calcium in rat colonic epithelium. J Clin Invest. 1990 Apr;85(4):1296–1303. doi: 10.1172/JCI114567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wali R. K., Bissonnette M., Khare S., Hart J., Sitrin M. D., Brasitus T. A. 1 alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol, a noncalcemic analogue of 1 alpha,25-dihydroxyvitamin D3, inhibits azoxymethane-induced colonic tumorigenesis. Cancer Res. 1995 Jul 15;55(14):3050–3054. [PubMed] [Google Scholar]
  39. Wali R. K., Bolt M. J., Tien X. Y., Brasitus T. A., Sitrin M. D. Differential effect of 1,25-dihydroxycholecalciferol on phosphoinositide turnover in the antipodal plasma membranes of colonic epithelial cells. Biochem Biophys Res Commun. 1992 Sep 16;187(2):1128–1134. doi: 10.1016/0006-291x(92)91314-g. [DOI] [PubMed] [Google Scholar]
  40. Warren S. L., Handel L. M., Nelson W. J. Elevated expression of pp60c-src alters a selective morphogenetic property of epithelial cells in vitro without a mitogenic effect. Mol Cell Biol. 1988 Feb;8(2):632–646. doi: 10.1128/mcb.8.2.632. [DOI] [PMC free article] [PubMed] [Google Scholar]

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