Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Apr 15;219(2):573–581. doi: 10.1042/bj2190573

Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-induced phosphorylation of proteins in purified microvillus membranes of rabbit ileum.

M Donowitz, M E Cohen, R Gudewich, L Taylor, G W Sharp
PMCID: PMC1153515  PMID: 6331391

Abstract

Evidence is available to suggest that Ca2+-calmodulin and cyclic nucleotides are involved in the regulation of ion transport in rabbit ileum. Since both Ca2+-calmodulin and cyclic nucleotides exert many of their effects by phosphorylation, the effects of Ca2+-calmodulin and cyclic nucleotides on phosphorylation of purified microvillus membrane from rabbit ileal mucosa were evaluated. Ca2+-calmodulin increased phosphorylation of five microvillus-membrane peptides, with Mr values of 137000, 77000, 58000, 53000 and 50000. The increases in phosphorylation caused by Ca2+-calmodulin were: Mr-137000 peptide, 111 +/- 26%; Mr-77000 peptide, 71 +/- 17%; Mr-58000 peptide, 51 +/- 8%; Mr-53000 peptide, 113 +/- 20%. These increases were maximal at 1 microM-calmodulin and 0.3-0.9 microM free Ca2+; concentrations of Ca2+ causing half-maximal effects on phosphorylation for the different peptides were 0.06-0.12 microM. Cyclic AMP and cyclic GMP increased phosphorylation of two peptides, of Mr 137000 and 85000. The concentrations of cyclic nucleotides giving half-maximal phosphorylation of the Mr-137000 peptide were 0.3 microM-cyclic AMP and 4.6 microM-cyclic GMP, and for the Mr-85000 peptide, 3.9 microM-cyclic AMP and 0.05 microM-cyclic GMP. The maximal increase in phosphorylation of the Mr-137000 peptide was 200% for cyclic AMP and 95% for cyclic GMP, and that of the Mr-85000 peptide was 220% for cyclic AMP and 120% for cyclic GMP. These studies demonstrate the existence of Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-dependent protein kinases and substrate proteins in purified rabbit ileal microvillus membranes and that Ca2+ can regulate phosphorylation of these proteins over the presumed physiological concentration range of cytosol free Ca2+.

Full text

PDF
573

Images in this article

576Fig. 1.
on p.576
578Fig. 4.
on p.578

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ALBERS R. W., RODRIGUEZDE LORES, DEROBERTIS E. SODIUM-POTASSIUM-ACTIVATED ATPASE AND POTASSIUM-ACTIVATED P-NITROPHENYLPHOSPHATASE: A COMPARISON OF THEIR SUBCELLULAR LOCALIZATIONS IN RAT BRAIN. Proc Natl Acad Sci U S A. 1965 Mar;53:557–564. doi: 10.1073/pnas.53.3.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Avruch J., Leone G. R., Martin D. B. Identification and subcellular distribution of adipocyte peptides and phosphopeptides. J Biol Chem. 1976 Mar 10;251(5):1505–1510. [PubMed] [Google Scholar]
  3. Bartfai T. Preparation of metal-chelate complexes and the design of steady-state kinetic experiments involving metal nucleotide complexes. Adv Cyclic Nucleotide Res. 1979;10:219–242. [PubMed] [Google Scholar]
  4. Beavo J. A., Bechtel P. J., Krebs E. G. Activation of protein kinase by physiological concentrations of cyclic AMP. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3580–3583. doi: 10.1073/pnas.71.9.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Becker G. L., Fiskum G., Lehninger A. L. Regulation of free Ca2+ by liver mitochondria and endoplasmic reticulum. J Biol Chem. 1980 Oct 10;255(19):9009–9012. [PubMed] [Google Scholar]
  6. Bolton J. E., Field M. Ca ionophore-stimulated ion secretion in rabbit ileal mucosa: relation to actions of cyclic 3',5'-AMP and carbamylcholine. J Membr Biol. 1977 Jun 30;35(2):159–173. doi: 10.1007/BF01869947. [DOI] [PubMed] [Google Scholar]
  7. Chrisman T. D., Vandenheede J. R., Khandelwal R. L., Gella F. J., Upton J. D., Krebs E. G. Purification and regulatory properties of liver phosphorylase kinase. Adv Enzyme Regul. 1980;18:145–159. doi: 10.1016/0065-2571(80)90013-8. [DOI] [PubMed] [Google Scholar]
  8. Costa M. R., Casnellie J. E., Catterall W. A. Selective phosphorylation of the alpha subunit of the sodium channel by cAMP-dependent protein kinase. J Biol Chem. 1982 Jul 25;257(14):7918–7921. [PubMed] [Google Scholar]
  9. Donowitz M., Asarkof N. Calcium dependence of basal electrolyte transport in rabbit ileum. Am J Physiol. 1982 Jul;243(1):G28–G35. doi: 10.1152/ajpgi.1982.243.1.G28. [DOI] [PubMed] [Google Scholar]
  10. Donowitz M., Asarkof N., Pike G. Calcium dependence of serotonin-induced changes in rabbit ileal electrolyte transport. J Clin Invest. 1980 Aug;66(2):341–352. doi: 10.1172/JCI109862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donowitz M. Ca2+ in the control of active intestinal Na and Cl transport: involvement in neurohumoral action. Am J Physiol. 1983 Aug;245(2):G165–G177. doi: 10.1152/ajpgi.1983.245.2.G165. [DOI] [PubMed] [Google Scholar]
  12. Donowitz M., Cusolito S., Battisti L., Fogel R., Sharp G. W. Dopamine stimulation of active Na and Cl absorption in rabbit ileum: interaction with alpha 2-adrenergic and specific dopamine receptors. J Clin Invest. 1982 Apr;69(4):1008–1016. doi: 10.1172/JCI110504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Donowitz M., Fogel R., Battisti L., Asarkof N. The neurohumoral secretagogues carbachol, substance P and neurotensin increase Ca++ influx and calcium content in rabbit ileum. Life Sci. 1982 Nov 1;31(18):1929–1937. doi: 10.1016/0024-3205(82)90031-5. [DOI] [PubMed] [Google Scholar]
  14. Forstner G. G., Sabesin S. M., Isselbacher K. J. Rat intestinal microvillus membranes. Purification and biochemical characterization. Biochem J. 1968 Jan;106(2):381–390. doi: 10.1042/bj1060381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frizzell R. A., Field M., Schultz S. G. Sodium-coupled chloride transport by epithelial tissues. Am J Physiol. 1979 Jan;236(1):F1–F8. doi: 10.1152/ajprenal.1979.236.1.F1. [DOI] [PubMed] [Google Scholar]
  16. Greengard P. Phosphorylated proteins as physiological effectors. Science. 1978 Jan 13;199(4325):146–152. doi: 10.1126/science.22932. [DOI] [PubMed] [Google Scholar]
  17. Hofmann F., Gensheimer H. P. Cyclic AMP-dependent protein kinase does not phosphorylate cyclic GMP-dependent protein kinase in vitro. FEBS Lett. 1983 Jan 10;151(1):71–75. doi: 10.1016/0014-5793(83)80345-7. [DOI] [PubMed] [Google Scholar]
  18. Hopfer U., Nelson K., Perrotto J., Isselbacher K. J. Glucose transport in isolated brush border membrane from rat small intestine. J Biol Chem. 1973 Jan 10;248(1):25–32. [PubMed] [Google Scholar]
  19. Ilundain A., Naftalin R. J. Role of Ca(2+)-dependent regulator protein in intestinal secretion. Nature. 1979 May 31;279(5712):446–448. doi: 10.1038/279446a0. [DOI] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lambin P. Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate. Anal Biochem. 1978 Mar;85(1):114–125. doi: 10.1016/0003-2697(78)90281-6. [DOI] [PubMed] [Google Scholar]
  23. Leavis P. C., Lehrer S. S. Intrinsic fluorescence studies on troponin C. Arch Biochem Biophys. 1978 Apr 15;187(1):243–251. doi: 10.1016/0003-9861(78)90030-9. [DOI] [PubMed] [Google Scholar]
  24. Lee C. O. Ionic activities in cardiac muscle cells and application of ion-selective microelectrodes. Am J Physiol. 1981 Oct;241(4):H459–H478. doi: 10.1152/ajpheart.1981.241.4.H459. [DOI] [PubMed] [Google Scholar]
  25. Lee C. O., Taylor A., Windhager E. E. Cytosolic calcium ion activity in epithelial cells of Necturus kidney. Nature. 1980 Oct 30;287(5785):859–861. doi: 10.1038/287859a0. [DOI] [PubMed] [Google Scholar]
  26. Lee C. O., Uhm D. Y., Dresdner K. Sodium-calcium exchange in rabbit heart muscle cells: direct measurement of sarcoplasmic Ca2+ activity. Science. 1980 Aug 8;209(4457):699–701. doi: 10.1126/science.7394527. [DOI] [PubMed] [Google Scholar]
  27. Messer M., Dahlqvist A. A one-step ultramicro method for the assay of intestinal disaccharidases. Anal Biochem. 1966 Mar;14(3):376–392. doi: 10.1016/0003-2697(66)90280-6. [DOI] [PubMed] [Google Scholar]
  28. Mircheff A. K., Wright E. M. Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities. J Membr Biol. 1976 Sep 17;28(4):309–333. doi: 10.1007/BF01869703. [DOI] [PubMed] [Google Scholar]
  29. Mooseker M. S., Howe C. L. The brush border of intestinal epithelium: a model system for analysis of cell-surface architecture and motility. Methods Cell Biol. 1982;25(Pt B):143–174. doi: 10.1016/s0091-679x(08)61424-7. [DOI] [PubMed] [Google Scholar]
  30. Omura T., Takesue S. A new method for simultaneous purification of cytochrome b5 and NADPH-cytochrome c reductase from rat liver microsomes. J Biochem. 1970 Feb;67(2):249–257. doi: 10.1093/oxfordjournals.jbchem.a129248. [DOI] [PubMed] [Google Scholar]
  31. Sharp G., Stiel D., Peters T. J. Cytochemical studies on the localization of alkaline phosphatase and HCO-3-activated adenosine triphosphatase in the brush border membrane of rat duodenal enterocytes. Histochem J. 1983 Nov;15(11):1131–1139. doi: 10.1007/BF01003976. [DOI] [PubMed] [Google Scholar]
  32. Shlatz L. J., Kimberg D. V., Cattieu K. A. Phosphorylation of specific rat intestinal microvillus and basal-lateral membrane proteins by cyclic nucleotides. Gastroenterology. 1979 Feb;76(2):293–299. [PubMed] [Google Scholar]
  33. Smith P. L., Blumberg J. B., Stoff J. S., Field M. Antisecretory effects of indomethacin on rabbit ileal mucosa in vitro. Gastroenterology. 1981 Feb;80(2):356–365. [PubMed] [Google Scholar]
  34. Stewart A. A., Ingebritsen T. S., Manalan A., Klee C. B., Cohen P. Discovery of a Ca2+- and calmodulin-dependent protein phosphatase: probable identity with calcineurin (CaM-BP80). FEBS Lett. 1982 Jan 11;137(1):80–84. doi: 10.1016/0014-5793(82)80319-0. [DOI] [PubMed] [Google Scholar]
  35. Tada M., Kirchberger M. A., Katz A. M. Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1975 Apr 10;250(7):2640–2647. [PubMed] [Google Scholar]
  36. Takai Y., Kishimoto A., Iwasa Y., Kawahara Y., Mori T., Nishizuka Y. Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J Biol Chem. 1979 May 25;254(10):3692–3695. [PubMed] [Google Scholar]
  37. Taylor L., Guerina V. J., Donowitz M., Cohen M., Sharp G. W. Calcium and calmodulin-dependent protein phosphorylation in rabbit ileum. FEBS Lett. 1981 Aug 31;131(2):322–324. doi: 10.1016/0014-5793(81)80395-x. [DOI] [PubMed] [Google Scholar]
  38. WURTMAN R. J., AXELROD J. A SENSITIVE AND SPECIFIC ASSAY FOR THE ESTIMATION OF MONOAMINE OXIDASE. Biochem Pharmacol. 1963 Dec;12:1439–1441. doi: 10.1016/0006-2952(63)90215-6. [DOI] [PubMed] [Google Scholar]
  39. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  40. Weiser M. M., Neumeier M. M., Quaroni A., Kirsch K. Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane. J Cell Biol. 1978 Jun;77(3):722–734. doi: 10.1083/jcb.77.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. de Jonge H. R. Cyclic GMP-dependent protein kinase in intestinal brushborders. Adv Cyclic Nucleotide Res. 1981;14:315–333. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES