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. 1985 Oct 1;101(4):1212–1218. doi: 10.1083/jcb.101.4.1212

Calmodulin-dependent protein phosphatase: immunocytochemical localization in chick retina

PMCID: PMC2113899  PMID: 3900087

Abstract

Calmodulin-dependent protein phosphatase, previously called CaM-BP80 or calcineurin, is present in high concentrations in the central nervous system. The level of the phosphatase has been shown by radioimmunoassay to increase during development in the retinas of embryonic and hatching chicks (Tallant, E.A., and W.Y. Cheung, 1983, Biochemistry, 22:3630- 3635). The aims of this study are to immunocytochemically localize the phosphatase in developing and mature retinas and to determine if the phosphatase is present in fractions of retinal synaptic membranes and synaptic junctions. Vibratome slices of fixed chick retina and Western blots of detergent-solubilized retinal fractions are both treated sequentially with rabbit primary antisera and goat anti-rabbit Fab fragments conjugated to peroxidase, and then reacted with hydrogen peroxide and diaminobenzidine. The tissue slices are further processed for electron microscopy. This paper demonstrates the presence of peroxidase reaction product in the retina just before synapse formation. In the outer plexiform layer the product is confined to photoreceptor synaptic terminals, whereas in the inner plexiform layer it is present in synaptic terminals of bipolar cells and in dendrites of ganglion cells. In this latter site the product is present postsynaptically at bipolar and amacrine synapses. The phosphatase is detected in Western blots of both synaptic plasma membrane and synaptic junction fractions.

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Selected References

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

  1. Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
  2. Cohen P. The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature. 1982 Apr 15;296(5858):613–620. doi: 10.1038/296613a0. [DOI] [PubMed] [Google Scholar]
  3. Cohen R. S., Blomberg F., Berzins K., Siekevitz P. The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition. J Cell Biol. 1977 Jul;74(1):181–203. doi: 10.1083/jcb.74.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. De Camilli P., Harris S. M., Jr, Huttner W. B., Greengard P. Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J Cell Biol. 1983 May;96(5):1355–1373. doi: 10.1083/jcb.96.5.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeLorenzo R. J. Calmodulin in neurotransmitter release and synaptic function. Fed Proc. 1982 May;41(7):2265–2272. [PubMed] [Google Scholar]
  6. Goldenring J. R., McGuire J. S., Jr, DeLorenzo R. J. Identification of the major postsynaptic density protein as homologous with the major calmodulin-binding subunit of a calmodulin-dependent protein kinase. J Neurochem. 1984 Apr;42(4):1077–1084. doi: 10.1111/j.1471-4159.1984.tb12713.x. [DOI] [PubMed] [Google Scholar]
  7. Grab D. J., Carlin R. K., Siekevitz P. Function of a calmodulin in postsynaptic densities. II. Presence of a calmodulin-activatable protein kinase activity. J Cell Biol. 1981 Jun;89(3):440–448. doi: 10.1083/jcb.89.3.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grab D. J., Carlin R. K., Siekevitz P. Function of calmodulin in postsynaptic densities. I. Presence of a calmodulin-activatable cyclic nucleotide phosphodiesterase activity. J Cell Biol. 1981 Jun;89(3):433–439. doi: 10.1083/jcb.89.3.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grab D. J., Carlin R. K., Siekevitz P. The presence and functions of calmodulin in the postsynaptic density. Ann N Y Acad Sci. 1980;356:55–72. doi: 10.1111/j.1749-6632.1980.tb29599.x. [DOI] [PubMed] [Google Scholar]
  10. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  11. Gupta R. C., Khandelwal R. L., Sulakhe P. V. Intrinsic phosphatase activity of bovine brain calcineurin requires a tightly bound trace metal. FEBS Lett. 1984 Apr 24;169(2):251–255. doi: 10.1016/0014-5793(84)80328-2. [DOI] [PubMed] [Google Scholar]
  12. Kelly W. G., Passaniti A., Woods J. W., Daiss J. L., Roth T. F. Tubulin as a molecular component of coated vesicles. J Cell Biol. 1983 Oct;97(4):1191–1199. doi: 10.1083/jcb.97.4.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kennedy M. B., Bennett M. K., Erondu N. E. Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7357–7361. doi: 10.1073/pnas.80.23.7357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kennedy M. B., McGuinness T., Greengard P. A calcium/calmodulin-dependent protein kinase from mammalian brain that phosphorylates Synapsin I: partial purification and characterization. J Neurosci. 1983 Apr;3(4):818–831. doi: 10.1523/JNEUROSCI.03-04-00818.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. King M. M., Huang C. Y., Chock P. B., Nairn A. C., Hemmings H. C., Jr, Chan K. F., Greengard P. Mammalian brain phosphoproteins as substrates for calcineurin. J Biol Chem. 1984 Jul 10;259(13):8080–8083. [PubMed] [Google Scholar]
  16. Klee C. B., Crouch T. H., Krinks M. H. Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6270–6273. doi: 10.1073/pnas.76.12.6270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kolb H. The inner plexiform layer in the retina of the cat: electron microscopic observations. J Neurocytol. 1979 Jun;8(3):295–329. doi: 10.1007/BF01236124. [DOI] [PubMed] [Google Scholar]
  18. Mahler H. R., Kleine L. P., Ratner N., Sorensen R. G. Identification and topography of synaptic phosphoproteins. Prog Brain Res. 1982;56:27–48. doi: 10.1016/S0079-6123(08)63767-X. [DOI] [PubMed] [Google Scholar]
  19. Pallen C. J., Wang J. H. Calmodulin-stimulated dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine by calcineurin. J Biol Chem. 1983 Jul 25;258(14):8550–8553. [PubMed] [Google Scholar]
  20. 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]
  21. Tallant E. A., Cheung W. Y. Calmodulin-dependent protein phosphatase: a developmental study. Biochemistry. 1983 Jul 19;22(15):3630–3635. doi: 10.1021/bi00284a014. [DOI] [PubMed] [Google Scholar]
  22. Tallant E. A., Wallace R. W., Cheung W. Y. Purification and radioimmunoassay of calmodulin-dependent protein phosphatase from bovine brain. Methods Enzymol. 1983;102:244–256. doi: 10.1016/s0076-6879(83)02025-x. [DOI] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wallace R. W., Lynch T. J., Tallant E. A., Cheung W. Y. An endogenous inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. Arch Biochem Biophys. 1978 Apr 30;187(2):328–334. doi: 10.1016/0003-9861(78)90042-5. [DOI] [PubMed] [Google Scholar]
  25. Wallace R. W., Tallant E. A., Cheung W. Y. High levels of a heat-labile calmodulin-binding protein (CaM-BP80) in bovine neostriatum. Biochemistry. 1980 Apr 29;19(9):1831–1837. doi: 10.1021/bi00550a016. [DOI] [PubMed] [Google Scholar]
  26. Winkler M. A., Merat D. L., Tallant E. A., Hawkins S., Cheung W. Y. Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A. Proc Natl Acad Sci U S A. 1984 May;81(10):3054–3058. doi: 10.1073/pnas.81.10.3054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wood J. G., Wallace R. W., Whitaker J. N., Cheung W. Y. Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain. J Cell Biol. 1980 Jan;84(1):66–76. doi: 10.1083/jcb.84.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wood J. G., Wallace R. W., Whitaker J. N., Cheung W. Y. Immunocytochemical localization of calmodulin in regions of rodent brain. Ann N Y Acad Sci. 1980;356:75–82. doi: 10.1111/j.1749-6632.1980.tb29601.x. [DOI] [PubMed] [Google Scholar]
  29. Yang S. D., Tallant E. A., Cheung W. Y. Calcineurin is a calmodulin-dependent protein phosphatase. Biochem Biophys Res Commun. 1982 Jun 30;106(4):1419–1425. doi: 10.1016/0006-291x(82)91272-4. [DOI] [PubMed] [Google Scholar]

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