Summary
Ectoderm explants from early gastrula stages of Xenopus laevis were induced with a neutralizing factor. The factor was isolated from Xenopus gastrulae and partially purified by chromatography on DEAE cellulose. The ectoderm was cultured for different periods of time and then homogenized. Protein kinase activity was determined in the homogenates from induced and control explants with histone H 1 or C-terminal peptide derived from histone H 1 as substrates. The C-terminal peptide is a more specific substrate for protein kinase C, whereas histoneH 1 is a substrate for cAMP/cGMP-dependent protein kinases as well protein kinase C. With both substrates the enzyme activity increases after induction. With the C-terminal peptide as the substrate the protein kinase activity is lower, but its relative increase after induction higher. This suggests that besides cAMP/cGMP dependent protein kinases protein kinase C or related enzymes are involved in the neural induction and differentiation processes. This corresponds to previous experiments which have shown that treatment of ectoderm with phorbol myristate acetate, an activator of protein kinase C and protein kinase C related enzymes, initiates neural differentiation. Endogeneous substrates, which are more intensively phosphorylated after induction are proteins with apparent molecular weights 21 kDa and 31 kDa. Addition of protein kinase C to the induced and control homogenates abolishes the difference in the phosphorylation rate of these proteins.
Similar content being viewed by others
References
Becker U, Tiedemann H, Tiedemann Hi (1959) Versuche zur Determination von embryonalem Amphibiengewebe durch Induktionsstoffe in Lösung. Z Naturforsch [B] 14:608–609
Born J, Hoppe P, Janeczek J, Tiedemann H (1986) Covalent coupling of neuralizing factors from Xenopus to sepharose beads:no decrease of inducing activity. Cell Differ 19:97–101
Bradford M (1976) A rapid and sensitive method for the quantitation of protein dye binding. Anal Biochem 72:248–254
Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y (1982) Direct activation of calcium-activated phospholipid-dependent protein kinase by tumor promoting phorbol esters. J Biol Chem 257:7847–7851
Coussens L, Parker PJ, Rhee L, Yang Feng TL, Chen E, Waterfield MD, Francke U, Ullrich A (1986) Multiple, distinct forms of bovine and human protein kinase C suggests diversity in cellular signaling pathways. Science 233:859–866
Davids M, Loppnow B, Tiedemann H, Tiedemann Hi (1987) Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester. Roux's Arch Dev Biol 196:137–140
Glynn B, Colliton J, McDermott J, Witters LA (1985) Assay of protein kinase C with an N-bromosuccinimide-cleavage fragment of histone H 1. Biochem J 231:489–492
Grinstein S, Cohen S, Goetz JD, Rothstein A, Gelfand EW (1985) Characterization of the activation of Na+/H+ exchange in lymphocytes by phorbolesters:Change in cytoplasmic pH dependence of the antiport. Proc Natl Acad Sci USA 82:1429–1433
Grunz H, Tiedemann Hi (1977) Influence of cyclic nucleotides on amphibian ectoderm. Roux's Arch Dev Biol 181:261–265
Hidaka H, Tanaka T, Onoda K, Hagiwara M, Watanabe M, Ohta H, Ito Y, Tsurudome M, Yoshida T (1988) Cell type-specific expression of protein kinase C isozymes in the rabbit cerebellum. J Biol Chem 263:4523–4526
Iwasa Y, Takai Y, Kikkawa U, Nishizuka Y (1980) Phosphorylation of calf thymus H 1 histone by calcium-activated phospholipid-dependent protein kinase. Biochem Biophys Res Commun 96:180–197
John M, Janeczek J, Born J, Hoppe P, Tiedemann H, Tiedemann Hi (1983) Neural induction in amphibians. Transmission of a neuralizing factor. Wilhelm Roux's Arch 192:45–47
Kaibuchi K, Takai Y, Sawamura M, Hoshijima M, Fujikura T, Nishizuka Y (1983) Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation. J Biol Chem 258:6701–6704
Kikkawa U, Minakuchis R, Takai Y, Nishizuka Y (1983) Calcium-activated, phospholipid-dependent protein kinase (protein kinase C from rat brain). Meth Enzymol 99:288–298
Kishimoto A, Takai Y, Mori T, Kikkawa U, Nishizuka Y (1980) Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem 255:2273–2276
Kishimoto A, Kajikawa N, Shiota M, Nishizuka Y (1983) Proteolytic activation of Ca-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease. J Biol Chem 258:1156–1164
Kitano T, Go M, Kikkawa U, Nishizuka Y (1986) Assay and purification of protein kinase C. Meth Enzymol 124:349–359
Knopf JL, Lee M-H, Sultzman LA, Kriz RW, Loomis CR, Hewick RM, Bell RM (1986) Cloning and expression of multiple protein kinase C cDNAs. Cell 46:491–502
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Moolenaar WH, Tertoolen LGJ, deLaat SW (1984) Phorbolester and diacylglycerol mimic growth factors in raising cytoplasmic pH. Nature 312:371–373
Niedel JE, Kuhn LJ, Vandenbank GR (1983) Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci USA 80:36–40
Nieuwkoop PD, Faber J (1956) Normal tables of Xenopus laevis (Daudin). North-Holland, Amsterdam
Nishizuka Y (1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308:693–698
O'Farrell PH (1975) High resolution two-dimensional elektrophoresis of proteins. J Biol Chem 250:4007–4021
Ohno S, Kawasaki H, Imajoh Sh, Suzuki K (1987) Tissue-specific expression of three distinct types of rabbit protein kinase C. Nature 325:161–166
Ohno S, Akita Y, Konno Y, Imajoh Sh, Suzuki K (1988) A novel phorbol ester Receptor/protein kinase, nPKC, distantly related to the protein kinase C family. Cell 53:731–741
Ono Y, Kurokawa T, Fujii T, Kawahara K, Igarashi K, Kikkawa U, Ogita K, Nishizuka Y (1986) Two types of complementary DNAs of rat brain protein kinase C. FEBS Lett 206:347–352
Ono Y, Kikkawa U, Ogita K, Fujii T, Kurokawa T, Asaoka Y, Sekiguchi K, Ase K, Igarashi K, Nishizuka Y (1987) Expression and properties of two types of protein kinase C: Alternative splicing from a single gene. Science 236:1116–1120
Parker PJ, Stabel S, Waterfield MD (1984) Purification to homogeneity of protein kinase C from bovine brain — identity with the phorbol ester receptor. EMBO J 3:953–959
Saxen L, Toivonen S (1962) Primary embryonic induction. Academic Press, London
Siffert W, Siffert G, Scheid P (1987) Activation of Na+/H+ exchange in human platelets stimulated by thrombin and a phorbolester. Biochem J 241:301–303
Spemann H (1938) Embryonic development and induction. Yale University Press, New Haven
Suzuki A, Kuwabara K (1974) Mitotic activity and cell proliferation in primary induction of newt embryo. Dev Growth Differ 16:29–36
Swann K, Whitaker M (1985) Stimulation of the Na/H exchanger of sea urchin eggs by phorbolester. Nature 314:374–377
Tiedemann H, Becker U, Tiedemann Hi (1963) Chromatographic separation of a hind-brain inducing substance into mesodermal and neural-inducing subfractions. Biochim Biophys Acta 74:557–560
Tiedemann Hi, Born J (1978) Biological activity of vegetalizing and neuralizing inducing factors after binding to BAC-cellulose and CNBr-sepharose. Wilhelm Roux's Arch 184:285–299
Waddell WJ, Chapel MD, Hill NC (1956) A simple ultraviolet spectrophotometric method for the determination of protein. J Lab Clin Med 48:311–314
Witters LA, Blackshear PJ (1987) Protein kinase C-mediated phosphorylation in intact cells. Methods Enzymol 141:412–424
Yamada T, Takata K (1961) A technique for testing macromolecular samples in solution for morphogenetic effects on the isolated ectoderm of the amphibian gastrula. Dev Biol 3:411–423
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Davids, M. Protein kinases in amphibian ectoderm induced for neural differentiation. Roux's Arch Dev Biol 197, 339–344 (1988). https://doi.org/10.1007/BF00375953
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00375953