Protein kinases in amphibian ectoderm induced for neural differentiation

Abstract

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.

Pluripotent cells (stem cells) and their determination and differentiation in early vertebrate embryogenesis†

“…The enzyme protein kinase C (PKC), a serine–threonine kinase, translocates to the plasma membrane in PMA‐treated explants, which correlates with its activation (Otte et al 1988). The activity of PKC is enhanced in X. laevis ectoderm explants treated with neuralizing factor (Davids 1988). PKC can upregulate or downregulate Ca 2+ channels in many systems.…”

Partial characterization of neural-inducing factors from Xenopus gastrulae Evidence for a larger protein complex containing the factor

“…The fractions were dissolved in 6 M urea, dialysed against distilled water and the pellets prepared for the test as described (Janeczek et al 1984b;Asahi et al 1979). The native proteins of the supernatant can be tested in solution on isolated ectoderm (Davids 1988). The other fractions contain denatured proteins which have a low solubility in buffer solutions at neutral pH.…”

“…The factor is present in oocytes in a masked biologically inactive form (Born et al 1989) and is in part activated after cleavage and secreted from the inducing mesoderm (SaxOn 1961;SaxOn 1989;John et al 1983). It acts on the plasma membrane of the reacting ectoderm by a signal transduction mechanism (Born et al 1986;Davids et al 1987;Davids 1988;Otte et al 1988;Tiedemann 1990). …”