Ganglioside-modulated protein phosphorylation. Partial purification and characterization of a ganglioside-inhibited protein kinase in brain.

Abstract

A novel protein kinase which could be inhibited specifically by gangliosides has been partially purified from the particulate fraction of guinea pig brain through extraction with nonionic detergent, ion-exchange chromatography, hydrophobic chromatography, hydroxylapatite chromatography, and gel filtration.The ganglioside-inhibited kinase activity was eluted with a Stokes radius of 29-30 A, corresponding to a globular protein of approximately 40,000 in molecular weight.Only gangliosides, especially polysialogangliosides, are potent inhibitors for this enzyme preparation.The modulatory action of the glycolipids on the kinase activity is not time-dependent, indicating that the mode of inhibition may not be mediated through a ganglioside-dependent proteolytic process.Calcium was not required for the inhibitory effects of the various gangliosides tested, suggesting that prior formation of Caa+.ganglioside complexes are not necessary.The partially purified ganglioside-inhibited protein kinase can phosphorylate exogenous substrates such as a synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly.The optimal pH for this reaction occurred between 7.0 and 7.4.M e + (6-10 mM) is required for the enzymic activity and cannot be substituted by Mn2+.Although the nature of the authentic substrates for this ganglioside-inhibited protein kinase is yet unknown, a search for other potential substrates revealed that the synthetic peptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val was the best phosphate acceptor tested so far.Other substrate specificity studies also showed that the ganglioside-inhibited protein kinase is distinct from either the ganglioside-stimulated protein kinase or protein kinase C. Thus, it is possible that gangliosides can act as bio-modulators which may confer a synchronistic action on these three different protein kinase systems.Gangliosides, a class of sialic acid-containing glycosphingolipids ubiquitous in eukaryotes, are found in particularly high concentrations in the central nervous system (see Refs. 1-5 for review).In neuronal plasma membranes, gangliosides comprise approximately 10% of the total lipids and two-thirds of the glycoconjugate sialic acid contents (6).These glycolipids also are found in myelin (7-9).Because of their unique and asymmetric distribution in the nervous system, gangliosides have been suggested to play important roles in neuronal functions such as modulation of synaptic transmission, ion permeability and fluxes, neuritogenesis, and the development