REGULATION OF GANGLIOSIDE METABOLISM BY PHOSPHORYLATION AND DEPHOSPHORYLATION

Cell differentiation is frequently accompanied by alterations in the c omposition of gangliosides in the plasma membrane resulting from a reg ulation of the enzyme activities involved. The regulation of CMP-NeuAc :GM1 alpha 2-3-sialyltransferase (ST-IV) and UDP-GalNAc:GM3 N-acetylga lactosaminyltransferase (Gal-NAc-T) by the degree of enzyme phosphoryl ation was analyzed by determination of the enzyme activity on incubati on of NG108-15 cells with various protein phosphatase inhibitors (okad aic acid and orthovanadate) or protein kinase activators (phorbol este r and forskolin), Incubation with okadaic acid, but not with orthovana date, inhibited the ST-IV activity to 45% of that of control cells wit h t(1/2) = 60 min for the inactivation reaction. This indicates a rapi d hyperphosphorylation of ST-IV due to the inhibition of a serine/thre onine-specific phosphatase. A similar rate of inactivation was found o n stimulation of protein kinase C with phorbol ester, In contrast to S T-IV, the activity of GalNAc-T was increased on stimulation of intrace llular phosphorylation systems. The fastest activation of GalNAc-T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up-regulation of GalNAc-T in con junction with down-regulation of ST-IV by stimulation of phosphorylati on is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation wi th the cell density. This assumption was corroborated by metabolic lab eling studies with radioactive ganglioside precursors indicating an en hancement of the relative amount of a-series gangliosides subsequent t o GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskol in. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108-15 cells can be specifically regulated by both protein kinase A- and protein kinase C-related phos phorylation systems.