Effect of okadaic acid on O-linked N-acetylglucosamine levels in a neuroblastoma cell line

• Published in: Biochimica et biophysica acta 1999-10, Vol.1472 (1), p.71-81
• Main Authors: Lefebvre, Tony, Alonso, Catherine, Mahboub, Saı̈d, Dupire, Marie-Joëlle, Zanetta, Jean-Pierre, Caillet-Boudin, Marie-Laure, Michalski, Jean-Claude
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - metabolism; Animals; COS Cells; Cytosolic glycoprotein; Galactose - metabolism; HeLa Cells; Humans; Neuroblastoma - metabolism; Neuroblastoma - pathology; Nuclear glycoprotein; Nuclear translocation; O- N-Acetylglucosamine; Okadaic acid; Okadaic Acid - pharmacology; Phosphorylation; Subcellular Fractions - metabolism; Tritium; Tumor Cells, Cultured
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(99)00105-1

O-Linked N-Acetylglucosamine ( O-GlcNAc) is a major form of post-translational modification found in nuclear and cytoplasmic proteins. Several authors have advanced the hypothesis according to which phosphorylation and O-GlcNAc glycosylation are reciprocally related to one another [1,2]. In order to test this hypothesis we have investigated the effect of a broad spectrum phosphatase inhibitor, okadaic acid (OA), generally used to induce protein hyperphosphorylation, on the GlcNAc content of cellular glycoproteins. We demonstrate that in neuronal cells lines OA decreases the level of O-GlcNAc in both nuclear and cytoplasmic proteins with a greater effect in the nuclear fraction. This phenomenon was demonstrated by the use of three different procedures for the detection of O-GlcNAc in conjunction with a systematic treatment with PNGase F. O-Linked GlcNAc was characterized using respectively lectin staining with WGA, galactosyltransferase labeling and metabolic labeling of cultured cells with [ 3H]glucosamine. Although the effects on individual proteins varied, a less pronounced effect was observed on HeLa or COS cell total homogenates. When Kelly cells were treated with OA, the major observation was a decrease in O-GlcNAc content of nuclear proteins. The measurement of the UDP-GlcNAc level clearly demonstrates that the decrease on the O-GlcNAc level in the neuroblastoma cell line after treatment with okadaic acid is not a consequence of the modification of the UDP-GlcNAc pool.