Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins—a role in nuclear localization

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Abstract

Both phosphorylation and O-GlcNAc glycosylation posttranslationally modify microtubule-associated Tau proteins. Whereas the hyperphosphorylation of these proteins that occurs in Alzheimer's disease is well characterized, little is known about the O-GlcNAc glycosylation. The present study demonstrates that a balance exists between phosphorylation and O-GlcNAc glycosylation of Tau proteins, and furthermore that a dysfunction of this balance correlates with reduced nuclear localization.

The affinity of Tau proteins for WGA lectin, together with evidence from [3H]-galactose transfer and analysis of beta-eliminated products, demonstrated the presence of O-GlcNAc residues on both cytosolic and nuclear Tau proteins. In addition, our data indicated the existence of a balance between phosphorylation and O-GlcNAc glycosylation events. Indeed, as demonstrated by 2D-electrophoresis and Western blotting, O-GlcNAc residues were mainly located on the less phosphorylated Tau 441 variants, whereas the more phosphorylated forms were devoid of O-GlcNAc residues. Furthermore, the Tau protein hyperphosphorylation induced by cellular okadaic acid treatment was correlated with reduced incorporation of O-GlcNAc residues into Tau proteins and with diminished Tau transfer into the nucleus. Hence, this paper establishes a direct relationship between O-GlcNAc glycosylation, phosphorylation and cellular localization of Tau proteins.

Introduction

Tau proteins belong to the family of brain microtubule-associated proteins involved in polymerisation and stability of neuronal microtubules. In adult brain, six different Tau isoforms (ranging from 352 to 441 amino acids in length) are present and arise from alternative splicing of a common, primary transcript [1]. A long Tau isoform (named big Tau), containing an additional insert in the middle part of the protein, is found only in the peripheral nervous tissue [2]. Another Tau protein, named small Tau because of its 26–30 kDa apparent molecular mass, has only been observed in the nuclei of neuroblastoma cells [3]. In contrast, the six adult Tau isoforms were predominantly found in the cytoplasmic compartment, although some of these isoforms were also found within the nucleus [4].

Tau proteins can be posttranslationally modified by events such as phosphorylation, N- and O-linked glycosylation, ubiquitination, glycation, proteolysis, etc. (reviewed in Ref. [5]). Under normal circumstances, phosphorylation of Tau proteins controls microtubule polymerisation, whereas abnormal phosphorylation of Tau proteins occurs during neurodegenerative diseases such as Alzheimer's disease, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) (reviewed in Ref. [5]). These abnormally phosphorylated Tau proteins are the main components of filaments that accumulate in degenerating neurons. A cellular model of Alzheimer-type Tau protein phosphorylation can be obtained by treating human neuroblastoma cells with okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A [6], [7], [8].

In addition to this hyperphosphorylation phenomenon, Tau proteins from Alzheimer brains (but not from normal ones) are glycosylated with N- and O-linked saccharides [9], [10]. In contrast, another type of glycosylation-O-GlcNAc glycosylation-was reported on normal Tau proteins [11], albeit in a bovine system. Despite a high degree of sequence homology, bovine Tau proteins differ from human ones in terms of (i) the number of major Tau isoforms (four and six, respectively) [12], (ii) a possible insert due to alternative splicing of exon 8 [13], (iii) their immunoreactivity [14] and (iv) their conformation [15]. As Tau proteins are involved in human neurodegenerative diseases such as Alzheimer's disease, we felt that it was important to determine whether human Tau proteins were modified by O-GlcNAc glycosylation.

O-GlcNAc glycosylation occurs on numerous cytoplasmic and nuclear proteins, such as cytoskeletal proteins [16], [17], [18], nuclear pore proteins [19], transcription factors [20], [21] and viral proteins [22], [23]. It has been suggested that this type of glycosylation shares certain features with protein phosphorylation [24], mainly by occupying the same or neighbouring sites on the peptide backbone. A global relationship between O-GlcNAc and O-phosphate has been demonstrated using different inhibitors. We have previously demonstrated that the O-GlcNAc level decreased in several Kelly cell neuroblastoma proteins following their hyperphosphorylation induced by OA treatment [25]. On the contrary, kinase inhibitors induced an increase in staining with an O-GlcNAc antibody following treatment of neuroblastoma cells [26]. Furthermore, a potent peptide O-GlcNAc-β-N-acetylglucosaminidase inhibitor increases O-GlcNAc levels and decreases incorporation of phosphate into the Sp1 transcription factor [27]. Thus, O-GlcNAc is reciprocal with phosphorylation on some sites. An increasing number of well-studied proteins are now identified to be submitted to such a balance O-GlcNAc glycosylation/phosphorylation (reviewed in Ref. [28]).

The occurrence of an O-GlcNAc/phosphorylation balance raises the question of its biological significance. While the role of phosphorylation is well documented, the role of O-GlcNAc glycosylation is still poorly understood. Some authors have suggested a role of O-GlcNAc residues in the nuclear transport of cytosolic proteins [29], [30]. In support of this hypothesis, we recently showed that the balance between phosphorylation and O-GlcNAc glycosylation in Kelly cells was strongly involved in the control of protein transfer to the nucleus [25]. We therefore decided to use this model to determine whether such a phosphorylation/O-GlcNAc glycosylation balance occurs for human Tau proteins, and whether this balance interferes with their transfer into the nucleus. To ensure a high level of Tau expression (and thus better detection of O-GlcNAc modified Tau proteins), the long Tau isoform (Tau 441) was overexpressed in human neuroblastoma Kelly cells. A stable clone (Kelly clone 16, K Cl16) was selected and used to study the effect of phosphorylation and O-GlcNAc levels on the cellular localization of Tau proteins.

Section snippets

Materials

The PcDNA3 vector was obtained from Invitrogen (Carlsbad, CA, USA) and the PRK172 plasmid was a kind gift from Dr. M. Goedert (Cambridge University, UK). Penicillin, streptomycin, okadaic acid, digitonin, leupeptin, pepstatin, DTT, CHAPS, nonidet P40, horseradish peroxidase-labelled wheat germ agglutinin (WGA), WGA immobilized on cross-linked 4% beaded agarose and bovine galactosyltransferase were all purchased from Sigma-Aldrich Chimie (St. Quentin-Fallavier, France). RPMI 1640, glutamine and

WGA staining of cytosolic and nuclear Tau proteins

The first stage of this work was to check the presence of O-GlcNAc residues on human Tau proteins and to determine whether glycosylated Tau proteins were present both in the cytosolic and nuclear compartments. First, the O-GlcNac glycosylated proteins in a cytosolic Kelly extract were selected by WGA–agarose precipitation. Next, the presence of Tau proteins in the precipitate was analysed by Western blotting (Fig. 1A). Anti Tau antibodies (Fig. 1A) recognized a faint band, which migrated at

Discussion

A dysfunction of Tau protein phosphorylation occurs in numerous neurodegenerative diseases. Over the last 10 years, a number of laboratories have studied Tau protein phosphorylation and have identified the phosphorylated sites and kinases involved (reviewed in Ref. [5]). In contrast, only one group has reported the presence of O-GlcNAc residues on normal Tau proteins [11], albeit of bovine origin. Our present work shows that Tau proteins of human origin are posttranslationally modified by O

Acknowledgements

This work was supported in part by the CNRS (Unité Mixte de Recherches CNRS no. 8576, director J.C. Michalski, the Université de Lille I, the INSERM (U422, director J.C. Beauvillain), and the Lille Génopole. We are very grateful to Dr. Michel Goedert for his generous gift of pRK 172 plasmid (hTau40). ADI294 antibody was obtained in collaboration with Immunotech (E. Rouvier and F. Jean in particular). We also thank Dr. David Fraser (SARL Biotech Communication) for helpful criticism of this

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