Influence of serine O-glycosylation or O-phosphorylation close to the vJun nuclear localisation sequence on nuclear import.
Nuclear import triggered by the nuclear-localisation sequence (NLS) of the viral Jun (vJun) protein is mediated by phosphorylation of a serine close to the NLS. Since phosphorylation and glycosylation of serine residues are often in a reciprocal "yin-yang" relationship, we investigated whether glycosylation of this serine with O-linked N-acetylglucosamine (O-GlcNAc) would also regulate nuclear import via the vJun NLS. Peptides containing the vJun NLS with an adjacent O-phosphorylated, O-GlcNAc-functionalised or unmodified serine, and equipped with an N-terminal biotin or a 7-nitrobenz-2-oxa-1,3-diazolyl (NBD) fluorescent label, were synthesised on the solid phase by means of an Fmoc/Boc strategy and a Pd0-sensitive HYCRON linker. Fluorescence-polarisation measurements on the NBD-labelled peptides indicated that modification with phosphate or O-GlcNAc leads to a decrease in affinity to the import-mediating adapter protein, importin alpha, of about one order of magnitude compared to the unmodified NLS. Microinjection of biotinylated NLS peptide conjugated with fluorescently labelled avidin into NIH/3T3 and MDCK cells, revealed that avidin-unmodified-NLS peptide was rapidly imported into the nucleus. However, either phosphate or O-GlcNAc next to the NLS caused almost complete exclusion of the protein conjugate from nuclear import. These findings indicate that nuclear import by the vJun NLS might not be regulated by a "yin-yang" modification of an adjacent serine with phosphate or O-GlcNAc. Rather, negative regulation of binding between the polybasic NLS and importin by a negatively charged or a bulky, uncharged residue appears likely.
Schlummer
Stefanie
S
Max-Planck-Institut für Molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Vetter
Robin
R
Kuder
Norman
N
Henkel
Andreas
A
Chen
Yong-Xiang
YX
Li
Yan-Mei
YM
Kuhlmann
Jürgen
J
Waldmann
Herbert
H
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001360:Avidin / Q000737:chemistry / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D006031:Glycosylation; D051379:Mice; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D016759:Oncogene Protein p65(gag-jun) / Q000737:chemistry*; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D019913:Nuclear Localization Signals; D016759:Oncogene Protein p65(gag-jun); D001360:Avidin; D012694:Serine