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Abstract No.: ThP-199
Session: Phosphoproteomics
Presentation date: Thu, Aug 31, 2006
Presentation time: 09:50 – 11:20

Analysis of Phosphorylation of the Nuclear Chaperone Nucleoplasmin

Miren J. Omaetxebarria1,2, Martin R. Larsen1, Isbaal Ramos2, Adeli Prado2, Arturo Muga2, Jesus M. Arizmendi2, Ole N. Jensen1

1 University of Southern Denmark, Odense, Denmark
2 University of the Basque Country, Leioa, Spain

Correspondence address: Miren Josu Omaetxebarria, University of the Basque Country, Biochemistry and Molecular Biology, Sarriena z/g, Leioa, 48940 Spain.

Keywords: Affinity, Chromatography; Mass Spectrometry; Phosphorylation; Surfactants.

Novel aspect: Application of TiO2 enrichment and mass spectrometry for the analysis of phosphorylation of the proteolysis-resistant Nucleoplasmin (NP) and involvement of phosphorylation in NP activity.


Multisite phosphorylation of proteins is a widely spread mechanism aimed to regulate protein function and protein-protein interactions.
Nucleoplasmin (NP), the first molecular chaperone ever described, is involved in sperm chromatin decondensation and exchanges sperm nuclear basic proteins by histones H2A and H2B in the process of pronuclear formation during fertilization. Binding of NP to basic proteins has been commonly assigned to the negatively charged region termed the poly-glu tract, which contains 20 aspartic and glutamic acid residues among the C-terminal 30 residues. However, it has been proved that the phosphorylated core domain, lacking this poly-glu tract, is also able to decondesate chromatin by its own, suggesting that the poly-glu tract is not the only region involved in NP activity and ligand binding.

Several studies have shown that chromatin decondensating activity of NP is modulated by phosphorylation at multiple sites. At oocyte stage, NP is believed to be poorly phosphorylated and although it is already active in histone binding, it is not till egg stage, when NP is phosphorylated at multiple sites, that sperm decondensation occurs efficiently (Hierro et al., 2001).

With the aim of identifying the phosphorylation sites present in NP isolated from eggs of Xenopus laevis, a strategy that combines TiO2 enrichment of phosphorylated peptides and mass spectrometry was used. NP resistance to enzymatic proteolysis needed to be tackled prior to the TiO2 enrichment approach. The results obtained in this study show that egg NP is highly phosphorylated at the stage where NP shows a high decondensating activity. The identified phosphorylation sites are located in the core and tail domains, indicating that both domains are involved in the fully activation of the decondensating activitiy of NP. Eight phosphorylation sites have been mapped using this strategy.