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Title

Differential phosphorylation patterns between the Cyclin-a2/CDK2 complex and their monomers

AuthorsCasado-Vela, Juan; Martínez-Torrecuadrada, Jorge Luis; Casal, J. Ignacio
KeywordsCyclin-A2
CDK2
Phosphorylation analysis
Baculovirus expression
Linear ion trap MS
Issue DateJul-2009
PublisherElsevier España
CitationProtein Expression and Purification, 66 (1) ; 15-21 (2009) ;
AbstractThe Cyclin-A2/CDK2 is a protein heterodimer with kinase activity that plays a key role in centrosome duplication and meiotic cell division. To check the suitability of the insect cells for the production and characterization of phosphorylated mammalian proteins, both proteins were expressed individually or as a complex using the baculovirus expression system. In this study, we used a linear ion trap mass spectrometer to identify the phosphorylated residues in mouse Cyclin-A2 and CDK2 recombinant proteins, after individual expression and after formation of the heterodimer complex, both in baculovirus. By using multi-protease digestion and data dependent neutral loss analysis, we identified a differential phosphorylation pattern before and after formation of the protein complex. The analysis of the monomeric proteins showed that Cyclin-A2 was phosphorylated on two Ser residues (Ser14 and Ser421) and CDK2 on a single residue (Thr160). After heterodimer formation, Cyclin-A2 was phosphorylated only on Ser14, whereas CDK2 contained two phosphorylated residues (Thr39 and Thr160). These findings may clarify relevant aspects of the functionality of the Cyclin-A2/CDK2 protein complex and its role in cell cycle and support the efficiency of the baculovirus system for the production of phosphorylated proteins mimicking the mammalian situation
Description7 páginas, 4 figuras, 2 tablas -- PAGS nros. 15-21
Publisher version (URL)http://dx.doi.org/10.1016/j.pep.2009.02.007
URIhttp://hdl.handle.net/10261/71935
DOI10.1016/j.pep.2009.02.007
ISSN1046-5928
E-ISSN1096-0279
Appears in Collections:(CIB) Artículos
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