Differential phosphorylation patterns between the Cyclin-A2/CDK2 complex and their monomers.
The 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 (Ser(14) and Ser(421)) and CDK2 on a single residue (Thr(160)). After heterodimer formation, Cyclin-A2 was phosphorylated only on Ser(14), whereas CDK2 contained two phosphorylated residues (Thr(39) and Thr(160)). 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.
Casado-Vela
Juan
J
Protein Technology Unit, Biotechnology Programme, Spanish National Cancer Centre, Madrid, Spain.
Martínez-Torrecuadrada
Jorge Luis
JL
Casal
J Ignacio
JI
D000595:Amino Acid Sequence; D000818:Animals; D019925:Cyclin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D007313:Insecta; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D019925:Cyclin A; D011994:Recombinant Proteins; D051357:Cyclin-Dependent Kinase 2