Abstract
Cell cycles, ordered series of events modulating cell growth and division, are tightly regulated by complexes containing cyclin-dependent kinases (CDKs) and cyclins. Cyclin O is a novel cyclin family protein which interacts with CDK2. However, the molecular effects of cyclin O on the activity of CDK2 have not been fully evaluated. In this study, an interaction between cyclin O and CDK2 was identified by co-immunoprecipitation and the effect of cyclin O on the kinase activity of CDK2 was investigated using cyclin O point mutants. Co-immunoprecipitation was achieved using using HEK293 human embryonic kidney cells which were transiently transfected with vectors expressing cyclin O and CDK2, which revealed that cyclin O interacted with CDK2, particularly with the active form of endogenous CDK2. Cyclin O was expressed as several different bands with molecular weights between 45 and 50 kDa, possibly due to different post-translational modifications. When co-expressed with CDK2, cyclin O appeared as a band with a molecular weight of 50 kDa. Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band. Mass spectroscopic analysis of cyclin O co-expressed with CDK2 revealed that the 81st serine residue of cyclin O was phosphorylated. The in vitro kinase activity of CDK2 phosphorylating histone H1 was markedly increased in the cells overexpressing cyclin O. This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A). These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.
Highlights
The cell cycle, a series of events regulating cell division and duplication, is a ubiquitous, complex process involved in the growth and proliferation of cells, organism development, regulation of DNA damage repair and diseases such as cancer
Western blot analysis using the anti‐flag signal of samples immunoprecipitated with anti‐c‐myc revealed that flag‐CDK2 was co‐immunoprecipitated by interacting with c‐myc‐CyO (Fig. 1A)
The interaction between cyclin O and CDK2 was further determined by co‐immunoprecipitation of endogenous CDK2 from HEK 293 cell extracts transfected with pCMV Tag3A/CyO (Fig. 1C and D)
Summary
The cell cycle, a series of events regulating cell division and duplication, is a ubiquitous, complex process involved in the growth and proliferation of cells, organism development, regulation of DNA damage repair and diseases such as cancer. The cell cycle is tightly regulated by complexes containing cyclin‐dependent kinases (CDKs) and cyclins. The catalytic activity of CDKs, phosphorylating proteins involved in diverse cell cycle processes, is tightly regulated by interactions with cyclin family proteins. CDK activity is differentially regulated by cyclins, a family of proteins that control the progression of cells through the cell cycle by activating CDKs [3]. Three subtypes of cyclin D (cyclin D1, cyclin D2 and cyclin D3) bind to CDK4 and CDK6 These CDK‐cyclin D complexes are essential for cell entry into G1 [4]. Mitosis is further regulated by the CDK1‐cyclin B complex, which is involved in the early stages of mitosis including chromosome condensation, nuclear envelope breakdown, and spindle pole assembly [10].
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