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Phosphorylation of cyclin O, a novel cyclin family protein containing a cyclin-like domain, is involved in the activation of cyclin-dependent kinase 2.

Kim DH, Park JH, Lee B, Jang KO, Chung IS, Han YS - Oncol Lett (2014)

Bottom Line: Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering and Graduate School of Biotechnology, Kyung Hee University, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus

Phosphorylation of c-myc-CyO was enhanced by the co-expression of CDK2. (A) HEK 293 human embryonic kidney cells were transiently transfected with vectors expressing c-myc-CyO and/or flag-CDK2. The expression levels of c-myc-CyO and flag-CDK2 were determined by western blot analysis. (B) HEK 293 cell lysates expressing c-myc-CyO were incubated with 5 U CIP in the presence or absence of 5 mm EDTA. The reaction mixtures were analyzed by western blotting using anti-c-myc. The arrow indicates the uppermost band that disappeared with CIP treatment, although this effect was reversed when EDTA was added. CyO, cyclin O; CDK 2 cyclin-dependent kinase 2; CIP, calf intestinal phosphatase; IB, immunoblotting.
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f2-ol-08-06-2769: Phosphorylation of c-myc-CyO was enhanced by the co-expression of CDK2. (A) HEK 293 human embryonic kidney cells were transiently transfected with vectors expressing c-myc-CyO and/or flag-CDK2. The expression levels of c-myc-CyO and flag-CDK2 were determined by western blot analysis. (B) HEK 293 cell lysates expressing c-myc-CyO were incubated with 5 U CIP in the presence or absence of 5 mm EDTA. The reaction mixtures were analyzed by western blotting using anti-c-myc. The arrow indicates the uppermost band that disappeared with CIP treatment, although this effect was reversed when EDTA was added. CyO, cyclin O; CDK 2 cyclin-dependent kinase 2; CIP, calf intestinal phosphatase; IB, immunoblotting.

Mentions: HEK 293 cells were transiently transfected with pCMV Tag3A/CyO in the presence or absence of pCMV Tag2C/CDK2, and the expression levels of c-myc-CyO were determined by western blot analysis. c-myc tagged cyclin O was expressed as three bands with molecular weights between 45 and 50 kDa. When co-expressed with flag-CDK2, c-myc-CyO was expressed as a single band with a molecular weight of 50 kDa (Fig. 2A). To determine whether the cyclin O was phosphorylated, the cell lysates of the HEK 293 cells transiently transfected with pCMV Tag3A/CyO were treated with 5 units of calf intestinal phosphatase (CIP) for 1 h at 30°C. The uppermost band of c-myc-CyO disappeared in CIP-treated cell lysates (Fig. 2B, lane 2), but not in cell lysates treated with CIP and EDTA (Fig. 2B, lane 3).


Phosphorylation of cyclin O, a novel cyclin family protein containing a cyclin-like domain, is involved in the activation of cyclin-dependent kinase 2.

Kim DH, Park JH, Lee B, Jang KO, Chung IS, Han YS - Oncol Lett (2014)

Phosphorylation of c-myc-CyO was enhanced by the co-expression of CDK2. (A) HEK 293 human embryonic kidney cells were transiently transfected with vectors expressing c-myc-CyO and/or flag-CDK2. The expression levels of c-myc-CyO and flag-CDK2 were determined by western blot analysis. (B) HEK 293 cell lysates expressing c-myc-CyO were incubated with 5 U CIP in the presence or absence of 5 mm EDTA. The reaction mixtures were analyzed by western blotting using anti-c-myc. The arrow indicates the uppermost band that disappeared with CIP treatment, although this effect was reversed when EDTA was added. CyO, cyclin O; CDK 2 cyclin-dependent kinase 2; CIP, calf intestinal phosphatase; IB, immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214489&req=5

f2-ol-08-06-2769: Phosphorylation of c-myc-CyO was enhanced by the co-expression of CDK2. (A) HEK 293 human embryonic kidney cells were transiently transfected with vectors expressing c-myc-CyO and/or flag-CDK2. The expression levels of c-myc-CyO and flag-CDK2 were determined by western blot analysis. (B) HEK 293 cell lysates expressing c-myc-CyO were incubated with 5 U CIP in the presence or absence of 5 mm EDTA. The reaction mixtures were analyzed by western blotting using anti-c-myc. The arrow indicates the uppermost band that disappeared with CIP treatment, although this effect was reversed when EDTA was added. CyO, cyclin O; CDK 2 cyclin-dependent kinase 2; CIP, calf intestinal phosphatase; IB, immunoblotting.
Mentions: HEK 293 cells were transiently transfected with pCMV Tag3A/CyO in the presence or absence of pCMV Tag2C/CDK2, and the expression levels of c-myc-CyO were determined by western blot analysis. c-myc tagged cyclin O was expressed as three bands with molecular weights between 45 and 50 kDa. When co-expressed with flag-CDK2, c-myc-CyO was expressed as a single band with a molecular weight of 50 kDa (Fig. 2A). To determine whether the cyclin O was phosphorylated, the cell lysates of the HEK 293 cells transiently transfected with pCMV Tag3A/CyO were treated with 5 units of calf intestinal phosphatase (CIP) for 1 h at 30°C. The uppermost band of c-myc-CyO disappeared in CIP-treated cell lysates (Fig. 2B, lane 2), but not in cell lysates treated with CIP and EDTA (Fig. 2B, lane 3).

Bottom Line: Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering and Graduate School of Biotechnology, Kyung Hee University, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus