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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

Cyclin O interacted with CDK2. (A) HEK 293 human embryonic kidney cells were transfected with different sets of vectors expressing c-myc-CyO and flag-CDK2. Following co-immunoprecipitation using anti-c-myc, flag-CDK2 interaction with c-myc-CyO was determined by western blot analysis using anti-flag. ‘3A’ signifies an empty vector, pCMV Tag3A. (B) Co-immunoprecipitation was also performed with anti-flag. The presence of c-myc-CyO in immunoprecipitates was determined by western blot analysis using anti-c-myc. (C) HEK 293 cell extracts transiently transfected with vector expressing c-myc-CyO were immunoprecipitated with rabbit IgG or anti-CDK2. The presence of c-myc-CyO in immunoprecipitates was determined by western blot analysis. The arrow indicates c-myc-CyO co-immunoprecipitated by anti-CDK2. (D) HEK 293 cell extracts were also immunoprecipitated with mouse IgG or anti-c-myc. The presence of endogenous CDK2 in the immunoprecipitates was determined by western blot analysis. CyO, cyclin O; CDK2, cyclin-dependent kinase 2; IP, immunoprecipitation; IB, immunoblotting.
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f1-ol-08-06-2769: Cyclin O interacted with CDK2. (A) HEK 293 human embryonic kidney cells were transfected with different sets of vectors expressing c-myc-CyO and flag-CDK2. Following co-immunoprecipitation using anti-c-myc, flag-CDK2 interaction with c-myc-CyO was determined by western blot analysis using anti-flag. ‘3A’ signifies an empty vector, pCMV Tag3A. (B) Co-immunoprecipitation was also performed with anti-flag. The presence of c-myc-CyO in immunoprecipitates was determined by western blot analysis using anti-c-myc. (C) HEK 293 cell extracts transiently transfected with vector expressing c-myc-CyO were immunoprecipitated with rabbit IgG or anti-CDK2. The presence of c-myc-CyO in immunoprecipitates was determined by western blot analysis. The arrow indicates c-myc-CyO co-immunoprecipitated by anti-CDK2. (D) HEK 293 cell extracts were also immunoprecipitated with mouse IgG or anti-c-myc. The presence of endogenous CDK2 in the immunoprecipitates was determined by western blot analysis. CyO, cyclin O; CDK2, cyclin-dependent kinase 2; IP, immunoprecipitation; IB, immunoblotting.

Mentions: The interaction between cyclin O and CDK2 was determined by co-immunoprecipitation using transiently transfected HEK 293 cells. Different sets of vectors (pCMV Tag3A/CyO, pCMV Tag2C/CDK2) expressing c-myc-tagged cyclin O (c-myc-CyO) and flag-tagged CDK2 (flag-CDK2), as described in Fig. 1A and B, were transiently transfected into HEK 293 cells. After 24 h of incubation, total cell lysates were harvested and immunoprecipitated with anti-c-myc or anti-flag. 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). In addition, western blot analysis using the anti-c-myc signal of samples immunoprecipitated with anti-flag also demonstrated that c-myc-CyO was co-immunoprecipitated by interacting with flag-CDK2 (Fig. 1B). 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). The total cell lysates obtained from HEK 293 cells transiently transfected with pCMV Tag3A/CyO were immunoprecipitated with rabbit IgG or anti-CDK2. Western blot analysis using anti-c-myc revealed that c-myc-CyO was co-immunoprecipitated by anti-CDK2, but not rabbit IgG (Fig. 1C). When the total cell lysates were immunoprecipitated with mouse IgG or anti-c-myc, the active form of endogenous CDK2 was also co-immunoprecipitated by anti-c-myc (Fig. 1D).


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)

Cyclin O interacted with CDK2. (A) HEK 293 human embryonic kidney cells were transfected with different sets of vectors expressing c-myc-CyO and flag-CDK2. Following co-immunoprecipitation using anti-c-myc, flag-CDK2 interaction with c-myc-CyO was determined by western blot analysis using anti-flag. ‘3A’ signifies an empty vector, pCMV Tag3A. (B) Co-immunoprecipitation was also performed with anti-flag. The presence of c-myc-CyO in immunoprecipitates was determined by western blot analysis using anti-c-myc. (C) HEK 293 cell extracts transiently transfected with vector expressing c-myc-CyO were immunoprecipitated with rabbit IgG or anti-CDK2. The presence of c-myc-CyO in immunoprecipitates was determined by western blot analysis. The arrow indicates c-myc-CyO co-immunoprecipitated by anti-CDK2. (D) HEK 293 cell extracts were also immunoprecipitated with mouse IgG or anti-c-myc. The presence of endogenous CDK2 in the immunoprecipitates was determined by western blot analysis. CyO, cyclin O; CDK2, cyclin-dependent kinase 2; IP, immunoprecipitation; IB, immunoblotting.
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Related In: Results  -  Collection

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f1-ol-08-06-2769: Cyclin O interacted with CDK2. (A) HEK 293 human embryonic kidney cells were transfected with different sets of vectors expressing c-myc-CyO and flag-CDK2. Following co-immunoprecipitation using anti-c-myc, flag-CDK2 interaction with c-myc-CyO was determined by western blot analysis using anti-flag. ‘3A’ signifies an empty vector, pCMV Tag3A. (B) Co-immunoprecipitation was also performed with anti-flag. The presence of c-myc-CyO in immunoprecipitates was determined by western blot analysis using anti-c-myc. (C) HEK 293 cell extracts transiently transfected with vector expressing c-myc-CyO were immunoprecipitated with rabbit IgG or anti-CDK2. The presence of c-myc-CyO in immunoprecipitates was determined by western blot analysis. The arrow indicates c-myc-CyO co-immunoprecipitated by anti-CDK2. (D) HEK 293 cell extracts were also immunoprecipitated with mouse IgG or anti-c-myc. The presence of endogenous CDK2 in the immunoprecipitates was determined by western blot analysis. CyO, cyclin O; CDK2, cyclin-dependent kinase 2; IP, immunoprecipitation; IB, immunoblotting.
Mentions: The interaction between cyclin O and CDK2 was determined by co-immunoprecipitation using transiently transfected HEK 293 cells. Different sets of vectors (pCMV Tag3A/CyO, pCMV Tag2C/CDK2) expressing c-myc-tagged cyclin O (c-myc-CyO) and flag-tagged CDK2 (flag-CDK2), as described in Fig. 1A and B, were transiently transfected into HEK 293 cells. After 24 h of incubation, total cell lysates were harvested and immunoprecipitated with anti-c-myc or anti-flag. 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). In addition, western blot analysis using the anti-c-myc signal of samples immunoprecipitated with anti-flag also demonstrated that c-myc-CyO was co-immunoprecipitated by interacting with flag-CDK2 (Fig. 1B). 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). The total cell lysates obtained from HEK 293 cells transiently transfected with pCMV Tag3A/CyO were immunoprecipitated with rabbit IgG or anti-CDK2. Western blot analysis using anti-c-myc revealed that c-myc-CyO was co-immunoprecipitated by anti-CDK2, but not rabbit IgG (Fig. 1C). When the total cell lysates were immunoprecipitated with mouse IgG or anti-c-myc, the active form of endogenous CDK2 was also co-immunoprecipitated by anti-c-myc (Fig. 1D).

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