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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 81st serine (S81) residue was phosphorylated following co-expression of cyclin O and CDK2. HEK 293 human embryonic kidney cells were transfected with vectors expressing c-myc-CyO and flag-CDK2. Phosphorylated and non-phosphorylated cyclin O were excised from a sodium dodecyl sulfate-polyacrylamide gel and analyzed using an LTQ linear ion-trap mass spectrometer. The collected MS/MS spectra were searched using the Sequest program. (A) and (B) indicate the identities of the non-phosphorylated and phosphorylated site of Ser81, respectively. CDK2, cyclin-dependent kinase 2; CyO, cyclin O; MS, mass spectroscopy.
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f3-ol-08-06-2769: Cyclin O 81st serine (S81) residue was phosphorylated following co-expression of cyclin O and CDK2. HEK 293 human embryonic kidney cells were transfected with vectors expressing c-myc-CyO and flag-CDK2. Phosphorylated and non-phosphorylated cyclin O were excised from a sodium dodecyl sulfate-polyacrylamide gel and analyzed using an LTQ linear ion-trap mass spectrometer. The collected MS/MS spectra were searched using the Sequest program. (A) and (B) indicate the identities of the non-phosphorylated and phosphorylated site of Ser81, respectively. CDK2, cyclin-dependent kinase 2; CyO, cyclin O; MS, mass spectroscopy.

Mentions: To determine which amino acid residues are phosphorylated by CDK2, total cell lysates were collected from the HEK 293 cells co-transfected with vectors expressing c-myc-CyO and flag-CDK2. Subsequently, 1 mg cell lysate was pre-cleared with 30 μl protein A/G-Sepharose beads and immunoprecipitated with anti-c-myc. The immunoprecipitated samples were resolved on SDS polyacrylamide gel and visualized by silver staining (data not shown). The regions with the phosphorylated and non-phosphorylated cyclin O bands were excised from the SDS polyacrylamide gel. The proteins were reduced, alkylated and digested with trypsin. MS analysis of the digested peptides revealed that sizes of the peptides including the 81st serine residue of phosphorylated c-myc-CyO were greater (~80 Da) than those obtained from non-phosphorylated c-myc-CyO (Fig. 3A and B). This result indicates that the 81st serine residue of cyclin O was phosphorylated, which may be caused by CDK2. To further examine whether the phosphorylation of the 81st serine residue of cyclin O is caused by CDK2, a cyclin O gene with a point mutation (cyclin O S81A) was generated by replacing the 81st serine residue with an alanine, and was transiently expressed in HEK 293 cells. As shown in Fig. 4, the c-myc-tagged cyclin O S81A (c-myc-CyO S81A) was expressed as two bands (lane 5). When co-expressed with flag-tagged CDK2, c-myc-CyOS81A was expressed as a band with a molecular weight of 48 kDa (lane 6), which was smaller than the band that appeared when cyclin O was co-expressed with CDK2 (lane 4).


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 81st serine (S81) residue was phosphorylated following co-expression of cyclin O and CDK2. HEK 293 human embryonic kidney cells were transfected with vectors expressing c-myc-CyO and flag-CDK2. Phosphorylated and non-phosphorylated cyclin O were excised from a sodium dodecyl sulfate-polyacrylamide gel and analyzed using an LTQ linear ion-trap mass spectrometer. The collected MS/MS spectra were searched using the Sequest program. (A) and (B) indicate the identities of the non-phosphorylated and phosphorylated site of Ser81, respectively. CDK2, cyclin-dependent kinase 2; CyO, cyclin O; MS, mass spectroscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ol-08-06-2769: Cyclin O 81st serine (S81) residue was phosphorylated following co-expression of cyclin O and CDK2. HEK 293 human embryonic kidney cells were transfected with vectors expressing c-myc-CyO and flag-CDK2. Phosphorylated and non-phosphorylated cyclin O were excised from a sodium dodecyl sulfate-polyacrylamide gel and analyzed using an LTQ linear ion-trap mass spectrometer. The collected MS/MS spectra were searched using the Sequest program. (A) and (B) indicate the identities of the non-phosphorylated and phosphorylated site of Ser81, respectively. CDK2, cyclin-dependent kinase 2; CyO, cyclin O; MS, mass spectroscopy.
Mentions: To determine which amino acid residues are phosphorylated by CDK2, total cell lysates were collected from the HEK 293 cells co-transfected with vectors expressing c-myc-CyO and flag-CDK2. Subsequently, 1 mg cell lysate was pre-cleared with 30 μl protein A/G-Sepharose beads and immunoprecipitated with anti-c-myc. The immunoprecipitated samples were resolved on SDS polyacrylamide gel and visualized by silver staining (data not shown). The regions with the phosphorylated and non-phosphorylated cyclin O bands were excised from the SDS polyacrylamide gel. The proteins were reduced, alkylated and digested with trypsin. MS analysis of the digested peptides revealed that sizes of the peptides including the 81st serine residue of phosphorylated c-myc-CyO were greater (~80 Da) than those obtained from non-phosphorylated c-myc-CyO (Fig. 3A and B). This result indicates that the 81st serine residue of cyclin O was phosphorylated, which may be caused by CDK2. To further examine whether the phosphorylation of the 81st serine residue of cyclin O is caused by CDK2, a cyclin O gene with a point mutation (cyclin O S81A) was generated by replacing the 81st serine residue with an alanine, and was transiently expressed in HEK 293 cells. As shown in Fig. 4, the c-myc-tagged cyclin O S81A (c-myc-CyO S81A) was expressed as two bands (lane 5). When co-expressed with flag-tagged CDK2, c-myc-CyOS81A was expressed as a band with a molecular weight of 48 kDa (lane 6), which was smaller than the band that appeared when cyclin O was co-expressed with CDK2 (lane 4).

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