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A variant form of cyclin-dependent kinase 2 (Cdk2) in a malignantly transformed rat thyroid (FRTL-Tc) cell line
1Third Department of Internal Medicine, University of Yamanashi Medical School, Japan.
Abstract:
Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.
Insights
Researchers identified a novel 39 kDa variant of Cyclin-dependent kinase 2 (Cdk2-beta) in rat cells. This Cdk2-beta protein, distinct from the standard Cdk2-alpha, appears crucial for cell cycle progression into the S phase.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
Background:
- Cyclin-dependent kinase 2 (Cdk2) is a key regulator of the G1 to S phase transition in the mammalian cell cycle.
- Immunoblotting revealed three Cdk2 protein forms (33, 34, and 39 kDa) in FRTL-5 and FRTL-Tc cells, with the 39 kDa form's identity being unknown.
Purpose of the Study:
- To elucidate the nature of the 39 kDa Cdk2 protein detected in FRTL-5 and FRTL-Tc cells.
- To characterize the novel Cdk2 variant and its role in cell cycle progression.
Main Methods:
- Screening of a FRTL-5 cDNA library to isolate rat homologues of human Cdk2.
- Isolation and sequencing of two cDNA clones: rat Cdk2-alpha and rat Cdk2-beta.
- Northern blot analysis to determine mRNA sizes.
- Partial proteolytic mapping to identify the 39 kDa protein.
- Analysis of protein-protein interactions (cyclin A, suc1).
- Western blot analysis to assess protein levels and phosphorylation during the cell cycle.
Main Results:
- Two rat Cdk2 homologues, Cdk2-alpha and Cdk2-beta, were identified.
- Rat Cdk2-beta cDNA encoded a 39 kDa protein, confirmed as the 39 kDa Cdk2 form by partial proteolytic mapping.
- Cdk2-beta protein interacts with cyclin A and suc1.
- Cdk2-alpha levels remained constant but phosphorylated during G1-S, while Cdk2-beta levels increased through S phase and decreased in early G2.
Conclusions:
- The 39 kDa protein is a novel variant, Cdk2-beta, differing from Cdk2-alpha by an insertion of 48 amino acids.
- Cdk2-beta protein expression and dynamics suggest its involvement in the G1 to S phase transition.
- A variant form of Cdk2 may be essential for initiating the S phase in FRTL-Tc cells.