A variant form of cyclin-dependent kinase 2 (Cdk2) in a malignantly transformed rat thyroid (FRTL-Tc) cell line.
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.
Kotani
S
S
Third Department of Internal Medicine, University of Yamanashi Medical School, Japan.
Endo
T
T
Kitagawa
M
M
Higashi
H
H
Onaya
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D002471:Cell Transformation, Neoplastic; D003001:Cloning, Molecular; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013961:Thyroid Gland / Q000201:enzymology
D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases