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 t o 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 cel ls). Although 33 kDa protein is a phosphorylated form of 34 kDa protei n previously reported, the nature of 39 kDa protein is unknown. In ord er 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 -bet a) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alp ha cDNA encoded a protein with 428 amino acids and has a high degree o f conservation with human Cdk2. The calculated molecular weight of Cdk 2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to C dk2-alpha cDNA except that it had extra 144 bp; this coincided with in sertion 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. Pa rtial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein bind s to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, C dk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S ph ase 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 ph ase of the cell cycle in FRTL-Tc cells.