The transcription factor of cyclin-dependent kinase 2 (cdk2) gene.

Cdk2是一种蛋白激酶，其活性在细胞周期中发生变化。据推测，这种活性的变化与基因转录速率的变化和翻译后修饰有关。我们发现cdk2 mRNA在G1/S期开始升高，在S期达到最大水平，之后下降。我们发现cdk2的转录量在细胞周期中发生了变化。为了确定参与细胞周期依赖性调控的DNA区域，我们克隆了小鼠cdk2基因。亚克隆+68bp至5'上游各位点片段，制备荧光素酶报告基因。将这些报告子转染到CHO细胞中，获得稳定的转化子。在这些稳定转化子的细胞周期同步后，检测荧光素酶的活性。我们发现，荧光素酶的细胞周期依赖性表达需要+68bp到- 419bp的区域。我们还研究了cdk2的翻译后修饰。这表明cdk家族蛋白的活性随着其磷酸化和去磷酸化而变化，然而，激酶和磷酸酶的活性尚不清楚。我们假设wee1就是其中一种激酶，并克隆了小鼠wee1的cDNA。该克隆全长2258bp，其开放阅读框对应646个氨基酸残基。将该小鼠wee1克隆插入空泡病毒转移载体，研究其磷酸化作用。结果表明：1)小鼠wee1蛋白的n端氨基酸残基磷酸化使其失活；2)小鼠wee1蛋白使cdc2激酶磷酸化。我们也研究了小鼠wee1是否磷酸化cdk2。

cdk2 is a protein kinase and its activity changes during the cell cycle. It is postulated that this change of activity follows the change of transcription rate of the gene and post-translational modification. We found the mRNA of cdk2 begun to increase at G1/S and reached to the largest level at S-phase then declined thereafter. We showed that the transcription amount of cdk2 changed during the cell cycle. To determine the DNA region which involved in the cell-cycle dependent regulation of the gene, mouse cdk2 gene was cloned. And fragments from+68bp to various sites of 5'upstream region were subcloned, and luciferase reporters were prepared. These reporters were transfected into CHO cells, and stable transformants were obtained. After synchronization of the cell cycle of these stable transformants, the activities of luciferase were assayd. We showed that the region from +68bp to- 419bp was required for cell-cycle dependent expression of luciferase. We also studied the post-translational modification of cdk2. It is suggested that the activity of cdk family proteins changes with their phospyorylation and dephosphorylation, however, the kinase (s) and phosphatase (s) are not well understood. wee1 is postulated as the one of such kinases, and we cloned cDNA of mouse wee1. This clone consisted with 2258bp and its open reading frame corresponds to a 646 amino acid residues. This mouse wee1 clone was inserted into vaculovirus transfer vector, and studied on phosphorylation. The results obtained were 1) mouse wee1 was inactivated by a phosphorylation of an amino acid residue of N-terminal region of the protein, 2) mouse wee1 phosphorylated the cdc2 kinase. We study if mouse wee1 phosphorylates cdk2, as well.