|Budget Amount *help
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
A unique Cdk (Cdc28) functions in the progression of yeast cell cycle, but there exists a Cdk family whose members, including Pho85 kinase, function in various cellular events. Among mammalian Cdk family members, Cdk5 is not yet demonstrated to be involved in cell proliferation. Cdk5, activated by binding of p35, plays an important role in control of neurogenesis. Cdk5 and Pho85 kinases share similarities in structure as well as in the regulation of their activity. They are not further activated by Cdk-activating kinase, whereas phosphorylation of Y15 of Cdk5 and Y18 of Pho85 appears to enhance binding of p35 and Pho80, respectively. We found that mouse Cdk5 produced in pho85Δ cells could suppress some of pho85Δ mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins, and that the immunocomplex could phosphorylate P
ho4, a native substrate of Pho85 kinase. Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase. This discovery will lead to further revelation of the function of the two kinases : yet unknown factors that associate with p35-Cdk5 to regulate neuronal developments, and yeast proteins interacting with cyclin-Pho85 complex to regulate cell-cycle progression and cell morphology will be identified.
To search more targets of Pho85 kinase, we carried out gene chip analysis to identify genes whose expression is affected by a pho85 null mutation. We found that some genes involved in the carbohydrate metabolism were affected, either positively or negatively. Among them, we focused on the UGP1 gene which is essential for growth, and catalyzes the synthesis of UDP-glucose from UTP and glucose 1-phosphate. By northern analysis and assaying the activity of a reporter composed of the UGP1 promoter and lacZ, we confirmed that UGP1 expression and its promoter activity were increased in the cells lacking PHO85. A deletion of putative Pho4 and Bas2 binding sites from the promoter or an introduction of a pho4 null mutation diminished an increase in the reporter activity in a pho85 null mutant, suggesting that Pho85 kinase acts through Pho4 to regulate UGP1 expression. Less