P34CDC2 AND CELL CYCLE CONTROL

在真核生物中，进入有丝分裂是由协同作用和 一系列不同的蛋白激酶和蛋白的相反活性 磷酸酶。这种级联反应集中在CDC2上，CDC2是一种丝氨酸/苏氨酸蛋白 细胞进入有丝分裂所需的激酶。两个相邻的 CDC2 N-末端的氨基酸(苏氨酸14和酪氨酸15) 已被证明是调节蛋白激酶活性的关键因素 CDC2。这些残基的磷酸化定量地抑制了 CDC2激酶的活性，从而阻止细胞进入 有丝分裂。这项资助的目标是描述 直接催化或间接调节这些蛋白的磷酸化 关键残留物。编码人类和非洲爪哇Thr14/Tyr 15蛋白的cDNA 将会被隔离。会产生特定的探针，每个激酶都会 在体外和体内都具有特征。此外，上游 这些酶的调节者将被确定。在分裂酵母中， 将研究Wee1、nim1/cdr1和mik1基因产物。最后， 将确定每个激酶的最佳多肽基序，以便 确定激酶的潜在底物(CDC2除外)，并 为每种酶开发特定的多肽抑制剂。这些抑制剂 将被用作体内蛋白质功能的探针，它们还可能 作为设计针对疾病的新药的模板 这是细胞异常增殖的结果。

In eukaryotes, entry into mitosis is regulated by the synergistic and opposing activities of a cascade of distinct protein kinases and phosphatases. This cascade converges on CDC2, a serine/threonine protein kinase required for the entry of cells into mitosis. Two neighboring amino acids within the N-terminus of CDC2 (threonine 14 and tyrosine 15) have been shown to be critical elements in regulating the kinase activity of CDC2. Phosphorylation of these residues quantatively inhibits the activity of the CDC2 kinase and thereby prevents cells from entering into mitosis. The goals of this grant are to characterize the kinases that either directly catalyze or indirectly regulate phosphorylation of these key residues. cDNAs encoding the human and Xenopus Thr14/Tyr 15 kinases will be isolated. Specific probes will be generated and each kinase will be characterized both in vitro and in vivo. In addition, upstream regulators of these enzymes will be identified. In fission yeast, the wee1, nim1/cdr1 and mik1 gene products will be studied. Finally, the optimal peptide motif for each kinase will be determined in order to identify potential substrates of the kinases (other that CDC2) and to develop specific peptide inhibitors for each kinase. These inhibitors will be used as in vivo probes of protein function and they may also serve as templates for the design of novel drugs targeted to diseases that arise as a result of aberrant cellular proliferation.