SIGNAL TRANSDUCTION BY PROTEIN PHOSPHORYLATION

我们建议继续对其结构和功能进行研究 两种类型的蛋白-磷酸酶参与调节 细胞过程与激素作用或其他类型的 细胞内信号传递。 具体地说，我们感兴趣的是： 1)Mn2+对磷酸化酶磷酸酶的调节机制 离子及其调节亚基(抑制物-2)的磷酸化 已知会引起催化剂构象的变化 亚单位。 2)该酶可能参与植物生长发育的调控 3T3细胞的碳水化合物代谢对激素或 增长因素。而胰岛素和EGF则会触发细胞内 直接参与合成或分解的酶的活性 糖原，导致这一现象的一系列事件是 不知道。荷尔蒙引起激活的可能性 作用于不同磷酸化的特定蛋白激酶 可以相互交互的站点将进一步 调查过了。 3)我们的主要工作将针对结构和 颗粒和胞质蛋白酪氨酸的调节 从人类中提纯到均一的磷酸酶 胎盘。虽然这两种酶在结构上看起来是相同的， 他们不同的本地化一定有原因。搁置一边 通过测定它们通常的结构和酶性质， 将获得寡核苷酸探针的序列信息 (用于克隆)，并合成用于产生抗体的多肽 (用于亚细胞和组织分布)。到目前为止，基本上 人们对这些酶的调节一无所知。这个 可能会通过修改它们的 亚细胞定位，或通过特定的激活剂和/或 将对抑制剂进行调查。我们将尝试 在适当的细胞类型中过度表达磷酸酶，以便 研究它们对生长因子和癌基因的反应性，以及 确定这可能会对细胞功能产生什么影响。

We propose to continue our studies on the structure and function of two types of protein-phosphatases involved in the regulation of cellular processes in relation to hormone action or other types of intracellular signalling. Specifically, we are interested in: 1) The mechanism of regulation of phosphorylase phosphatase by Mn2+ ions and phosphorylation of its regulatory subunit (inhibitor-2) known to bring about changes in conformation in the catalytic subunit. 2) The possible involvement of this enzyme in the control of carbohydrate metabolisms in 3T3 cells in response to hormones or growth factors. While insulin and EGF trigger changes in the activity of enzymes directly involved in the synthesis or breakdown of glycogen, the chain of events by which this is brought about is not known. The possibility that the hormones cause the activation of specific protein kinases that act on different phosphorylation sites that may interact with one another will be further investigated. 3) Our main effort will be directed toward the structure and regulation of particulate and cytosolic protein tyrosine phosphatases that have been purified to homogeneity from human placenta. While both enzyme species appear structurally identical, something must account for their different localization. Aside from determining their usual structural and enzymatic properties, sequence information will be obtained for oligonucleotide probes (for cloning), and synthesis of peptides for antibody production (for subcellular and tissue distribution). As of now, essentially nothing is known about the regulation of these enzymes. The possibility that it might be exercised by modifying their subcellular localization, or through specific activators and/or inhibitors will be investigated. Attempts will be made to overexpress the phosphatases in appropriate cell types in order to study their responsiveness to growth factors and oncogenes, and determine what effect this might have on cell functions.