PHOSPHOPROTEIN PHOSPHATASE PP2A IN EUKARYOTIC SIGNAL TRANSDUCTION

我们建议通过分子遗传学的方法，研究可逆蛋白质磷酸化在信号转导和发育中的作用。磷酸化蛋白磷酸酶PP2A是真核生物中主要的磷酸酶。该酶被认为在信号转导中发挥重要作用，要么是通过结构性地去磷酸化蛋白质来响应细胞外刺激，要么是在这种刺激下直接被激活。然而，这一结论是基于该酶在体外对许多蛋白质进行去磷酸化的能力，在这种情况下，其特异性较低，或者基于影响许多磷酸酶的抑制剂的作用。我们提出的假设是，PP2A在体内对特定蛋白质的去磷酸化起重要作用，这些蛋白质在信号转导和发育过程中被磷酸化激活。真核微生物盘基网柄菌特别适合于这些研究，因为它的基因组样本，发育周期短，定义明确，易于通过反向遗传学产生突变。我们以前已经证明，胞外cAMP激活Dictyostelials中的细胞表面受体，通过信号转导诱导基因。参与这些途径的蛋白质是高度保守的。在初步研究中，我们克隆了PP2A催化亚单位(PP2Ac)基因，并将其导入野生型细胞。我们通过转基因技术发现了显性负突变，并通过定点突变构建了其他突变。突变体似乎在发育过程中存在缺陷。我们计划确认和扩展这些研究，寻找可能被PP2A去磷酸化的底物蛋白，或者可能与该酶相互作用的蛋白以响应信号。我们还计划通过PP2Ac的定点突变来构建更多的冈田酸抗性突变体。我们建议利用这些突变来准确地确定PP2A参与发育或生长的点以及被激活的途径。PP2A被认为是一种重要的信号转导酶，即蛋白激酶B(PKB)，它被认为是一种被广泛研究的酶。哺乳动物的PKB基因是一种原癌基因，也参与了细胞凋亡的释放。我们已经敲除了网柄菌PKB基因，并观察到它影响发育。我们建议使用上述显性负性和冈田酸突变体来检验PP2A调节PKB信号转导活性的假设。这些研究应该更清楚地定义体内涉及PP2A信号传递的相互作用。此外，该机构应该从这项研究与学生和教职员工以及所提供的技术之间的互动中受益。

We propose to investigate, by molecular genetic approaches, the role of reversible protein phosphorylation in signal transduction and development. The phospho-protein phospho-protein phosphatase PP2A is the major ser/thre phosphatase in eukaryotes. The enzyme is assumed to play an important role in signal transduction either by constitutively dephosphorylating proteins that are phosphorylated in response to extracellular stimuli, or becoming activated directly in response to such stimulation. However, this conclusion is based on the ability of the enzyme to dephosphorylate many proteins in vitro, where its specificity is low, or on the action of inhibitors that affect many phosphatases. We propose to test the hypothesis that PP2A play an important role in vivo in the dephosphorylation of specific proteins which are activated by phosphorylation during signal transduction and development. The eukaryotic microorganism Dictyostelium discoideum, is particularly suitable for these studies, because of its sample genome, short, well- defined developmental cycle and ease of making mutants by reverse genetics. We had previously shown that extracellular cAMP, which activates cell surface receptors in Dictyostelium, induces genes by signal transduction. The proteins involved in the pathways are highly conserved. In preliminary studies we cloned the gene for the catalytic subunit PP2A (PP2Ac) and transfected it intro wild type cells. We discovered dominant negative mutations by the transfection technique, and constructed others by site specific mutagenesis. The mutants appear to be defective in development. We plan to confirm and extend these studies, to look for substrate proteins that may dephosphorylated by PP2A or proteins that may interact with the enzyme in response to signaling. We also plan to construct more okadaic acid resistant mutants by site specific mutagenesis of PP2Ac. We propose to exploit these mutants to determine precisely the points at which PP2A is involved in development or growth and the pathways that are activated. PP2A is presumed to dephosphorylate an important and signaling enzyme, protein kinase B (PKB) which is intensively studied. The mammalian PKB gene is a proto-oncogene and is also involved in the release from apoptosis. We have knocked out the Dictyostelium PKB gene and observed that it affects development. We propose to test the hypothesis that PP2A regulates the signal transduction activities of PKB by use of the dominant negative and okadaic acid mutants described above. The studies should define more clearly the interactions that involve PP2A in signaling in vivo. In addition the institution should benefit from the interactions that quality arises from this research with students and faculty and the technologies provided.