INTERACTIONS OF ANCHORING PROEINS WITH THEIR TARGETS

蛋白质磷酸化控制可能包括激素在内的细胞过程 反应性、细胞生长、分化和凋亡。“瞄准” “假说”认为磷酸化事件是受控制的，部分地， 通过蛋白激酶和磷酸酶的亚细胞定位， 通过酶上的对接结构域与特定的 锚定蛋白这种局部化假设背后的原理是 蛋白激酶将在接近其目标时， 激活信号被释放。因此，一个紧迫的问题是定义 这些靶向相互作用的分子基础。因此，长期目标 这项提议的一个重要目的是确定蛋白质：蛋白质相互作用如何控制 蛋白激酶在细胞中的定位。 蛋白激酶和磷酸酶的正确细胞内靶向 赋予特异性，部分是通过将它们放置在靠近它们的位置， 优选的基质。也许哺乳动物最好的特征 靶向蛋白是A-激酶介导蛋白（AKAP）家族。 AKAP维持环AMP依赖性蛋白激酶（PKA）的特异性 亚细胞区室，从而确保激酶的可及性， 在特定位置的有限数量的衬底。这 通过对接结构域的相互作用发生区室化 蛋白激酶A的调节亚基与一个保守的“锚定位点”上， 各种AKP新的证据表明，这些AKAP也参与了 定位磷酸酶以及蛋白激酶C（PKC）在一个单一的 站点，从而提供了一种可能的机制， 转导途径。本提案的目标是 了解负责本地化的相互作用， 蛋白激酶PKA和PKC。我们将在我们的 具体目标： 1)我们将求解PKA二聚化结构域的溶液结构 其负责将酶与各种AKAP对接。 2)我们将研究PKA与蛋白质相互作用的分子本质， 通过确定“锚定”的溶液构象， 具有PKA的野生型和突变体二聚化结构域的“位点”肽。 3)我们将研究负责靶向的结构基序， PKC的突变和光谱技术的组合。

Protein phosphorylation controls may cellular processes including hormone responsiveness, cell growth, differentiation and apoptosis. The "targeting hypothesis" processes that phosphorylation events are controlled, in part, by subcellular localization of protein kinases and phosphatases in the cell via the interaction of docking domains on the enzymes with specific anchoring proteins. The principle behind this localization hypothesis is that the protein kinase will be in close proximity to its target when the activation signal is discharged. A pressing issue then is to define the molecular basis for these targeting interactions. Thus, the long term goal of this proposal is to determine how protein:protein interactions control the localization of protein kinases in the cell. The correct intracellular targeting of protein kinases and phosphatases confers specificity, in part, by placing them in close proximity to their preferred substrates. Perhaps the best characterized of the mammalian targeting proteins is the family of A-Kinase Anchoring Proteins (AKAPs). AKAPs maintain the cyclic AMP dependent protein kinases (PKA) in specific subcellular compartments, thereby ensuring accessibility of the kinase to a limited number of substrates in a particular location. This compartmentalization occurs through the interaction of the docking domains of the regulatory subunit of PKA with a conserved "anchoring site" on the various AKAPs. New evidence suggests these AKAPs are also involved in localizing phosphatases as well as protein kinase C (PKC) at a single site, thus affording a possible mechanism for integrating signal transduction pathways. The goals of this proposal are directed towards understanding of the interactions responsible for the localization of the protein kinases PKA and PKC. We will accomplish the following in our Specific Aims: 1) We will solve the solution structure of the dimerization domain of PKA which is responsible for docking the enzyme to various AKAPs. 2) We will investigate the molecular nature of the interaction between PKA and various AKAPs by determining the solution conformation of "anchoring site" peptides with the wild-type and mutant dimerization domain of PKA. 3) We will investigate the structural motifs responsible for targeting of PKC by a combination of mutagenic and spectroscopic techniques.