The Role of Pka Anchoring in Cell Differentiation

描述（由申请人提供）：我们假设AKAP在细胞内的丰度和定位可能通过调节PKA全酶活性在细胞分化过程中起作用。我们将通过确定候选AKAP的表达和分布的变化以及PKA全酶在细胞分化过程中的活性和定位的相应变化来验证这一假设。作为模型系统，我们将使用L6成肌细胞系，因为它们的特征在于肌肉特异性基因的协调激活和细胞增殖的停滞的良好模式。 具体目标1。我们将鉴定AKAP，其表达随着肌源性分化的发生而变化。为了表征那些可能参与分化过程的AKAP，我们将使用细胞分级法检查L6成肌细胞分化过程中AKAP表达的变化，并通过传统的蛋白质印迹方法鉴定那些差异表达的AKAP。为了确定AKAP定位和分布的变化是否与成肌细胞向肌管的转变相关，我们将使用选定AKAP和相关PKA的免疫标记，结合使用荧光显微镜与亚细胞区室的共定位。我们还将检测分化过程中的mRNA表达。 具体目标2。我们将检验AKAP表达和定位的变化影响肌源性分化进展的假设。通过用PKA阻断肽Ht31阻断成肌细胞，可以确定阻断PKA定位对发育的影响。然后，我们将使用定点诱变突变候选AKAP上的PKA结合结构域，并将这些突变体转染到成肌细胞中。与PKA定位变化相关的分化模式的改变将在研究肌球蛋白重链表达和多核肌管形成中进行。determined.by 支架或锚定蛋白如何有助于细胞分化程序的知识将提供新的机会，研究PKA的AKAP锚定如何影响细胞分化。该项目还将为服务不足的本科研究人员提供新的机会。

DESCRIPTION (provided by applicant): We hypothesize that AKAP abundance and localization within the cell may play a role in cellular differentiation processes through regulation of PKA holoenzyme activity. We will test this hypothesis by determining the changes in expression and distribution of candidate AKAPs and the corresponding changes in the activity and localization of PKA holoenzymes during cell differentiation. As a model system we will use L6 myoblast cell lines, because they are characterized by a well-established pattern of coordinate activation of muscle specific genes and the arrest of cell proliferation. Specific Aim 1. We will identify AKAPs whose expression changes as myogenic differentiation occurs. To characterize those AKAPs that are potentially involved in the differentiation process, we will examine changes in AKAP expression during L6 myoblast differentiation using cell fractionation, and identify those differentially expressed AKAPs through traditional western blotting methods. To determine whether changes in AKAP localization and distribution are associated with the transition from myoblasts to myotubes, we will use immunolabeling of selected AKAPs and associated PKA, combined with co-localization with subcellular compartments using fluorescence microscopy. We will also examine mRNA expression during differentiation. Specific Aim 2. We will test the hypothesis that changes in AKAP expression and localization influence the progression of myogenic differentiation. By transfecting myoblasts with the PKA blocking peptide Ht31, the effects of blocking PKA localization on development can be determined. We will then mutate the PKA binding domain on candidate AKAPs using site directed mutagnesis, and transfect these mutants into myoblasts. Alterations in differentiation patterns associated with the changes in PKA localization will be determined.by examining myosin heavy chain expression and formation of multinucleate myotubes. Knowledge of how scaffolding or anchoring proteins contribute to cellular differentiation programs will provide new opportunities to investigate how AKAP anchoring of PKA can influence cell differentiation. The project will also provide new opportunities for an underserved population of undergraduate researchers.