PKA and PAK in Adhesion Dependent Signal Transduction

描述(申请人提供)：输入信号的正确积分 从生长因子和细胞黏附到细胞外基质(ECM) 控制正常的细胞增殖、运动和分化，而 异常整合导致许多病理状态，包括 我是一种恶性肿瘤。了解这一过程的机制 整合的发生对于我们理解转化的生物学是至关重要的 细胞。生长因子激活MAPK级联反应的能力，a 信号转导通路在细胞增殖调控中的作用 运动性，在很大程度上依赖于细胞的粘附性和 皮质肌动蛋白细胞骨架，是黏附依赖信号的原型 转导。最近有证据表明，锚地依赖 生长因子介导的MAPK激活受 CAMP依赖的蛋白激酶(PKA)，一种已知可抑制 生长因子信号转导以及破坏肌动蛋白细胞骨架，以及p21- 激活的激酶-1(PAK)--皮质肌动蛋白和细胞的重要调节因子 运动性，通过一种可能涉及直接磷酸化的机制 PKA对PAK的失活作用。这项提案旨在调查 进一步阐述了PKA和PAK之间的生化联系及其相互作用 Ro L在锚定依赖的信号转导调节中的作用。 目标1中提出的实验将通过以下方式定义PAK抑制的机制 并将研究PKA在PAK中的作用。 对形态和运动的依赖调节。目标2包括实验 这将决定PKA和PAK控制锚地的机制- 依赖于MAPK的信号转导，以及评估该信号转导的能力 促进下游事件，导致细胞周期进入和增殖。两者都有 我是S，需要包括电离质量在内的生化技术 光谱分析、免疫共沉淀和体外激酶和磷酸酶 检测，以及包括免疫荧光在内的细胞生物技术， 迁移试验和软琼脂集落形成。评估PKA的作用 而PAK在整合胞外信号时会拓宽电流 了解依赖锚定的生长的复杂性，并可能提供 恶性疾病治疗干预的新途径。

DESCRIPTION (provided by applicant): Correct integration of signals coming from growth factors and from cell adhesion to the extracellular matrix (ECM) controls normal cellular proliferation, movement and differentiation, while aberrant integration contributes to many pathological states, including m a lignant neoplasia. Understanding the mechanism through which this integration occurs is crucial to our understanding the biology of transformed cells. The ability of growth factors to activate the MAPK cascade, a signaling pathway important for the regulation of cell proliferation and motility, is profoundly dependent on cell adhesion and the integrity of the cortical actin cytoskeleton, and is an archetype of adhesion-dependent signal transduction. It has recently been demonstrated that the anchorage-dependence of growth factor-mediated MAPK activation is coordinately regulated by the cAMP-dependent protein kinase (PKA), a venerable protein known to inhibit growth factor signaling as well as disrupt the actin cytoskeleton, and p21- activated kinase-1 (PAK), an important regulator of cortical actin and cell motility, through a mechanism that is likely to involve direct phosphorylation and inactivation of PAK by PKA. This proposal is designed to investigate further the newly described biochemical connection between PKA and PAK and its r o l e in the regulation of anchorage-dependent signal transduction. Experiments proposed in Aim 1 will define the mechanism of PAK inhibition by PKA-mediated phosphorylation and will examine the role of PKA in the PAK- dependent regulation of morphology and motility. Aim 2 comprises experiments that will determine the mechanism through which PKA and PAK control anchorage- dependent signaling to MAPK as well as assess the ability of this signaling to promote downstream events leading to cell cycle entry and proliferation. Both a i m s w ill entail biochemical techniques including ionization mass spectrometry, co-immunoprecipitation and in vitro kinase and phosphatase assays, as well as cell biological techniques including immunofluorescence, migration assays, and soft agar colony formation. Evaluating the role of PKA and PAK in the integration of extracellular signals will broaden the current understanding of the complexity of anchorage-dependent growth and may provide new avenues for therapeutic intervention for malignant disease.