Hsp90 Chaperone Machine Structure and Function

描述（由申请人提供）：Hsp90是一种分子伴侣，其功能主要局限于信号转导蛋白的折叠，如蛋白激酶和转录因子。Hsp90不是单独起作用，而是与许多不同的同伴或助手一起起作用。我们研究的目的是了解Hsp90作为参与蛋白激酶折叠的伴侣机器的作用。过去十年的研究已经证明了Hsp90功能和组织的一般原理，但对它们在蛋白激酶折叠中的功能知之甚少，蛋白激酶折叠也需要称为Cdc37的伴侣。此外，还有许多新的共同伴侣在最初的Hsp90作用描述中没有被分配角色。我们的研究在三个具体目标中解决了这些知识缺乏的领域。在目标1中，我们将确定蛋白激酶折叠所需的伴侣。我们将首先通过物理结合和功能分析确定哪些酵母激酶与Hsp90和Cdc37相互作用。结合将通过与单个激酶相关的Hsp90和Cdc37的Western blot分析来确定。需要Cdc37激活的激酶将在Cdc37突变酵母菌株中筛选。这一分析将揭示伴侣蛋白与酵母激酶相互作用的程度。然后，我们将确定与Hsp90一起起作用的共伴侣是否对所有激酶都以相同的方式起作用，或者它们是否对不同的激酶表现出特异性。压力对伴侣互动的影响也将被确定。在目标2中，我们将确定Hsp90伴侣机的组成。Hsp90存在于一系列含有不同共伴侣的亚复合物中。在本目标的第一部分，我们将描述这些亚复合物的组成，在本目标的第二部分，我们将确定哪些Hsp90共伴侣存在于与蛋白激酶的复合物中。然后，我们将验证我们的假设，即Stil和Cdc37参与不同Hsp90：激酶复合物的组装。在目标3中，我们分析了伴侣蛋白和辅伴侣蛋白在蛋白激酶折叠和激活中的作用。在第一个子目标中，我们将通过有限蛋白水解和脉冲追踪分析来测定不同共伴侣突变株的蛋白激酶构象和稳定性。在第二个子目标中，我们将检测对蛋白激酶折叠很重要的新型共伴侣的伴侣活性。最后，我们将区分Cdc37在Cdk折叠和细胞周期蛋白结合中的作用。

DESCRIPTION (provided by applicant): Hsp90 is a molecular chaperone whose function is largely restricted to folding of signal transducing proteins, such as protein kinases and transcription factors. Hsp90 does not function by itself, but in association with many different co-chaperones or helpers. The goal of our studies is to understand the role of Hsp90 as a chaperone machine involved in protein kinase folding. Studies over the past decade have demonstrated general principles of Hsp90 function and organization, but little is known about their function in protein kinase folding, which also requires the chaperone called Cdc37. In addition, there are many novel co-chaperones that were not assigned roles in the original description of Hsp90 action. Our studies address these areas where knowledge is lacking in three specific aims. In aim 1 we will determine the chaperone requirements for protein kinase folding. We will first determine which yeast kinases interact with Hsp90 and Cdc37 using physical binding and functional assays. Binding will be determined by Western blot analysis of Hsp90 and Cdc37 that associate with individual kinases. Kinases that require Cdc37 for activity will be screened in a cdc37 mutant yeast strain. This analysis will reveal the extent to which chaperones interact with the yeast kinome. We will then determine whether co-chaperones that function with Hsp90 act in the same way for all kinases or whether they exhibit specificity for different kinases. The effect of stress on chaperone interactions will also be determined. In aim 2 we will determine the composition of the Hsp90 chaperone machine. Hsp90 exists in a series of sub-complexes containing different co-chaperones. In the first part of this aim we will characterize the composition of these sub-complexes and in the second part of the aim we will determine which Hsp90 co-chaperones exist in complexes with a protein kinase. We will then test our hypothesis that Stil and Cdc37 participate in assembly of different Hsp90:kinase complexes. In aim 3 we analyze the function of chaperones and co-chaperones in protein kinase folding and activation. In the first sub-aim we will assay protein kinase conformation and stability in different co-chaperone mutant strains by limited proteolysis and pulse chase analysis. In the second sub-aim we will assay for chaperone activity of novel co-chaperones that are important for protein kinase folding. Finally, we will distinguish between the role of Cdc37 in Cdk folding and cyclin binding.