Hormonal regulation of MAP kinase via Rap1 and B-Raf

描述（由申请人提供）：与第二信使环腺苷酸（cAMP）偶联的激素对细胞生长和分化具有细胞类型特异性作用。这些作用反映了cAMP对促分裂原活化蛋白（MAP）激酶（也称为细胞外信号调节激酶，或ERK）级联的激活，这是正常和恶性细胞中刺激生长的主要介质。从我们以前的资助期间的工作已经确定，cAMP的细胞类型特异性是由MAP激酶B-Raf的表达决定的。B-Raf最近因其在人类恶性肿瘤中极高的突变/激活率及其在特定人类癌症中的主要作用而受到关注。B-Rafs致癌潜力与其在正常激素信号传导中的作用之间的关系尚不清楚。B-Raf和更好地研究的Raf-1都可以响应来自小G蛋白Ras的信号而激活ERK，但只有B-Raf可以响应来自相关G蛋白Rap 1的信号而激活ERK。Rap 1已被证明通过其对ERK的激活在一些表达B-Raf的细胞中加强恶性进展。我们以前已经确定Rap 1/B-Raf是cAMP激活ERK的关键介质。本申请的一个主要主题是证明Rapt通过cAMP调节ERK是由所用Rap 1鸟嘌呤核苷酸交换因子（GEF）的选择决定的。在具体目标1中，我们将研究cAMP激活Rap 1/B-Raf/ERK的生化机制，并测试蛋白激酶A（PKA），酪氨酸激酶Src和RaplGEF C3 G是必需的假设。在具体目标2中，我们将测试cAMP激活至少两个不同的Rap 1库的假设，每个库由不同的RaplGEF介导，其中只有一个能够激活B-Raf/ERK信号通路。新的细胞原则，强调这种隔离的信号将被测试。我们将在Specific Aim 3中确定Rap 1对B-Raf的这种特异性的生化基础。为此，我们还将建立新的小鼠模型来验证B-Raf在体内具有不可替代的功能的假设。

DESCRIPTION (provided by applicant): Hormones that couple to the second messenger cyclic AMP (cAMP) have cell-type specific effects on cell growth and differentiation. These effects mirror cAMP's activation of the mitogen-activated protein (MAP) kinase (also called extracellular signal-regulated kinase, or ERK) cascade, the principal mediator of stimulated growth in normal and malignant cells. Work from our previous funding period has established that the cell-type specificity of cAMP is dictated by the expression of the MAP kinase kinase kinase B-Raf. B-Raf has received recent attention for its extremely high rate of mutation/activation in human malignancies and its primary role in specific human cancers. The relationship between B-Rafs oncogenic potential and its role in normal hormonal signaling is unknown. Both B-Raf and the better-studied Raf-1 can activate ERKs in response to signals from the small G protein Ras, but only B-Raf can activate ERKs in response to signals from the related G protein Rap1. Rap1 has been shown to potentiate malignant progression in some B-Raf- expressing cells via its activation of ERKs. We have previously identified Rap 1/B-Raf as a critical mediator of cAMP's activation of ERKs. A major theme of this application is to demonstrate that Rapt regulation of ERKs via cAMP is dictated by the choice of Rap1 guanine nucleotide exchange factor (GEF) utilized. In Specific Aim 1, we will examine the biochemical mechanism by which cAMP activates Rap1/B-Raf/ERK and test the hypothesis that protein kinase A (PKA), the tyrosine kinase Src, and the RaplGEF C3G are required. In Specific Aim 2, we will test the hypothesis that cAMP activates at least two distinct pools of Rap1, each mediated by a distinct RaplGEF, only one of which is capable of activating B-Raf/ERK signaling pathways. Novel cellular principles that underscore this segregation of signaling will be tested. We will determine the biochemical basis for this specificity of Rap1 for B-Raf in Specific Aim 3. In this aim, we will also establish novel mouse models to test the hypothesis that B-Raf has irreplaceable functions in vivo.