Hormonal regulation of MAP kinase via Rap1 and B-Raf

描述(申请人提供)：与第二信使环磷酸腺苷(CAMP)偶联的激素对细胞生长和分化有特定细胞类型的影响。这些效应反映了cAMP对丝裂原活化蛋白(MAP)激酶(也称为细胞外信号调节激酶，ERK)级联的激活，该通路是正常和恶性肿瘤细胞刺激生长的主要中介。我们上一个资助期的工作已经确定了cAMP的细胞类型特异性是由MAP激酶B-Raf的表达决定的。B-Raf因其在人类恶性肿瘤中极高的突变/激活率以及在特定人类癌症中的主要作用而受到关注。B-RAF的致癌潜能与其在正常激素信号中的作用之间的关系尚不清楚。B-Raf和研究较好的Raf-1都可以激活ERKs，以响应小G蛋白RAS的信号，但只有B-Raf可以激活ERKs，以响应相关G蛋白Rap1的信号。研究表明，RAP1通过激活ERKs促进某些B-Raf表达细胞的恶性进展。我们先前已经确定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通过cAMP对ERKs的JAPT调控是由所利用的Rap1鸟嘌呤核苷酸交换因子(Global)的选择决定的。在特定的目标1中，我们将研究cAMP激活Rap1/B-Raf/ERK的生化机制，并检验蛋白激酶A(PKA)、酪氨酸激酶Src和Raplgef C3G是必需的假设。在特定的目标2中，我们将检验这一假设，即cAMP至少激活两个不同的Rap1池，每个池都由不同的Raplgef介导，其中只有一个能够激活B-Raf/ERK信号通路。强调这种信号分离的新的细胞原理将得到测试。我们将在特定的目标3中确定Rap1对B-Raf的这种特异性的生化基础。在这个目标中，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说。

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.