Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation
Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation
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DOI：
10.1128/mcb.26.6.2130-2145.2006
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发表时间：
2006-03-01
影响因子：
5.3
通讯作者：
Stork, PJS
中科院分区：
文献类型：
--
作者：
Wang, ZP;Dillon, TJ;Stork, PJS
与Ras超家族的其他小G蛋白一样，Rap 1被不同的鸟嘌呤核苷酸交换因子（GEF）激活，以响应不同的信号，引发细胞反应。环腺苷酸（cAMP）激活Rapt可以通过cAMP依赖性蛋白激酶A（PKA）非依赖性和PKA依赖性机制发生。cAMP对Rapt 1的PKA非依赖性激活是通过cAMP与Rap 1-鸟嘌呤核苷酸交换因子（Rap 1-GEF）Epac 1（由cAMP 1直接激活的交换蛋白）和Epac 2（Epac 1和Epac 2也称为cAMP-GEFI和-GEFI I）的直接结合介导的。选择性激活Epacs而不是PKA的cAMP类似物的可用性提供了激活Rap 1的特定工具。有人认为，尽管激活Rap 1，但这些类似物不能调节细胞外信号调节激酶（ERK）信号，这提供了Rapt不能调节ERK的证据。我们证实，PKA独立激活Rap 1的Epac 1激活Rapt的核周池，这不会导致ERK激活。然而，我们证明了这种无法调节ERK的能力不是Rapt的属性，而是Epacs本身的属性。在Epac 1上添加膜靶向基序（Epac-CAAX）可将Epac 1从其正常的核周区域重新定位到质膜。在这个新的区域，它能够以Rap 1和cAMP依赖的方式激活ERK。Rapt 1激活Epac-CAAX，而不是野生型Epac，触发其与B-Raf的关联。因此，我们认为Epac 1的细胞内定位阻止了Epac 1激活ERK。C3 G（Crk SH 3结构域鸟嘌呤核苷酸交换器）是一种Rapt交换器，激活后靶向质膜。我们表明，C3 G可以定位于质膜cAMP/PKA，可以Rapt时，激活cAMP/PKA。使用小干扰RNA的方法，我们证明，C3 G是必需的激活ERK和Rap 1的cAMP/PKA。这种激活需要Rap 1与B-Raf的GTP依赖性缔合。这些数据表明，B-Raf是Rap 1的生理靶标，但其作为Rap 1效应子的利用是GEF特异性的。我们提出了一个模型，特定的GEFs激活不同的Rap 1池，差异耦合到下游效应。
Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rapt by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rapt1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rapt is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rapt and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rapt but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rapt1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rapt exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rapt when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.