cAMP-Dependent Protein Kinases: Mediators of Receptors

许多激素和神经递质通过动员无处不在的第二信使阵营发挥作用。细胞内转导系统接收这些信号，并快速而准确地传输它们，从而放大适当的生物反应。特异性是通过组装多蛋白信号复合体来实现的，这些复合体产生了酶活性的焦点。因此，蛋白激酶和/或磷酸酶的时空激活对于控制底物磷酸化发生的地点和时间是重要的。锚定蛋白和靶向亚基证明了一种将蛋白激酶和磷酸酶定向到选定底物的分子框架。这些“信号导向分子”的典型例子是A-激酶锚定蛋白(AKAP)，它支持由cAMP依赖的蛋白激酶(PKA)和其他酶组成的多组分信号复合体。这项建议侧重于了解AKAP介导的PKA靶向参与肌动蛋白重组事件和细胞运动控制的底物的功能分支。这些实验是在最近发现Wiskott-Aldrich综合征蛋白(WASP)支架蛋白家族的成员Wave-1是AKAP的基础上发展起来的。WAVE通过将GTP酶RAC偶联到Arp2/3复合体的动员来协调肌动蛋白重组事件。初步研究表明，WAGE-1还与两种PKA底物结合，一种新的GTP酶激活蛋白WINE和MENA，一种参与细胞运动控制的蛋白质。该提议有两个特定的目标。AIM 1将定位WAW-1上的WARP结合位点，确定WARP是否是RAC选择性缝隙，并确定WARP是否具有减弱RAC介导的肌动蛋白重塑事件的功能。AIM 2将定位WAVE-1上的MENA结合位置，确定MENA是否优先被锚定的PKA磷酸化，并确定WAVE-1是否在迁移成纤维细胞的前缘招募MENA来控制细胞运动速度。

Many hormones and neurotransmitters exert their action through the mobilization of the ubiquitous second messenger cAMP. Intracellular transduction systems receive these signals and transmit them quickly and precisely resulting in the amplification of appropriate biological responses. Specificity is achieved by the assembly of multi-protein signaling complexes that create focal points of enzyme activity. Accordingly, the spatiotemporal activation of protein kinases and/orphosphatases is important in controlling where and when substrate phosphorylation occurs. Anchoring proteins and targeting subunits prove a molecular framework that orients protein kinases and phosphatases towards selected substrates. Prototypic examples of these "signal directing molecules" are A-kinases Anchoring Proteins (AKAPs) which sustain multi-component signaling complexes of the cAMP-dependent protein kinase (PKA) and other enzymes. This proposal focuses on understanding the functional ramifications of AKAP-mediated targeting of PKA to substrates that participate in the control of actin reorganization events and cell movement. These experiments have developed from a recent finding that WAVE-1, a member of the Wiskott-Aldrich Syndrome Protein (WASP) family of scaffolding proteins, is an AKAP. WAVE coordinates actin reorganization events by coupling the GTPase Rac to the mobilization of the Arp2/3 complex. Preliminary studies have demonstrated that WAVE-1 also binds to two PKA substrates" a novel GTPase activating protein called WARM and Mena, a protein involved in the control of cell motility. There are two specific aims of this proposal. Aim 1 will map the WARP binding site on WAVE-1, establish if WARP is a Rac selective GAP and determine if WARP functions to attenuate Rac-mediated actin remodeling events. Aim 2 will map the Mena binding site on WAVE-1, establish whether Mena is preferentially phosphorylated by anchored PKA and determine if WAVE-1 functions to recruit Mena at the leading edg3e of migrating fibroblasts to control the rate of cell motility.