CAP1 binds and activates adenylyl cyclase in mammalian cells.
CAP1 binds and activates adenylyl cyclase in mammalian cells.
复制标题
CAP1 结合并激活哺乳动物细胞中的腺苷酸环化酶。
DOI：
10.1073/pnas.2024576118
复制
发表时间：
2021
影响因子：
11.1
通讯作者：
Altschuler,DanielL
中科院分区：
文献类型：
--
作者：
Zhang,Xuefeng;Pizzoni,Alejandro;Hong,Kyoungja;Naim,Nyla;Qi,Chao;Korkhov,Volodymyr;Altschuler,DanielL
CAP 1（Cyclase-Associated Protein 1）是一种高度保守的蛋白质。最初在酵母中被鉴定为参与Ras-腺苷酸环化酶和F-肌动蛋白动力学调节的双功能蛋白，腺苷酸环化酶组分似乎在哺乳动物细胞中丢失。通过我们最近鉴定的Ras样小GT-Ras Rap 1作为GTP独立的但香叶基香叶基特异性的CAP 1伴侣，我们假设CAP 1-Rap 1，类似于酵母中的CAP-Ras环化酶，可能在哺乳动物细胞中的cAMP动力学中发挥关键作用。在这项研究中，我们报告，CAP 1结合和激活哺乳动物腺苷酸环化酶在体外，调节cAMP在活细胞中的Rap 1依赖的方式，并影响cAMP依赖的增殖。利用缺失和诱变方法，我们绘制了CAP 1-环化酶与CAP的N-末端结构域的相互作用，该结构域涉及保守RLE基序中的关键亮氨酸残基和腺苷酸环化酶的保守催化环（例如，C1 a和/或C2 a）。当与基于FRET的cAMP传感器，CAP 1过表达敲低策略，以及Rap 1的组成性活性和负调节剂的使用相结合时，我们的研究强调了CAP 1-Rap 1在活细胞中腺苷酸环化酶调节中的关键作用。同样，我们发现CAP 1的调制显着影响cAMP介导的增殖在RLE基序依赖性的方式。综合研究表明，CAP 1-cyclase-Rap 1代表了cAMP动力学和生物学的调节单位。由于Rap 1是一个既定的下游效应的cAMP，我们提出的假设，CAP 1环化酶Rap 1代表一个积极的反馈回路，可能参与cAMP微域的建立和本地化的信号。
CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.