The small GTPase RhoA is a central signaling enzyme that is involved in various cellular processes such as cytoskeletal dynamics, transcription, and cell cycle progression. Many signal transduction pathways activate RhoA—for instance, Gα_q-coupled Histamine 1 Receptor signaling via Gα_q-dependent activation of RhoGEFs such as p63. Although multiple upstream regulators of RhoA have been identified, the temporal regulation of RhoA and the coordination of different upstream components in its regulation have not been well characterized. In this study, live-cell measurement of RhoA activation revealed a biphasic increase of RhoA activity upon histamine stimulation. We showed that the first and second phase of RhoA activity are dependent on p63 and Ca²⁺/PKC, respectively, and further identified phosphorylation of serine 240 on p115 RhoGEF by PKC to be the mechanistic link between PKC and RhoA. Combined approaches of computational modeling and quantitative measurement revealed that the second phase of RhoA activation is insensitive to rapid turning off of the receptor and is required for maintaining RhoA-mediated transcription after the termination of the receptor signaling. Thus, two divergent pathways enable both rapid activation and persistent signaling in receptor-mediated RhoA signaling via intricate temporal regulation.

小 GTPase RhoA 是一种中央信号酶，参与各种细胞过程，如细胞骨架动力学、转录和细胞周期进程。许多信号转导通路激活 RhoA——例如，Gα _q偶联的组胺 1 受体信号通过 Gα _q依赖性激活 RhoGEF（如 p63）。尽管已经确定了 RhoA 的多个上游监管机构，但 RhoA 的时间监管及其监管中不同上游组件的协调尚未得到很好的表征。在这项研究中，RhoA 激活的活细胞测量揭示了组胺刺激后 RhoA 活性的双相增加。我们发现 RhoA 活动的第一阶段和第二阶段依赖于 p63 和 Ca ²⁺/PKC，并进一步确定 PKC 对 p115 RhoGEF 上丝氨酸 240 的磷酸化是 PKC 和 RhoA 之间的机制联系。计算建模和定量测量的组合方法表明，RhoA 激活的第二阶段对受体的快速关闭不敏感，并且是在受体信号传导终止后维持 RhoA 介导的转录所必需的。因此，两种不同的途径能够通过复杂的时间调节在受体介导的 RhoA 信号传导中实现快速激活和持续信号传导。