Spatiotemporal regulation of intracellular signaling molecules, such as the 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

细胞内信号分子（例如 3',5'-环磷酸腺苷 (cAMP) 依赖性蛋白激酶 (PKA)）的时空调节可确保正常的细胞功能。普遍存在的 PKA 调节亚基 RIα 的液-液相分离 (LLPS) 可促进 cAMP 区室化和信号传导特异性。然而，RIα LLPS 的分子决定因素仍不清楚。在这里，我们揭示了两个独立的二聚化界面，与cAMP诱导的假底物抑制序列中PKA催化亚基（PKA-C）的释放相结合，驱动哺乳动物细胞胞质溶胶中RIα缩合物的形成，这通过对接来拮抗。 A-激酶锚定蛋白。引人注目的是，我们发现 RIα 假底物区域在形成非典型 R:C 复合物中发挥着关键作用，该复合物将活性 PKA-C 募集到 RIα 缩合物中，以维持细胞质中较低的基础 PKA 活性。我们的结果表明，RIα LLPS 不仅促进 cAMP 区室化，而且在空间上抑制活性 PKA-C，从而突出了生物分子缩合物在驱动信号特异性方面的功能多功能性。