Reversible protein phosphorylation is an important mechanism for regulating (dis)assembly of biomolecular condensates. However, condensate-specific phosphosites remain largely unknown, thereby limiting our understanding of the underlying mechanisms. Here, we combine solubility proteome profiling with phosphoproteomics to quantitatively map several hundred phosphosites enriched in either soluble or condensate-bound protein subpopulations, including a subset of phosphosites modulating protein–RNA interactions. We show that multi-phosphorylation of the C-terminal disordered segment of heteronuclear ribonucleoprotein A1 (HNRNPA1), a key RNA-splicing factor, reduces its ability to locate to nuclear clusters. For nucleophosmin 1 (NPM1), an essential nucleolar protein, we show that phosphorylation of S254 and S260 is crucial for lowering its partitioning to the nucleolus and additional phosphorylation of distal sites enhances its retention in the nucleoplasm. These phosphorylation events decrease RNA and protein interactions of NPM1 to regulate its condensation. Our dataset is a rich resource for systematically uncovering the phosphoregulation of biomolecular condensates.

可逆的蛋白质磷酸化是调节（解）生物分子缩合物的重要机制。然而，冷凝物特异性磷酸位点仍然很大程度上未知，从而限制了我们对潜在机制的理解。在这里，我们将溶解度蛋白质组分析与磷酸蛋白质组学相结合，以定量绘制数百个富含可溶性或凝聚结合蛋白质亚群的磷酸位点，包括调节蛋白质-RNA 相互作用的磷酸位点子集。我们表明，异核核糖核蛋白 A1 (HNRNPA1) 的 C 末端无序片段是一种关键的 RNA 剪接因子，它的多磷酸化降低了其定位到核簇的能力。对于核磷蛋白 1 (NPM1)，一种必需的核仁蛋白，我们表明 S254 和 S260 的磷酸化对于降低其向核仁的分配至关重要，并且远端位点的额外磷酸化增强了其在核质中的保留。这些磷酸化事件减少了 NPM1 的 RNA 和蛋白质相互作用以调节其凝聚。我们的数据集是系统揭示生物分子缩合物磷酸调节的丰富资源。