Systematic perturbation of amino acids at endogenous loci provides diverse insights into protein function. Here, we performed a genome-wide screen to globally assess the cell fitness dependency of serine, threonine and tyrosine residues. Using an adenine base editor, we designed a whole-genome library comprising 817,089 single guide RNAs to perturb 584,337 S, T and Y sites. We identified 3,467 functional substitutions affecting cell fitness and 677 of them involving phosphorylation, including numerous phosphorylation-mediated gain-of-function substitutions that regulate phosphorylation levels of itself or downstream factors. Furthermore, our findings highlight that specific substitution types, notably serine to proline, are crucial for maintaining domain structure broadly. Lastly, we demonstrate that 309 enriched hits capable of initiating cell overproliferation might be potential cancer driver mutations. This study represents an extensive functional profiling of S, T and Y residues and provides insights into the distinctive roles of these amino acids in biological mechanisms and tumor progression.

内源位点氨基酸的系统扰动提供了对蛋白质功能的不同见解。在这里，我们进行了全基因组筛选，以全面评估丝氨酸、苏氨酸和酪氨酸残基的细胞适应性依赖性。使用腺嘌呤碱基编辑器，我们设计了一个包含 817,089 个单引导 RNA 的全基因组文库，可干扰 584,337 个 S、T 和 Y 位点。我们鉴定了 3,467 个影响细胞适应性的功能取代，其中 677 个涉及磷酸化，包括许多磷酸化介导的功能获得性取代，可调节自身或下游因子的磷酸化水平。此外，我们的研究结果强调，特定的取代类型，特别是丝氨酸到脯氨酸，对于广泛维持结构域结构至关重要。最后，我们证明能够启动细胞过度增殖的 309 个富集命中可能是潜在的癌症驱动突变。这项研究代表了 S、T 和 Y 残基的广泛功能分析，并深入了解这些氨基酸在生物机制和肿瘤进展中的独特作用。