Amino acids are known regulators of cellular signaling and physiology， but how they are sensed intracellularly is not fully understood. Herein， we report that each aminoacyl-tRNA synthetase (ARS) senses its cognate amino acid sufficiency through catalyzing the formation of lysine aminoacylation (K-AA) on its specific substrate proteins. At physiologic levels， amino acids promote ARSs bound to their substrates and form K-AAs on the ?-amine of lysines in their substrates by producing reactive aminoacyl adenylates. The K-AA marks can be removed by deacetylases， such as SIRT1 and SIRT3， employing the same mechanism as that involved in deacetylation. These dynamically regulated K-AAs transduce signals of their respective amino acids. Reversible leucylation on ras-related GTP-binding protein A/B regulates activity of the mammalian target of rapamycin complex 1. Glutaminylation on apoptosis signal-regulating kinase 1 suppresses apoptosis. We discovered non-canonical functions of ARSs and revealed systematic and functional amino acid sensing and signal transduction networks.