To test the hypothesis that tRNA Tyr recognition differs between bacterial and human tyrosyl-tRNA synthetases, we sequenced several clones identified as human tyrosyl-tRNA synthetase cDNAs by the Human Genome Project. We found that human tyrosyl-tRNA synthetase is composed of three domains: 1) an aminoterminal Rossmann fold domain that is responsible for formation of the activated E⅐Tyr-AMP intermediate and is conserved among bacteria, archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that has not been conserved between bacteria and eukaryotes; and 3) a carboxyl-terminal domain that is unique to the human tyrosyl-tRNA synthetase and whose primary structure is 49% identical to the putative human cytokine endothelial monocyte-activating protein II, 50% identical to the carboxyl-terminal domain of methionyl-tRNA synthetase from Caenorhabditis elegans, and 43% identical to the carboxyl-terminal domain of Arc1p from Saccharomyces cerevisiae. The first two domains of the human tyrosyl-tRNA synthetase are 52, 36, and 16% identical to tyrosyl-tRNA synthetases from S. cerevisiae, Methanococcus jannaschii, and Bacillus stearothermophilus, respectively. Nine of fifteen amino acids known to be involved in the formation of the tyrosyl-adenylate complex in B. stearothermophilus are conserved across all of the organisms, whereas amino acids involved in the recognition of tRNA Tyr are not conserved. Kinetic analyses of recombinant human and B. stearothermophilus tyrosyl-tRNA synthetases expressed in Escherichia coli indicate that human tyrosyl-tRNA synthetase aminoacylates human but not B. stearothermophilus tRNA Tyr , and vice versa, supporting the original hypothesis. It is proposed that like endothelial monocyte-activating protein II and the carboxyl-terminal domain of Arc1p, the carboxyl-terminal domain of human tyrosyltRNA synthetase evolved from gene duplication of the carboxyl-terminal domain of methionyl-tRNA synthetase and may direct tRNA to the active site of the enzyme.Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNA by their cognate amino acid. For most aminoacyl-tRNA synthetases (E), tRNA aminoacylation can be separated into two steps: formation of a stable enzyme-bound aminoacyladenylate intermediate (E⅐AA-AMP, Equation 1), followed by transfer of the amino acid (AA) from the aminoacyl-adenylate intermediate to the 3Ј end of the tRNA substrate (Equation 2).The 20 different aminoacyl-tRNA synthetases fall into two distinct structural classes (1, 2). Class I aminoacyl-tRNA synthetases (of which tyrosyl-tRNA synthetase is a member) are characterized by a structurally well conserved amino-terminal Rossmann fold domain which contains the signature sequences "HIGH" and "KMSKS" (3, 4). In contrast, the carboxyl-terminal domains of class I aminoacyl-tRNA synthetases are structurally diverse suggesting that the primordial aminoacyl-tRNA synthetase consisted solely of the amino-terminal Rossmann fold (5-8). Both x-ray crystallography and site-directed mutagenesis of class I aminoacyl-tRNA synthet...

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the ␤12-␤13 loop attenuated the inhibition of the resulting PP1␣ catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with doseresponse characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1␣, the PP1␣ catalytic core, and CRHM2 with identical IC 50 . Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the ␤12-␤13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the ␤12-␤13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.Type-1 protein serine/threonine phosphatases (PP1) 1 are expressed in all eukaryotic cells and have been implicated in the control of a variety of physiological processes, including carbohydrate and lipid metabolism, protein synthesis, and gene transcription (1, 2). PP2A, the major type-2 protein serine/ threonine phosphatase, shares nearly 50% sequence identity with the PP1 catalytic subunit with most of this being in sequences that organize the three-dimensional structure of the catalytic site (3). Microcystin-LR and other toxins that inhibit PP1 activity also inhibit PP2A, emphasizing the shared structural determinants at or near the catalytic site that mediate phosphatase inhibition by these natural compounds (4).Despite these similarities between the two phosphatases, cellular mechanisms that regulate PP1 and PP2A show a high degree of specificity. For instance, the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and the nuclear inhibitor NIPP-1 uniquely inhibit PP1 activity. Moreover, these PP1 inhibitors are regulated by reversible phosphorylation so that they can modulate PP1 activity in response to hormonal stimuli. Physiological studies suggest that PP1 inhibitors function as molecular switches to control cellular signaling pathways (5). For example, I-1 and its structural homologue, DARPP-32 (dopamine-and cAMP-regulated phosphoprotein of apparent M r 32,000), once phosphorylated by cAMP-dependent protein kinase, inhibit PP1 activity to elevate and maintain cellular proteins in their phosphorylated state. In this manner, I-1 and DARPP-32 amplify and prolong cAMP signals. The importance of PP1 inhibitors in cAMP signaling was highlighted by the disruption of the mouse DARPP-32 gene (6), which severely atten...