PubMed · 8900273
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L Lin, S P Hale, P Schimmel. 1996-11-07. Aminoacylation error correction.. https://doi.org/10.1038/384033b0
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The Src-related tyrosine kinase p56(lck) (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.
The N73 nucleotide at the end of the tRNA acceptor stem is commonly used by tRNA synthetases for discrimination. Because only a few synthetase-tRNA cocrystal structures have been determined, understanding of the molecular basis for N73 discrimination is limited. Here we investigated the possibility that, for at least some synthetases, the capacity to recognize different N73 nucleotides resides in the variable sequence of the loop of motif 2, a motif found in all class II enzymes. In the cocrystal of the class II yeast aspartyl-tRNA synthetase, atomic groups of the G73 discriminator of tRNAAsp interact with three side chains of the enzyme. We examined lysyl-tRNA synthetase, a close structural homologue of the aspartyl enzyme. Different substitutions were introduced into the Escherichia coli enzyme (A73 discriminator) to make its loop more like that of the human enzyme (G73 discriminator). Our data show that the loop of motif 2 of the lysine enzyme makes tRNA functional contacts, as predicted from the structural comparison. And yet, the E. coli enzyme with the "humanized" loop sequence had the same quantitative kinetic preference for A73 versus G as the wild-type enzyme. We conclude that discriminator base selectivity in the lysine enzyme requires residues in addition to or other than those in the loop of motif 2. Thus, even tRNA synthetases that are close structural homologues may use the same RNA binding element to make functional contacts with places (in the acceptor stem) that are idiosyncratic to each synthetase-tRNA pair.
The first 10 residues within the Src homology domain (SH)-4 domain of the Src family kinase Fyn are required for binding to the immune receptor tyrosine-based activation motif (ITAM) of T cell receptor (TCR) subunits. Recently, mutation of glycine 2, cysteine 3, and lysines 7 and 9 was shown to block binding of Fyn to TCR zeta chain ITAMs, prompting the designation of these residues as an ITAM recognition motif (Gauen, L.K.T., M.E. Linder, and A.S. Shaw. 1996. J. Cell Biol. 133:1007-1015). Here we show that these residues do not mediate direct interactions with TCR ITAMs, but rather are required for efficient myristoylation and palmitoylation of Fyn. Specifically, coexpression of a K7,9A-Fyn mutant with N-myristoyltransferase restored myristoylation, membrane binding, and association with the cytoplasmic tail of TCR zeta fused to CD8. Conversely, treatment of cells with 2-hydroxymyristate, a myristoylation inhibitor, blocked association of wild-type Fyn with zeta. The Fyn NH2 terminus was necessary but not sufficient for interaction with zeta and both Fyn kinase and SH2 domains were required, directing phosphorylation of zeta ITAM tyrosines and binding to zeta ITAM phosphotyrosines. Fyn/zeta interaction was sensitive to octylglucoside and filipin, agents that disrupt membrane rafts. Moreover, a plasma membrane bound, farnesylated Fyn construct, G2A,C3S-FynKRas, was not enriched in the detergent insoluble fraction and did not associate with zeta. We conclude that the Fyn SH4 domain provides the signals for fatty acylation and specific plasma membrane localization, stabilizing the interactions between the Fyn SH2 domain and phosphotyrosines in TCR zeta chain ITAMs.