Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.

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SUMMARY Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207-phosphorylation provokes a new conformer of LysRS that inactivates its translational, but activates its transcriptional function. The crystal structure of an MSC sub-complex established that LysRS is held in the MSC by binding to the N-terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap4A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.

MITF and its related family members TFE3 and TFEB heterodimerize with each other, recognize the same DNA sequences, and are subject to many of the same post-translational modifications. We show that lysine residues within conserved small ubiquitin-like modifier (SUMO) consensus sites in these family members are subject to SUMO modification. Mutation of these sites significantly affects the transcriptional activity of MITF but does not alter dimerization, DNA binding, stability, or nuclear localization. Mutagenesis reducing the number of MITF binding sites in the promoter of TRPM1 from three to one eliminated the difference in transcriptional activity between the MITF mutants. Among other MITF target gene promoter constructs, differences in transcriptional activity between wild type and nonsumoylatable MITF were only seen in promoters with multiple MITF binding sites. These data support a synergy control model in which the functional consequences of MITF sumoylation depend on promoter context. Sumoylation, thus, provides a possible mechanism for altering the effects of MITF by affecting the target genes that it activates.MITF is a tissue-restricted, basic helix-loop-helix leucine zipper dimeric transcription factor. It is encoded by the mitf locus in mice (1) and when mutated leads to defects in melanocytes, the retinal pigment epithelium, mast cells, and osteosclasts. Mitf mutant mice are white due to a complete lack of melanocytes, whereas heterozygotes have a white belly spot (1, 2), demonstrating a requirement for mitf in production of this lineage. MITF continues to be necessary in the adult based on the existence of hypomorphic alleles in mice which cause postnatal melanocyte death and premature graying (3, 4). As a transcriptional mediator of differentiation, MITF acts downstream of the melanizing hormone ␣-melanocyte-stimulating hormone (5) and transcriptionally regulates the expression of the enzymes necessary for melanin production in differentiated melanocytes (for review, see Ref. 6). Although these data implicate MITF in both the survival and differentiation of melanocytes, little is known about biochemical regulatory pathways that control MITF in these different roles.MITF is part of the MiT transcription factor family whose members share significant homology and recognize the same DNA elements. Functionally, MITF binds to the canonical E-box promoter sequence CACGTG as well as to the nonpalindromic sequence CACATG (7,8). MITF functions as either a homodimer or as a heterodimer with the related MiT family transcription factors TFE3, TFEB, and TFEC (62). The related factor TFEB was recently identified as a translocated oncogene in papillary renal cell carcinoma in humans (10, 11). The structural features of these family members are so similar that MITF and TFE3 have been shown to genetically compensate for one another in regulation of osteoclast development in mice (12).Several post-translational modifications affect members of the MiT family. In melanocytes, activation of the mitogenactivated protei...

Although human lysyl-tRNA synthetase (KRS), an enzyme for protein synthesis, is often highly expressed in various cancer cells, its pathophysiological implications have not been understood. Here we found that KRS induces cancer cell migration through interaction with the 67-kDa laminin receptor (67LR) that is converted from ribosomal subunit p40. On laminin signal, KRS was phosphorylated at the T52 residue by p38MAPK and dissociated from the cytosolic multi-tRNA synthetase complex for membrane translocation. The importance of T52 phosphorylation for membrane translocation of KRS was confirmed by site-directed mutagenesis. In the membrane, turnover of 67LR was controlled by Nedd4-mediated ubiquitination, and KRS inhibited ubiquitin-dependent degradation of 67LR, thereby enhancing laminin-induced cell migration. This work thus unveiled a unique function of KRS in the control of cell migration and its pathological implication in metastasis.