A secreted tyrosine kinase acts in the extracellular environment.

Although tyrosine phosphorylation of extracellular proteins has been reported to occur extensively in vivo, no secreted protein tyrosine kinase has been identified. As a result, investigation of the potential role of extracellular tyrosine phosphorylation in physiological and pathological tissue regulation has not been possible. Here, we show that VLK, a putative protein kinase previously shown to be essential in embryonic development, is a secreted protein kinase, with preference for tyrosine, that phosphorylates a broad range of secreted and ER-resident substrate proteins. We find that VLK is rapidly and quantitatively secreted from platelets in response to stimuli and can tyrosine phosphorylate coreleased proteins utilizing endogenous as well as exogenous ATP sources. We propose that discovery of VLK activity provides an explanation for the extensive and conserved pattern of extracellular tyrosine phosphophorylation seen in vivo, and extends the importance of regulated tyrosine phosphorylation into the extracellular environment.

Aminoacyl-tRNA synthetase inhibition activates a pathway that branches from the canonical amino acid response in mammalian cells.

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.

Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase.

Febrifugine, the bioactive constituent of one of the 50 fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity, though its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of T(H)17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response (AAR) pathway. Here we show that HF binds glutamyl-prolyl-tRNA synthetase (EPRS), inhibiting prolyl-tRNA synthetase activity; this inhibition is reversed by the addition of exogenous proline or EPRS. We further show that inhibition of EPRS underlies the broad bioactivities of this family of natural product derivatives. This work both explains the molecular mechanism of a promising family of therapeutics and highlights the AAR pathway as an important drug target for promoting inflammatory resolution.

Delayed clearance of the pro-carcinogen benzo[a]pyrene in PLHC-1 cells when co-exposed to the antifungal drug clotrimazole and effects on the CYP1A biomarker.

Cytochrome P450 1 A (CYP1A) is a key enzyme in the metabolism of the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) in animals, and a biomarker for environmental PAH exposure. The common antimycotic imidazole drug clotrimazole (CLO) has been detected in the aquatic environment and likely co-exists with BaP. Like BaP, CLO can bind to CYP1A enzymes and can act as a CYP1A inhibitor. Co-exposure of BaP with CLO significantly delayed BaP elimination in a fish liver cell line (PLHC-1). Intracellular BaP concentration was 2.4 times higher after 6 h in co-exposed cells, compared to cells exposed to BaP alone. Higher BaP concentrations in cells co-exposed to CLO positively correlated with CLO dose, indicating CLO-mediated delays in BaP clearance. After 24 h, BaP was undetectable irrespective of CLO co-exposure. In contrast, intracellular CLO concentrations remained constant over the 72 h experimental period. Co-exposure of BaP with CLO caused synergistic and time-dependent increases on the CYP1A biomarker both on CYP1A mRNA levels and on CYP1A enzyme activity, in accordance with an apparent delayed BaP elimination in the presence of CLO. These results indicate a toxicokinetic interaction between BaP and CLO on the CYP1A enzyme that delays metabolic clearance of BaP.