The immunosuppressant FK506 (Tacrolimus, Prograf) has increased the survival rates of organ transplantation. FK506 exerts its immunosuppressive effect by inhibition of the protein phosphatase calcineurin in activated T-cells. Unfortunately, FK506 therapy is associated with undesired non-therapeutic effects involving targets other than calcineurin. To identify these targets we have addressed FK506 cellular toxicity in budding yeast. We show that FK506 increased cell sensitivity upon osmotic challenge independently of calcineurin and the FK506-binding proteins Fpr1p, 2p, 3p and 4p. FK506 also induced strong amino acid starvation and activation of the general control (GCN) pathway. Tryptophan prototrophy or excess tryptophan overcame FK506 toxicity, showing that tryptophan deprivation mediated this effect. Mutation of the GCN3 and 4 genes partially alleviated FK506 toxicity, suggesting that activation of the GCN pathway by FK506 was also involved in osmotic tolerance. FK506 enhanced osmotic stress-dependent Hog1p kinase phosphorylation that was not accompanied by induction of a Hog1p-dependent reporter. Interestingly, deletion of the GCN2 gene suppressed FK506-dependent Hog1p hyperphosphorylation and restored Hog1p-dependent reporter activity. Conversely, deletion of the HOG1 gene impaired FK506-dependent activation of Gcn2p kinase and translation of a GCN4-lacZ reporter, highlighting functional cross-talk between the Gcn2p and Hog1p protein kinases. Taken together, these data demonstrate that both FK506-induced amino acid starvation and activation of the GCN pathway contribute to cell sensitivity to osmotic stress and reveal a positive regulatory loop between the Hog1p and Gcn2p pathways.

Given the conserved nature of Gcn2p and Hog1p pathways, this mechanism of FK506 toxicity could be relevant to the non-therapeutic effects of FK506 therapy. In order to assess this issue in depth, we studied FK506 toxicity in mammalian cells. FK506 regulated p38 activation by osmotic stress, and decreased viability in osmostressed cells. In addition, FK506 treatment strongly increased the phosphorylation of the eukaryotic initiation factor-2alpha (eIF2alpha) subunit. eIF2alpha phosphorylation, p38 inhibition and cell lethality were relieved by addition of excess amino acids to the medium, suggesting that amino acid availability mediated FK506 toxicity also in mammalian cells.
 
Post-transplantation diabetes mellitus is one of the most relevant non-therapeutic effects of FK506. Last part of this work is focused on the mechanism of action of tungstate, a small molecule that modulates FK506 effects. This inorganic salt, used as an antidiabetic agent, rescued FK506-dependent growth defect in yeast. This rescue was calcineurin independent. Tungstate augmentated FK506-dependent GCN pathway activation and exacerbated FK506-induced osmosensitivity. In addition, tungstate modulated Hog1p activation and Hog1p-dependent gene expression, although no phenotype was observed. Induction of GCN pathway by tungstate occurs only when other inducer is present. That suggests tungstate modulation of a pathway regulator. Tungstate inhibited in vitro Glc7p phosphatase, a GCN pathway negative regulator, and its mammalian orthologue, PP1. Furthermore, a mutant with low Glc7p activity simulated FK506 tolerance caused by tungstate, and genetic interaction data wiht gcn2-507 mutant are consistent with an in vivo inhibition of the phosphatase. Other phenotypes induced in yeast by tungstate are related to ionic homeostasis. In this tungstate effect could be involved Glc7p, as well as Ppz1p phosphatase, that is inhibited in vitro by tungstate, and even the GCN pathway itself, whose constitutive activation showed regulation of ionic homeostasis.