Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

Abstract

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the response to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, -13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBPI (for rapamycin-binding protein). The RBPI protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.Agents that inhibit T-cell activation include cyclosporin A (CsA) (19) and the recently discovered macrolide FK506 (31, 36). CsA was originally discovered as an antifungal agent, and FK506 was identified as an inhibitor of interleukin-2 (IL-2) production (20). Despite the structural dissimilarity between these two immunosuppressive drugs, recent reports suggest that the targets for both agents, cyclophilin and FK506-binding protein (FKBP), respectively, are peptidylprolyl cis-trans isomerases (PPlases), enzymes that promote protein folding in vitro (12,15,34,35,37,40,41). Although the endogenous function of PPlases is not known, the fact that the immunosuppressive action of CsA and FK506 is linked to inhibition of PPIase activity suggests that they may be required in the regulation of intracellular signaling events leading to T-cell activation (8, 12, 37).Rapamycin, a macrolide antifungal agent with structural similarity to FK506 (32, 42), also exhibits immunosuppressive (3, 26, 38) as well as antineoplastic (9, 18) properties. Rapamycin and FK506 act as reciprocal antagonists in vivo (murine T cell activation [6]) and compete for binding to FKBP (15). Given these similarities, the mechanism of action of rapamycin remains enigmatic because, whereas FK506 (like CsA) acts to inhibit IL-2 transcription, rapamycin has no effe...

Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression

“…FKBP12 is an abundant and ubiquitously expressed peptidyl-prolyl cis/trans isomerase that may function in protein folding (reviewed in Schreiber, 1991). While rapamycin inhibits the isomerase activity of FKBP12 (Heitman et al, 1991;Koltin et al, 1991;Wiederrecht et al, 1991), it appears that inhibition of this activity is not responsible for rapamycin sensitivity: Deletion of FPR1 (FKBP12) (or deletion of all four FKBP12 genes (FPR1-4)) in S. cerevisiae is not lethal; rather, the yeast are viable and exhibit resistance to the toxic effects of rapamycin (Heitman et al, 1991;Koltin et al, 1991;Dolinski et al, 1997). Therefore, it is the presence of FKBP12, not its activity, that is required for the toxic, antiproliferative action of rapamycin in yeast.…”

The rapamycin-sensitive signal transduction pathway as a target for cancer therapy

“…Although there are many members of the FKBP family, a large body of biochemical and genetic studies suggest that FKBP12 is the most important binding protein with respect to the rapamycin-sensitive signal transduction pathway (Heitman et al, 1991;Koltin et al, 1991;Fruman et al, 1995). The resultant rapamycin-FKBP12 complex interacts with and inhibits the activity of a 290 kd kinase, termed mammalian target of rapamycin (mTOR) (also known as FRAP, RAFT1, and RAP1) (Figure 1) (Sabatini et al, 1994;Sabers et al, 1995;Brown et al, 1994;Chiu et al, 1994).…”

“…Rapamycin binds intracellularly to members of the immunophilin family of FK506 binding proteins (FKBPs), inhibiting their enzymatic activity as prolyl isomerases (Heitman et al, 1991;Koltin et al, 1991;Fruman et al, 1995). Although there are many members of the FKBP family, a large body of biochemical and genetic studies suggest that FKBP12 is the most important binding protein with respect to the rapamycin-sensitive signal transduction pathway (Heitman et al, 1991;Koltin et al, 1991;Fruman et al, 1995).…”

Rapamycin Blocks Sexual Development in Fission Yeast through Inhibition of the Cellular Function of an FKBP12 Homolog

“…Similar to the effect of rapamycin in T cells and certain non-lymphoid cells, rapamycin treatment of S. cerevisiae cells results in a G 1 cell cycle arrest (34). S. cerevisiae cells contain one FKBP12 homolog, named FPR1 (34), also known as RBP1 (35). Disruption of FPR1 results in slightly slowly growing but viable cells that are completely resistant to rapamycin.…”