Isolation of a Protein Target of the FKBP12-Rapamycin Complex in Mammalian Cells

Abstract

The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.

IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK

“…Similar results were obtained using wortmannin at 50 nM (results not shown). The MEK inhibitor PD98059 [20] at 25 µM blocked IGF-I-stimulated Rapamycin has been shown to inhibit the mTOR (mammalian target of rapamycin) [21], which is downstream of Akt and upstream of p70S6 kinase [22]. Consistent with this, rapamycin at 5 nM blocked the phosphorylation of p70S6 kinase ( Figure 3A) without affecting the phosphorylation of ERK ( Figure 3C).…”

Urolithin B, a newly identified regulator of skeletal muscle mass

“…Here, we did not detect any change in the phosphorylation state of Akt with urolithin B ( Figure 1F). Nevertheless, the phosphorylation states of mTOR, rpS6, and 4E‐BP1 were higher, and rapamycin, a well‐known inhibitor of mTORC1,16 completely blunted the effects ( Figure 1G–1I). These results strongly suggest that the mTORC1 pathway was activated independently of Akt in response to urolithin B treatment.…”

The role of ATM in DNA damage responses and cancer

“…Functionally, PI-3 kinase-related proteins can be divided into two groups. The ®rst group includes the S. cerevisiae TOR1 and TOR2 proteins and mammalian homologs FRAP/RAFT1/mTOR, all of which participate in G1/S cell cycle progression and are targets for the rapamycin-FKBP12 complex (Kunz et al, 1993;Helliwell et al, 1994;Brown et al, 1994;Sabatini et al, 1994;Sabers et al, 1995). In general, the other family members are necessary for proper responses to DNA damage and cells individually lacking expression of several of these gene products share many of the same phenotypes as AT cells.…”