Missense mutations at the FKBP12-rapamycin-binding site of TOR1.
The TOR genes were first identified in Saccharomyces cerevisiae by the isolation of mutants which exhibit dominant resistance to the immunosuppressive and antifungal drug rapamycin (Rm). The originally characterized Rm-resistant (RmR) TOR1-1 and TOR2-1 alleles contain an Arg in place of a conserved Ser residue, which lies adjacent to the phosphatidylinositol (PI) kinase-related domain of TOR (Ser1972 in TOR1; Ser1975 in TOR2). Additional spontaneous RmR mutants containing Lys, Ile or Asn substitutions were subsequently isolated. As this Ser is a potential site for protein kinase C phosphorylation, we were interested in determining whether the observed RmR is due to steric hindrance of the FKBP12-Rm-TOR interaction or whether phosphorylation at this site is required to mediate the interaction. Using site-directed mutagenesis, we replaced the Ser1972 residue of TOR1 with either a conservative residue, Ala, an alternative potential phosphorylation site, Thr, or Asp to mimic phosphorylation. The TOR1 (S1972A) mutant protein retained Rm sensitivity (RmS), whereas both the Thr and Asp substitutions conferred RmR. RmS correlated with the ability to interact with FKBP12-Rm in a two-hybrid assay: both wild-type TOR1 and the S1972A mutant retained the ability to interact with FKBP12-Rm, whereas the S1972T, S1972D and S1972R mutants failed to interact. All mutant TOR1 proteins were able to complement the growth defect of tor1 null alleles, suggesting that the Ser1972 residue may not be required for TOR1 function in cycling cells. Since a TOR1(S1972A) mutant protein confers a RmS phenotype, interacts with FKBP12-Rm in a two-hybrid assay, and functions in vivo, we conclude that phosphorylation at Ser1972 is not necessary for the interaction between TOR1 and FKBP12-Rm.
Freeman
K
K
Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
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D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011090:Polyenes / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D011090:Polyenes; D029701:Saccharomyces cerevisiae Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C083324:TOR1 protein, S cerevisiae; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus