The FKBP12-Rapamycin-binding Domain Is Required for FKBP12-Rapamycin-associated Protein Kinase Activity and G1 Progression

• Published in: The Journal of biological chemistry 1999-02, Vol.274 (7), p.4266-4272
• Main Authors: Vilella-Bach, M, Nuzzi, P, Fang, Y, Chen, J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Carrier Proteins; Catalysis; Cell Line; G1 Phase; Humans; Immunophilins - genetics; Immunophilins - metabolism; Immunosuppressive Agents - metabolism; Microinjections; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphatidylinositol 3-Kinases - metabolism; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases - metabolism; S Phase; Sequence Homology, Amino Acid; Serine - metabolism; Sirolimus - metabolism; Tacrolimus Binding Proteins; TOR Serine-Threonine Kinases; Tryptophan - metabolism; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.7.4266

The immunosuppressant rapamycin, in complex with its cellular receptor FKBP12, targets the cellular protein FKBP12-rapamycin-associated protein/mammalian target of rapamycin/rapamycin and FKBP12 target 1 (FRAP/mTOR/RAFT1) and inhibits/delays G 1 cell cycle progression in mammalian cells. As a member of the novel phosphatidylinositol kinase-related kinase family, FRAP’s kinase activity is essential for its signaling function. The FKBP12-rapamycin binding (FRB) domain in FRAP is also speculated to play an important role in FRAP function and signaling. However, the biochemical and physiological functions of FRB, as well as the mechanism for rapamycin inhibition, have been unclear. The present study focuses on investigation of FRB’s role and the functional relationship between FRB domain and kinase domain in FRAP. Microinjection of purified FRB protein into human osteosarcoma MG63 cells results in a drastic blockage of the G 1 to S cell cycle progression; such a dominant negative effect is reversed by a point mutation (Trp 2027 → Phe). The same mutation also abolishes kinase activity of FRAP without affecting ATP binding, and truncation studies suggest that upstream sequences including FRB are required for kinase activity in vitro . Given these data, we propose a model for FRAP function, in which the FRB domain is required for activation of the kinase domain, possibly through the interaction with an upstream activator. In addition, our observations provide direct evidence linking FRAP function to G 1 cell cycle progression.