title: Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue.
abstract: Complexed with its intracellular receptor, FKBP12, the natural product rapamycin inhibits G1 progression of the cell cycle in a variety of mammalian cell lines and in the yeast Saccharomyces cerevisae. Previously, a mammalian protein that directly associates with FKBP12-rapamycin has been identified and its encoding gene has been cloned from both human (designated FRAP) [Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S. & Schreiber, S.L. (1994) Nature (London) 369, 756-758] and rat (designated RAFT) [Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S.H. (1994) Cell 78, 35-43]. The full-length FRAP is a 289-kDa protein containing a putative phosphatidylinositol kinase domain. Using an in vitro transcription/translation assay method coupled with proteolysis studies, we have identified an 11-kDa FKBP12-rapamycin-binding domain within FRAP. This minimal binding domain lies N-terminal to the kinase domain and spans residues 2025-2114. In addition, we have carried out mutagenesis studies to investigate the role of Ser2035, a potential phosphorylation site for protein kinase C within this domain. We now show that the FRAP Ser2035-->Ala mutant displays similar binding affinity when compared with the wild-type protein, whereas all other mutations at this site, including mimics of phosphoserine, abolish binding, presumably due to either unfavorable steric interactions or induced conformational changes.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: X F
authors.initials: XF
authors.lastname: Zheng
authors.forename: E J
authors.initials: EJ
authors.lastname: Brown
authors.forename: S L
authors.initials: SL
authors.lastname: Schreiber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011090:Polyenes / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012694:Serine; D020123:Sirolimus; D016559:Tacrolimus / Q000378:metabolism; D022021:Tacrolimus Binding Proteins; D014158:Transcription, Genetic
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D010446:Peptide Fragments; D011090:Polyenes; D011993:Recombinant Fusion Proteins; D012694:Serine; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus; D016559:Tacrolimus