Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue

Jie Chen, Xiao Feng Zheng, Eric J. Brown, Stuart L. Schreiber

Research output: Contribution to journalArticlepeer-review

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. and Schreiber, S. L. (1994) Nature (London) 369, 756-758] and rat (designated RAFT) [Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P. and 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.

Original languageEnglish (US)
Pages (from-to)4947-4951
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number11
DOIs
StatePublished - May 23 1995

ASJC Scopus subject areas

  • General

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