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

Identifieur interne : 001B18 ( Main/Exploration ); précédent : 001B17; suivant : 001B19

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

Auteurs : J. Chen [États-Unis] ; X F Zheng ; E J Brown ; S L Schreiber

Source :

RBID : pubmed:7539137

Descripteurs français

English descriptors

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.

DOI: 10.1073/pnas.92.11.4947
PubMed: 7539137
PubMed Central: PMC41824


Affiliations:


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Le document en format XML

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<term>Carrier Proteins (chemistry)</term>
<term>Carrier Proteins (metabolism)</term>
<term>Circular Dichroism (MeSH)</term>
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<term>DNA-Binding Proteins (biosynthesis)</term>
<term>DNA-Binding Proteins (chemistry)</term>
<term>DNA-Binding Proteins (metabolism)</term>
<term>Heat-Shock Proteins (biosynthesis)</term>
<term>Heat-Shock Proteins (chemistry)</term>
<term>Heat-Shock Proteins (metabolism)</term>
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<term>Immunosuppressive Agents (metabolism)</term>
<term>Kinetics (MeSH)</term>
<term>Mammals (MeSH)</term>
<term>Molecular Sequence Data (MeSH)</term>
<term>Molecular Weight (MeSH)</term>
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<term>Peptide Fragments (chemistry)</term>
<term>Peptide Fragments (metabolism)</term>
<term>Polyenes (metabolism)</term>
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<term>Recombinant Fusion Proteins (chemistry)</term>
<term>Recombinant Fusion Proteins (metabolism)</term>
<term>Saccharomyces cerevisiae (metabolism)</term>
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<term>Sirolimus (MeSH)</term>
<term>Tacrolimus (metabolism)</term>
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<term>Données de séquences moléculaires (MeSH)</term>
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<term>Fragments peptidiques (métabolisme)</term>
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<term>Immunosuppresseurs (métabolisme)</term>
<term>Mammifères (MeSH)</term>
<term>Masse moléculaire (MeSH)</term>
<term>Mutagenèse dirigée (MeSH)</term>
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<term>Protéines de fusion recombinantes (biosynthèse)</term>
<term>Protéines de fusion recombinantes (composition chimique)</term>
<term>Protéines de fusion recombinantes (métabolisme)</term>
<term>Protéines de liaison au tacrolimus (MeSH)</term>
<term>Protéines de liaison à l'ADN (biosynthèse)</term>
<term>Protéines de liaison à l'ADN (composition chimique)</term>
<term>Protéines de liaison à l'ADN (métabolisme)</term>
<term>Protéines de transport (biosynthèse)</term>
<term>Protéines de transport (composition chimique)</term>
<term>Protéines de transport (métabolisme)</term>
<term>Protéines du choc thermique (biosynthèse)</term>
<term>Protéines du choc thermique (composition chimique)</term>
<term>Protéines du choc thermique (métabolisme)</term>
<term>Rats (MeSH)</term>
<term>Saccharomyces cerevisiae (métabolisme)</term>
<term>Sirolimus (MeSH)</term>
<term>Sites de fixation (MeSH)</term>
<term>Séquence d'acides aminés (MeSH)</term>
<term>Sérine (MeSH)</term>
<term>Tacrolimus (métabolisme)</term>
<term>Transcription génétique (MeSH)</term>
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<term>DNA-Binding Proteins</term>
<term>Heat-Shock Proteins</term>
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<term>DNA-Binding Proteins</term>
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<term>Immunosuppressive Agents</term>
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<term>Fragments peptidiques</term>
<term>Immunosuppresseurs</term>
<term>Polyènes</term>
<term>Protéines de fusion recombinantes</term>
<term>Protéines de liaison à l'ADN</term>
<term>Protéines de transport</term>
<term>Protéines du choc thermique</term>
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<term>Animals</term>
<term>Binding Sites</term>
<term>Circular Dichroism</term>
<term>Cloning, Molecular</term>
<term>Humans</term>
<term>Kinetics</term>
<term>Mammals</term>
<term>Molecular Sequence Data</term>
<term>Molecular Weight</term>
<term>Mutagenesis, Site-Directed</term>
<term>Protein Biosynthesis</term>
<term>Protein Conformation</term>
<term>Rats</term>
<term>Serine</term>
<term>Sirolimus</term>
<term>Tacrolimus Binding Proteins</term>
<term>Transcription, Genetic</term>
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<term>Conformation des protéines</term>
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<term>Masse moléculaire</term>
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<term>Protéines de liaison au tacrolimus</term>
<term>Rats</term>
<term>Sirolimus</term>
<term>Sites de fixation</term>
<term>Séquence d'acides aminés</term>
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<div type="abstract" xml:lang="en">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.</div>
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<AbstractText>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.</AbstractText>
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