Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex

G. Wiederrecht, S. Hung, H. K. Chan, A. Marcy, M. Martin, J. Calaycay, D. Boulton, N. Sigal, R. L. Kincaid, John Siekierka

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Abstract

The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12·FK- 506·calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807- 815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506- binding protein. Our characterization of the FKBP12·FK-506·CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12·FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12·FK-506, FKBP51·FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.

Original languageEnglish
Pages (from-to)21753-21760
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number30
StatePublished - 11 Nov 1992

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Tacrolimus Binding Proteins
Tacrolimus Binding Protein 1A
Tacrolimus
Molecular Weight
Molecular weight
Peptidylprolyl Isomerase
Proteins
Phosphoric Monoester Hydrolases
Brain
Gels
IgE Receptors
Signal transduction
T-cells
Steroid Receptors
Poisons
Exocytosis
Molecular mass
Calmodulin
Immunosuppressive Agents
Lymphocyte Activation

Cite this

Wiederrecht, G. ; Hung, S. ; Chan, H. K. ; Marcy, A. ; Martin, M. ; Calaycay, J. ; Boulton, D. ; Sigal, N. ; Kincaid, R. L. ; Siekierka, John. / Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 30. pp. 21753-21760.
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abstract = "The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12·FK- 506·calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807- 815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506- binding protein. Our characterization of the FKBP12·FK-506·CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12·FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12·FK-506, FKBP51·FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.",
author = "G. Wiederrecht and S. Hung and Chan, {H. K.} and A. Marcy and M. Martin and J. Calaycay and D. Boulton and N. Sigal and Kincaid, {R. L.} and John Siekierka",
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Wiederrecht, G, Hung, S, Chan, HK, Marcy, A, Martin, M, Calaycay, J, Boulton, D, Sigal, N, Kincaid, RL & Siekierka, J 1992, 'Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex', Journal of Biological Chemistry, vol. 267, no. 30, pp. 21753-21760.

Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex. / Wiederrecht, G.; Hung, S.; Chan, H. K.; Marcy, A.; Martin, M.; Calaycay, J.; Boulton, D.; Sigal, N.; Kincaid, R. L.; Siekierka, John.

In: Journal of Biological Chemistry, Vol. 267, No. 30, 11.11.1992, p. 21753-21760.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex

AU - Wiederrecht, G.

AU - Hung, S.

AU - Chan, H. K.

AU - Marcy, A.

AU - Martin, M.

AU - Calaycay, J.

AU - Boulton, D.

AU - Sigal, N.

AU - Kincaid, R. L.

AU - Siekierka, John

PY - 1992/11/11

Y1 - 1992/11/11

N2 - The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12·FK- 506·calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807- 815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506- binding protein. Our characterization of the FKBP12·FK-506·CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12·FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12·FK-506, FKBP51·FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.

AB - The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12·FK- 506·calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807- 815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506- binding protein. Our characterization of the FKBP12·FK-506·CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12·FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12·FK-506, FKBP51·FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.

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M3 - Article

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SP - 21753

EP - 21760

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 30

ER -