The Role of the Regulatory Subunit of Fission Yeast Calcineurin for in Vivo Activity and Its Relevance to FK506 Sensitivity

Abstract

Calcineurin, a protein phosphatase required for Ca2+signaling in many cell types, is a heterodimer composed of catalytic and regulatory subunits. The fission yeast genome encodes a single set of catalytic (Ppb1) and regulatory (Cnb1) subunits, providing an ideal model system to study the functions of these subunits in vivo. Here, we cloned the cnb1+ gene and showed that the cnb1 knock-out (Δcnb1) exhibits identical phenotypes with Δppb1 and that overexpression of Ppb1 failed to suppress the phenotypes of Δcnb1. Interestingly, overexpression of the C-terminal-deleted Ppb1 (Ppb1ΔC), the constitutively active form of Ppb1, also failed to suppress the phenotypes of Δcnb1. FK506 caused MgCl2 sensitivity to the wild-type cells in an FKBP12-dependentmanner. Co-overexpression of Ppb1 and Cnb1 suppressed the FK506-induced MgCl2 sensitivity, but the suppression was only partial, suggesting that an excess amount of the Ppb1-Cnb1 complex cannot compete out the FKBP12-FK506 complex. Although overexpression of Ppb1ΔC alone had little effect on cell growth, co-overexpression of Ppb1ΔC and Cnb1 caused a distinct growth defect. FK506 suppressed the growth defect when Cnb1 was co-expressed using the attenuated nmt1 promoter, but it failed to suppress the defect when Cnb1 was co-expressed using the wild-type nmt1 promoter. Knock-out of the prz1+ gene, encoding a downstream target transcription factor of calcineurin, suppressed the growth defect irrespective of the promoter potency. These results suggest that Cnb1 is essential for the activation of calcineurin and that the activated calcineurin is the pharmacological target of the FKBP12-FK506 complex in vivo. Calcineurin, a protein phosphatase required for Ca2+signaling in many cell types, is a heterodimer composed of catalytic and regulatory subunits. The fission yeast genome encodes a single set of catalytic (Ppb1) and regulatory (Cnb1) subunits, providing an ideal model system to study the functions of these subunits in vivo. Here, we cloned the cnb1+ gene and showed that the cnb1 knock-out (Δcnb1) exhibits identical phenotypes with Δppb1 and that overexpression of Ppb1 failed to suppress the phenotypes of Δcnb1. Interestingly, overexpression of the C-terminal-deleted Ppb1 (Ppb1ΔC), the constitutively active form of Ppb1, also failed to suppress the phenotypes of Δcnb1. FK506 caused MgCl2 sensitivity to the wild-type cells in an FKBP12-dependentmanner. Co-overexpression of Ppb1 and Cnb1 suppressed the FK506-induced MgCl2 sensitivity, but the suppression was only partial, suggesting that an excess amount of the Ppb1-Cnb1 complex cannot compete out the FKBP12-FK506 complex. Although overexpression of Ppb1ΔC alone had little effect on cell growth, co-overexpression of Ppb1ΔC and Cnb1 caused a distinct growth defect. FK506 suppressed the growth defect when Cnb1 was co-expressed using the attenuated nmt1 promoter, but it failed to suppress the defect when Cnb1 was co-expressed using the wild-type nmt1 promoter. Knock-out of the prz1+ gene, encoding a downstream target transcription factor of calcineurin, suppressed the growth defect irrespective of the promoter potency. These results suggest that Cnb1 is essential for the activation of calcineurin and that the activated calcineurin is the pharmacological target of the FKBP12-FK506 complex in vivo. Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, plays key roles in various Ca2+-mediated cellular processes (1.Aramburu J. Heitman J. Crabtree G.R. EMBO Rep. 2004; 5: 343-348Crossref PubMed Scopus (131) Google Scholar, 2.Sugiura R. Kita A. Kuno T. Cell Cycle. 2004; 3: 286-288Crossref PubMed Scopus (1) Google Scholar), and inhibition of calcineurin by specific immunosuppressive drugs such as cyclosporin A or tacrolimus (FK506) underlies the molecular mechanisms of these drugs used in organ transplantation (3.Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3633) Google Scholar). Calcineurin is a heterodimer of a catalytic (calcineurin A) subunit and a Ca2+-binding regulatory (calcineurin B) subunit. In known mammalian cells, three genes encode the catalytic subunits, and two genes encode the regulatory subunits (4.Rusnak F. Mertz P. Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1117) Google Scholar). The physiological significance of the gene multiplicity encoding these subunits is unknown, and this makes it very difficult to study the role of each subunit of calcineurin in vivo. Genes encoding calcineurin subunit proteins are highly conserved among species from yeast to man (5.Cyert M.S. Annu. Rev. Genet. 2001; 35: 647-672Crossref PubMed Scopus (150) Google Scholar). The fission yeast Schizosaccharomyces pombe genome contains the smallest number of protein-coding genes yet recorded for a eukaryote (6.Wood V. Gwilliam R. Rajandream M.A. Lyne M. Lyne R. Stewart A. Sgouros J. Peat N. Hayles J. Baker S. Basham D. Bowman S. Brooks K. Brown D. Brown S. Chillingworth T. Churcher C. Collins M. Connor R. Cronin A. Davis P. Feltwell T. Fraser A. Gentles S. Goble A. Hamlin N. Harris D. Hidalgo J. Hodgson G. Holroyd S. Hornsby T. Howarth S. Huckle E.J. Hunt S. Jagels K. James K. Jones L. Jones M. Leather S. McDonald S. McLean J. Mooney P. Moule S. Mungall K. Murphy L. Niblett D. Odell C. Oliver K. O'Neil S. Pearson D. Quail M.A. Rabbinowitsch E. Rutherford K. Rutter S. Saunders D. Seeger K. Sharp S. Skelton J. Simmonds M. Squares R. Squares S. Stevens K. Taylor K. Taylor R.G. Tivey A. Walsh S. Warren T. Whitehead S. Woodward J. Volckaert G. Aert R. Robben J. Grymonprez B. Weltjens I. Vanstreels E. Rieger M. Schafer M. Muller-Auer S. Gabel C. Fuchs M. Dusterhoft A. Fritzc C. Holzer E. Moestl D. Hilbert H. Borzym K. Langer I. Beck A. Lehrach H. Reinhardt R. Pohl T.M. Eger P. Zimmermann W. Wedler H. Wambutt R. Purnelle B. Goffeau A. Cadieu E. Dreano S. Gloux S. Lelaure V. Mottier S. Galibert F. Aves S.J. Xiang Z. Hunt C. Moore K. Hurst S.M. Lucas M. Rochet M. Gaillardin C. Tallada V.A. Garzon A. Thode G. Daga R.R. Cruzado L. Jimenez J. Sanchez M. del Rey F. Benito J. Dominguez A. Revuelta J.L. Moreno S. Armstrong J. Forsburg S.L. Cerutti L. Lowe T. McCombie W.R. Paulsen I. Potashkin J. Shpakovski G.V. Ussery D. Barrell B.G. Nurse P. Cerrutti L. Nature. 2002; 415: 871-880Crossref PubMed Scopus (1251) Google Scholar) and has only a single gene encoding the catalytic subunit of calcineurin, Ppb1 (7.Yoshida T. Toda T. Yanagida M. J. Cell Sci. 1994; 107: 1725-1735Crossref PubMed Google Scholar), whereas the budding yeast Saccharomyces cerevisiae has two genes encoding the catalytic subunit (8.Liu Y. Ishii S. Tokai M. Tsutsumi H. Ohki O. Akada R. Tanaka K. Tsuchiya E. Fukui S. Miyakawa T. Mol. Gen. Genet. 1991; 227: 52-59Crossref PubMed Scopus (143) Google Scholar, 9.Cyert M.S. Kunisawa R. Kaim D. Thorner J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7376-7380Crossref PubMed Scopus (241) Google Scholar). The small genome size and gene number of fission yeast as well as its relevance to a higher system makes it a highly suitable model system in which to study the calcineurin signaling pathway by a molecular genetic approach. We have previously shown that Ppb1 plays an essential role in maintaining chloride ion homeostasis (10.Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar). We have also shown that calcineurin acts antagonistically with the Pmk1 MAP 1The abbreviations used are: MAP, mitogen-activated protein; GFP, green fluorescent protein; GST, glutathione S-transferase; Ppb1ΔC, C-terminal-deleted Ppb1; FKBP12, 12-kDa FK506-binding protein; YPD, yeast extract/peptone/dextrose; EMM, Edinburgh minimal medium. kinase in chloride ion homeostasis (10.Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar, 11.Sugiura R. Toda T. Dhut S. Shuntoh H. Kuno T. Nature. 1999; 399: 479-483Crossref PubMed Scopus (72) Google Scholar, 12.Sugiura R. Kita A. Shimizu Y. Shuntoh H. Sio S.O. Kuno T. Nature. 2003; 424: 961-965Crossref PubMed Scopus (61) Google Scholar). In addition, analyses of mutants that require calcineurin activity for their growth revealed that calcineurin is implicated in cytokinesis, the septation initiation network, and membrane trafficking in fission yeast (13.Zhang Y. Sugiura R. Lu Y. Asami M. Maeda T. Itoh T. Takenawa T. Shuntoh H. Kuno T. J. Biol. Chem. 2000; 275: 35600-35606Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 14.Yada T. Sugiura R. Kita A. Itoh Y. Lu Y. Hong Y. Kinoshita T. Shuntoh H. Kuno T. J. Biol. Chem. 2001; 276: 13579-13586Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 15.Lu Y. Sugiura R. Yada T. Cheng H. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 1009-1019Crossref PubMed Scopus (30) Google Scholar, 16.Cheng H. Sugiura R. Wu W. Fujita M. Lu Y. Sio S.O. Kawai R. Takegawa K. Shuntoh H. Kuno T. Mol. Biol. Cell. 2002; 13: 2963-2976Crossref PubMed Scopus (80) Google Scholar, 17.Kita A. Sugiura R. Shoji H. He Y. Deng L. Lu Y. Sio S.O. Takegawa K. Sakaue M. Shuntoh H. Kuno T. Mol. Biol. Cell. 2004; 15: 2920-2931Crossref PubMed Google Scholar). Recently, we cloned the prz1+ gene, which encodes a Crz1/Tcn1 transcription factor homolog, and results have suggested that calcineurin activates at least two distinct signaling branches, i.e. the Prz1-dependent branch that regulates the expression of the Pmc1 Ca2+ pump and an unknown pathway that functions antagonistically with the Pmk1 MAP kinase pathway (18.Hirayama S. Sugiura R. Lu Y. Maeda T. Kawagishi K. Yokoyama M. Tohda H. Giga-Hama Y. Shuntoh H. Kuno T. J. Biol. Chem. 2003; 278: 18078-18084Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Biochemical studies using recombinant mammalian proteins have suggested that the regulatory subunit as well as calmodulin is required for the high calcineurin activity in vitro (19.Perrino B.A. Ng L.Y. Brickey D.A. Saitoh Y. Ushio Y. Fukunaga K. Miyamoto E. Soderling T.R. J. Biol. Chem. 1992; 267: 15965-15969Abstract Full Text PDF PubMed Google Scholar, 20.Perrino B.A. Ng L.Y. Soderling T.R. J. Biol. Chem. 1995; 270: 340-346Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and that the regulatory subunit, but not calmodulin, is required for the of calcineurin to the complex Crabtree G.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). is in molecular genetic study on the functions of calcineurin subunits. S. pombe has a single set of catalytic and regulatory subunits, it an ideal model system to study the role of these subunits in using the molecular genetic approach. Here, we cloned the fission yeast cnb1+ gene, which encodes the regulatory subunit of results suggest that the regulatory subunit is essential for the activation of calcineurin in and that the activated calcineurin is the pharmacological target of the FKBP12-FK506 complex in vivo. and pombe used in this study are in I. The and the minimal have previously T. Dhut S. G. Y. E. Sugiura R. Kuno T. Mol. Cell. Biol. PubMed Scopus Google Scholar). for S. pombe to Moreno S. A. Nurse P. 1991; PubMed Scopus Google Scholar). FK506 was by the pombe used in this T. C. PubMed Scopus Google study in a are by the gene by two and the wild-type gene used for gene are by an of the gene by are by and only the is using the for and the S. pombe and cnb1+ gene was by with the of S. pombe as a The used for was and the was The the cnb1+ gene was with and and the was expression of we used the nmt1 promoter G. E. K. PubMed Scopus Google Scholar). was by the of to and was by and the cells in Genes or the or to the gene at various expression of the gene was using whereas the attenuated of the nmt1 promoter G. E. K. PubMed Scopus Google Scholar). the and cnb1+ genes the gene was the and the cnb1+ gene was in the the or genes of the the genes with the nmt1 promoter at various the the and the at the gene of T. C. PubMed Scopus Google Scholar). of the gene by PubMed Scopus Google Scholar) was The was as The cloned of the cnb1+ gene in the was with and and the the cnb1+ gene was the of an the gene was the of the the of the The the cnb1+ gene was on and of the gene was by not Cell and the of the expression of the from of cells at to in of and a of A and and the cells using a at for which the on for and which the and cellular by at for to with or to fission yeast protein was by with the protein and was used for the of as a cells with for at in to the expression of in and using as was with an as previously Y. Sugiura R. T. S. Lu Y. K. Shuntoh H. Kuno T. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). The three with and the of from the cells Ppb1 and Ppb1 or Ppb1ΔC alone with of calmodulin and and the was for at The was by three with and and by in an for the by to in or and to various as in the with with or to the or and cells in in for D. PubMed Scopus Google Scholar), and was to of the cell at the excess was with the an with a with the of the S. pombe using the of Cnb1 the regulatory subunit of calcineurin of S. cerevisiae T. Tanaka H. H. K. Miyakawa T. Tanaka C. 1991; PubMed Scopus Google Scholar, M.S. Thorner J. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar), the S. pombe protein at the revealed an which for the and exhibits a to the budding yeast Cnb1 we the gene shown in the cnb1+ gene encodes a protein of that contains The cnb1 Knock-out to of the Knock-out of the the cnb1+ gene we the cells for phenotypes by the knock-out cells of the catalytic subunit, Ppb1, such as to the ion and cell (7.Yoshida T. Toda T. Yanagida M. J. Cell Sci. 1994; 107: 1725-1735Crossref PubMed Google Scholar, R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar). The growth of the or Δppb1 cells was by the of MgCl2 in the cells and Δppb1 cells in the of and failed to in the of MgCl2 on the the sensitivity of the cells was to that of the Δppb1 the sensitivity of the knock-out cells was also to their single knock-out cells These results that cnb1+ gene is required for the calcineurin activity in vivo. also revealed that the and cells showed identical cell with and cells as shown in the MgCl2 sensitivity of the cells was suppressed by expression of the gene encoding a MAP kinase phosphatase, which and also suppressed by expression of the form of an kinase of to the as that of the Δppb1 cells (10.Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar). These results also the that Cnb1 is required for the calcineurin activity in and that calcineurin acts antagonistically with the Pmk1 MAP kinase in ion homeostasis (10.Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar, 11.Sugiura R. Toda T. Dhut S. Shuntoh H. Kuno T. Nature. 1999; 399: 479-483Crossref PubMed Scopus (72) Google Scholar, 12.Sugiura R. Kita A. Shimizu Y. Shuntoh H. Sio S.O. Kuno T. Nature. 2003; 424: 961-965Crossref PubMed Scopus (61) Google Scholar). Cnb1 Δppb1 cells the protein and the cells the protein in suggesting that these proteins are In the with but not with a these two subunits. of the of Calcineurin or to the of the cnb1 the of the cnb1 knock-out we overexpression of the catalytic subunit Ppb1 or its has effect on the cnb1 knock-out phenotypes in vivo. We the expression of Ppb1 and Cnb1 by a of expression with the nmt1 or the transcription is in the of K. PubMed Scopus Google Scholar). contains the wild-type nmt1 promoter with a very the and in their promoter in an and a attenuated promoter a gene is the expression in the of is to higher that of the gene in the of G. E. K. PubMed Scopus Google Scholar). of the Ppb1 or Cnb1 proteins by A and shown in Δppb1 cells that the the gene on an with MgCl2 in the of promoter the cells that the the gene in the of MgCl2 in the of promoter and failed to on the growth defect in the of MgCl2 was identical with of the cells that the amount of the catalytic Ppb1, has physiological its regulatory is that the of the catalytic subunit has an to the phosphatase the C-terminal-deleted calcineurin is used as a constitutively active (10.Sugiura R. Toda T. Shuntoh H. Yanagida M. Kuno T. EMBO J. 1998; 17: 140-148Crossref PubMed Scopus (111) Google Scholar, 15.Lu Y. Sugiura R. Yada T. Cheng H. Sio S.O. Shuntoh H. Kuno T. Genes Cells. 2002; 7: 1009-1019Crossref PubMed Scopus (30) Google Scholar, S. Sugiura R. Lu Y. Maeda T. Kawagishi K. Yokoyama M. Tohda H. Giga-Hama Y. Shuntoh H. Kuno T. J. Biol. Chem. 2003; 278: 18078-18084Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, M. Sugiura R. Lu Y. L. Y. Shuntoh H. Kuno T. 2002; PubMed Google Scholar). We the C-terminal-deleted Ppb1ΔC suppress the MgCl2 sensitivity of the shown in the cells Ppb1ΔC by the in the of and failed to on the The growth defect in the of MgCl2 was identical with of the cells or that Ppb1ΔC has physiological activity in the of the regulatory subunit in vivo. overexpression of Ppb1 or Ppb1ΔC failed to the cnb1 the of the calcineurin (18.Hirayama S. Sugiura R. Lu Y. Maeda T. Kawagishi K. Yokoyama M. Tohda H. Giga-Hama Y. Shuntoh H. Kuno T. J. Biol. Chem. 2003; 278: 18078-18084Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) was when it was with Ppb1 and Cnb1 in the calmodulin and but this was not when it was with Ppb1 or Ppb1ΔC alone in the Co-overexpression of Ppb1 and Cnb1 the of we the effect of co-overexpression of Ppb1 and Cnb1 on the FK506-induced sensitivity of wild-type shown in the wild-type cells the gene the of the nmt1 promoter not on an FK506 and Cnb1 was co-expressed with Ppb1 using the or expression the cells to in the of FK506 and MgCl2 in a Cnb1 The of the co-overexpression of Ppb1 and Cnb1 to suppress the FK506-induced sensitivity suggested that the complex the calcineurin the suppression was only cells failed to in the of FK506 and MgCl2 when Ppb1 and Cnb1 using the the growth of cells was not by the of FK506 in the medium. These results suggest that an excess amount of the Ppb1-Cnb1 complex cannot compete out the FKBP12-FK506 complex. of Calcineurin and Cell the it is that the effect of calcineurin was when the catalytic and the regulatory subunits at the this the wild-type cells with the or genes the of the nmt1 promoter with or in the of and the to the to gene that was only a the of the Ppb1 and Ppb1ΔC protein in the cells Co-overexpression of Cnb1 and Ppb1ΔC caused a distinct growth defect as shown in A and C. FK506 the growth defect when Cnb1 was using an attenuated nmt1 promoter but FK506 failed to the growth defect when Cnb1 was using the wild-type nmt1 promoter These results suggest that an excess amount of the complex compete out the FKBP12-FK506 complex. the that an excess amount of the complex cannot compete out the FKBP12-FK506 complex and that an excess amount of the complex compete out the FKBP12-FK506 complex suggest that the FKBP12-FK506 complex to calcineurin in its active co-overexpression of Cnb1 and Ppb1 caused growth suggesting that the activity of the is by calmodulin and the Ca2+ ion in its in physiological shown in the cells showed such as or cells Although the was the only that was by in wild-type cells not the from the cell was in the cells In addition, the cells showed of and an that contains The caused by co-overexpression of Ppb1ΔC and Cnb1 was suppressed by the of FK506 to the suggesting that of calcineurin also the the results of study have suggested that calcineurin activates at least two distinct signaling branches, of which is Prz1-dependent (18.Hirayama S. Sugiura R. Lu Y. Maeda T. Kawagishi K. Yokoyama M. Tohda H. Giga-Hama Y. Shuntoh H. Kuno T. J. Biol. Chem. 2003; 278: 18078-18084Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). the growth caused by the of calcineurin is by the activation of the transcription factor (18.Hirayama S. Sugiura R. Lu Y. Maeda T. Kawagishi K. Yokoyama M. Tohda H. Giga-Hama Y. Shuntoh H. Kuno T. J. Biol. Chem. 2003; 278: 18078-18084Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), cells with the gene the of the nmt1 promoter with or in the of and the to the to gene Co-overexpression of Ppb1ΔC and Cnb1 in cells, as shown in not the growth that was in the wild-type cells, that it is by the activation of this defect was not suppressed by the FK506 these results suggest that of calcineurin caused the growth defect and the that the FKBP12-FK506 complex to calcineurin in its active In the a molecular genetic was to the in functions of the calcineurin subunits in fission yeast as a model we that the regulatory subunit is essential for the activation of calcineurin in and results suggest that the FKBP12-FK506 complex calcineurin in its active and the activity in vivo. Calcineurin, a protein phosphatase, is a heterodimer of the catalytic (calcineurin Ppb1 in S. and the regulatory (calcineurin Cnb1 in S. subunits. The catalytic subunit of calcineurin a high to the catalytic subunit of protein and and of these catalytic subunits are to have activity in the of their regulatory subunits Sci. Full Text PDF PubMed Scopus Google Scholar). S. J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) that a recombinant catalytic A subunit of in was by calmodulin and was shown to have a phosphatase activity to that of the calcineurin suggest that the catalytic subunit of the calcineurin high activity in the of its regulatory subunit. the studies using a mammalian catalytic subunit showed that the catalytic subunit alone has very and the regulatory subunit the activity by the (19.Perrino B.A. Ng L.Y. Brickey D.A. Saitoh Y. Ushio Y. Fukunaga K. Miyamoto E. Soderling T.R. J. Biol. Chem. 1992; 267: 15965-15969Abstract Full Text PDF PubMed Google Scholar, 20.Perrino B.A. Ng L.Y. Soderling T.R. J. Biol. Chem. 1995; 270: 340-346Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). it is to the of the catalytic subunit has activity in the of the regulatory subunit in vivo. we for the that the regulatory subunit is required for the activation of calcineurin in by that the overexpression of Ppb1 that of the constitutively active Ppb1ΔC, suppress the phenotypes of the cnb1 the or of Ppb1 was not by the or of Cnb1 not results suggest that the regulatory subunit is required for the to the active of Ppb1ΔC in the phenotypes of the cnb1 knock-out that the of the PubMed Scopus Google Scholar) is not to the and to its physiological as well as have using the C-terminal-deleted catalytic subunit of calcineurin in the as a constitutively active form to the with the activation of calcineurin in various and cells, and this has very well S.J. J. Nature. 1992; PubMed Scopus Google Scholar). results that these are and by the and amount of the regulatory subunit, the of the calcineurin catalytic subunit only when it makes a complex with the regulatory subunit. the catalytic subunit the wild-type catalytic subunit, and of the regulatory subunit form a constitutively active complex with the catalytic subunit. complex for the effect with overexpression of the constitutively active In the the wild-type cells with FK506 showed MgCl2 sensitivity, whereas the cells not by the of FK506 in the medium. These results that the complex and calcineurin that is required for the FK506-induced results also suggest that Ppb1 Cnb1 alone the FKBP12-FK506 complex and that only the Ppb1-Cnb1 complex the complex. wild-type cells Ppb1 and Cnb1 failed to in the of FK506 and whereas cells in the that the amount of Ppb1 and Cnb1 higher that of the excess amount of Ppb1 and Cnb1 cannot compete out the suggesting that the FKBP12-FK506 complex and the active complex in vivo. knock-out of the transcription factor but not FK506 suppressed the growth defect caused by co-overexpression of Ppb1ΔC and Cnb1 using and a wild-type nmt1 promoter. These results suggest that an excess amount of the complex compete out the FKBP12-FK506 complex. A study by Crabtree G.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) showed that the regulatory subunit but not calmodulin is required for the of calcineurin to the and calcineurin activity has not with its to the complex. in in vitro study using a we showed that the FKBP12-FK506 complex the complex but not to the complex M. Kuno T. H. Tanaka C. PubMed Scopus Google Scholar), suggesting the calcineurin activity and its to the FKBP12-FK506 complex. In addition, we showed that the complex the FKBP12-FK506 its was that of the complex M. Kuno T. H. Tanaka C. PubMed Scopus Google Scholar). these results suggest that the in pharmacological target of the FKBP12-FK506 complex is the active complex. Although the of a complex was by studies A. Nature. 1995; PubMed Scopus Google Scholar), studies suggest that the of the complex to to the molecular mechanisms of We Yanagida and Nurse for their of and