The flexible loop of Bcl-2 is required for molecular interaction with immunosuppressant FK-506 binding protein 38 (FKBP38)

Bcl-2 contains an unusually long loop between the first and the second helices. This loop has been shown to be highly flexible based on NMR and X-ray crystallographic analyses of this region. Bcl-2 is regulated at the posttranslational level through phosphorylation of specific residues within the flexible loop. The biological role and posttranslational modifications of the loop of Bcl-2 is currently unclear. FK-506 binding protein 38 (FKBP38) has been reported to interact with Bcl-2, suggesting that FKBP38 could act as a docking molecule to localize Bcl-2 at the mitochondrial membrane [Shirane, M. and Nakayama, K.I. (2003) Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis. Nat. Cell Biol. 5, 28-37]. Here, we investigated the molecular interaction between FKBP38 and Bcl-2, and demonstrated that Bcl-2 interacts with FKBP38 through the unstructured loop, and the interaction appears to regulate phosphorylation in the loop of Bcl-2.     

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

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.     

Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis

The mitochondrial localization of the membrane proteins Bcl-2 and Bcl-x(L) is essential for their anti-apoptotic function. Here we show that mitochondrial FK506-binding protein 38 (FKBP38), unlike FKBP12, binds to and inhibits calcineurin in the absence of the immunosuppressant FK506, suggesting that FKBP38 is an inherent inhibitor of this phosphatase. FKBP38 is associated with Bcl-2 and Bcl-x(L) in immunoprecipitation assays and colocalizes with these proteins in mitochondria; in addition, the expression of FKBP38 mutant proteins induces a marked redistribution of Bcl-2 and Bcl-x(L). Overexpression of FKBP38 blocks apoptosis, whereas functional inhibition of this protein by a dominant-negative mutant or by RNA interference promotes apoptosis. Thus, FKBP38 might function to inhibit apoptosis by anchoring Bcl-2 and Bcl-x(L) to mitochondria.     

A novel calmodulin-Ca2+ target recognition activates the Bcl-2 regulator FKBP38

The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290-Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.     

Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A.

The immunosuppressive complexes cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 inhibit calcineurin, a heterodimeric Ca(2+)-calmodulin-dependent protein phosphatase that regulates signal transduction. We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK506-sensitive Saccharomyces cerevisiae strain. Three mutations that confer dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1. One mutation that confers dominant FK506 resistance alters a single residue (W430C) in the calcineurin A catalytic subunit CMP2. In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have reduced affinity for cyclophilin A-CsA. When introduced into the CMP1 subunit, the FK506 resistance mutation (W388C) blocks binding by FKBP12-FK506, but not by cyclophilin A-CsA. Co-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and to FK506 but not to CsA plus FK506. Double mutant calcineurin A subunits (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506. These studies identify cyclophilin A-CsA and FKBP12-FK506 binding targets as distinct, highly conserved regions of calcineurin A that overlap the binding domain for the calcineurin B regulatory subunit.     

Hepatitis C virus non-structural protein NS5A interacts with FKBP38 and inhibits apoptosis in Huh7 hepatoma cells

Hepatitis C virus non-structural protein NS5A plays an important role in viral replication and various cellular events. To gain further insight into the function of NS5A, we screened a human fetal liver cDNA library for its interacting proteins using the yeast two-hybrid system. FKBP38, a 38 kDa immunosuppressant FK506-binding protein, was identified and its interaction with NS5A was confirmed by both in vitro and in vivo. The interaction was mapped to the amino acids 148-236 of NS5A containing a BH domain (Bcl-2 homology domain). Besides, both NS5A and FKBP38 were found to localize in mitochondria and endoplasmic reticulum. Moreover, NS5A stably expressing Huh7 hepatoma cells showed more resistance to apoptosis and such inhibition of apoptosis could specifically be abrogated by depletion of FKBP38 using RNA interference. These results indicate that HCV NS5A inhibits apoptosis through interaction with FKBP38.     

A reassessment of the inhibitory capacity of human FKBP38 on calcineurin

The microbial peptidomacrolide FK506 affects many eukaryotic developmental and cell signaling programs via calcineurin inhibition. Prior formation of a complex between FK506 and intracellular FK506-binding proteins (FKBPs) is the precondition for the interaction with calcineurin. A puzzling difference has emerged between the mammalian multidomain protein hFKBP38 and other FKBPs. It was shown that hFKBP38 not only binds to calcineurin but also inhibits the protein phosphatase activity of calcineurin on its own [Shirane, M. and Nakayama, K.I. (2003) Nature Cell Biol. 5, 28-37]. Inherent calcineurin inhibition by hFKBP38 would completely eliminate the need for FK506 in controlling many signal transduction pathways. To address this issue, we have characterized the functional and physical interactions between calcineurin and hFKBP38. A recombinant hFKBP38 variant and endogenous hFKBP38 were tested both in vitro and in vivo. The proteins neither directly inhibited calcineurin activity nor affected NFAT reporter gene activity in SH-SY5Y and Jurkat cells. In addition, a direct physical interaction between calcineurin and hFKBP38 was not detected in co-immunoprecipitation experiments. However, hFKBP38 indirectly affected the subcellular distribution of calcineurin by interaction with typical calcineurin ligands, as exemplified by the anti-apoptotic protein Bcl-2. Our data suggest that hFKBP38 cannot substitute for the FKBP/FK506 complex in signaling pathways controlled by the protein phosphatase activity of calcineurin.     

Rheb GTPase Controls Apoptosis by Regulating Interaction of FKBP38 with Bcl-2 and Bcl-XL

FKBP38 is a member of the family of FK506-binding proteins that acts as an inhibitor of the mammalian target of rapamycin (mTOR). The inhibitory action of FKBP38 is antagonized by Rheb, an oncogenic small GTPase, which interacts with FKBP38 and prevents its association with mTOR. In addition to the role in mTOR regulation, FKBP38 is also involved in binding and recruiting Bcl-2 and Bcl-X_L, two anti-apoptotic proteins, to mitochondria. In this study, we investigated the possibility that Rheb controls apoptosis by regulating the interaction of FKBP38 with Bcl-2 and Bcl-X_L. We demonstrate in vitro that the interaction of FKBP38 with Bcl-2 is regulated by Rheb in a GTP-dependent manner. In cultured cells, the interaction is controlled by Rheb in response to changes in amino acid and growth factor conditions. Importantly, we found that the Rheb-dependent release of Bcl-X_L from FKBP38 facilitates the association of this anti-apoptotic protein with the pro-apoptotic protein Bak. Consequently, when Rheb activity increases, cells become more resistant to apoptotic inducers. Our findings reveal a novel mechanism through which growth factors and amino acids control apoptosis.     

FKBP38 Protects Bcl-2 from Caspase-dependent Degradation

The cellular processes that regulate Bcl-2 at the posttranslational levels are as important as those that regulate bcl-2 synthesis. Previously we demonstrated that the suppression of FK506-binding protein 38 (FKBP38) contributes to the instability of Bcl-2 or leaves Bcl-2 unprotected from degradation in an unknown mechanism. Here, we studied the underlying molecular mechanism mediating this process. We first showed that Bcl-2 binding-defective mutants of FKBP38 fail to accumulate Bcl-2 protein. We demonstrated that the FKBP38-mediated Bcl-2 stability is specific as the levels of other anti-apoptotic proteins such as Bcl-X^L and Mcl-1 remained unaffected. FKBP38 enhanced the Bcl-2 stability under the blockade of de novo protein synthesis, indicating it is posttranslational. We showed that the overexpression of FKBP38 attenuates reduction rate of Bcl-2, thus resulting in an increment of the intracellular Bcl-2 level, contributing to the resistance of apoptotic cell death induced by the treatment of kinetin riboside, an anticancer drug. Caspase inhibitors markedly induced the accumulation of Bcl-2. In caspase-3-activated cells, the knockdown of endogenous FKBP38 by small interfering RNA resulted in Bcl-2 down-regulation as well, which was significantly recovered by the treatment with caspase inhibitors or overexpression of FKBP38. Finally we presented that the Bcl-2 cleavage by caspase-3 is blocked when Bcl-2 binds to FKBP38 through the flexible loop. Taken together, these results suggest that FKBP38 is a key player in regulating the function of Bcl-2 by antagonizing caspase-dependent degradation through the direct interaction with the flexible loop domain of Bcl-2, which contains the caspase cleavage site.     

FK-506 binding protein from Tolypocladium inflatum: resistance of FKBP/FK-506 complex against proteolysis.

A 12-kDa peptidyl-prolyl-cis/trans-isomerase was purified 225-fold to homogeneity from the cyclosporin producing fungus Tolypocladium inflatum. The enzyme is highly sensitive to the immunosuppressant FK-506 but not to cyclosporin and thus belongs to the class of FK-506 binding proteins (FKBP). Interestingly the FKBP/FK-506 complex is resistant against proteolytic digestion by the endoproteases GluC and LysC, in contrast to the free FKBP, which is readily cleaved by these proteases. This protection may play a role in the effects of FK-506 in the living cell.     

FKBP, the binding protein for the immunosuppressive drug, FK-506, is not an inhibitor of protein kinase C activity.

Recently, the amino acid sequence of a 12 Kd endogenous protein inhibitor of protein kinase C (PKC-I 2) has been shown to be identical to that of the 12 KDa receptor for the immunosuppressive drug, FK-506. In view of this observation we examined the effects of recombinant and native human FKBP on protein kinase C (PKC) activity. FKBP, at molar concentrations up to 1900-fold over that of PKC, failed to inhibit PKC phosphorylation of histone H1 and failed to block the auto-phosphorylation of PKC. Interestingly, FKBP is phosphorylated by PKC in these reactions. The phosphorylation of FKBP by PKC appears to be specific since the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate the binding protein. Our results fail to support a role for FKBP as an inhibitor of protein kinase C.     

FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein which is distinct from the cyclosporin A-binding protein, cyclophilin

A novel macrolide antibiotic, FK-506, isolated from Streptomyces tsukubaensis, has been shown to be a potent immunosuppressive agent in vivo and in vitro. FK-506 shares a number of immunosuppressive properties with the cyclic peptide, cyclosporin A (CsA), although 10 to 100 times more potent in this regard. These similarities suggest that both agents may share a similar mechanism(s) of action at the biochemical level. We have identified a cytoplasmic binding protein for FK-506 in the human T cell line, JURKAT, using [3H]FK-506. The FK-506 binding protein has a mr of 10 to 12 kDa (as determined by gel filtration), is heat stable and does not bind CsA. This contrasts with the CsA binding protein, cyclophilin, in that cyclophilin is heat labile and has a mr of 15 to 17 kDa. Our data suggest that FK-506 binds to a low m.w. protein(s) in JURKAT cells, which is distinct from cyclophilin. This protein may mediate the immunosuppressive effects of FK-506 in T cells. In addition, our results suggest that the immunosuppressive activity of FK-506, as with CsA, is mediated by an intracellular mechanism.     

The FKBP38 catalytic domain binds to Bcl-2 via a charge-sensitive loop

FKBP38 is a regulator of the prosurvival protein Bcl-2, but in the absence of detailed structural insights, the molecular mechanism of the underlying interaction has remained unknown. Here, we report the contact regions between Bcl-2 and the catalytic domain of FKBP38 derived by heteronuclear NMR spectroscopy. The data reveal that a previously identified charge-sensitive loop near the putative active site of FKBP38 is mainly responsible for Bcl-2 binding. The corresponding binding epitope of Bcl-2 could be identified via a peptide library-based membrane assay. Site-directed mutagenesis of the key residues verified the contact sites of this electrostatic protein/protein interaction. The derived structure model of the complex between Bcl-2 and the FKBP38 catalytic domain features both electrostatic and hydrophobic intermolecular contacts and provides a rationale for the regulation of the FKBP38/Bcl-2 interaction by Ca(2+).     

The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase.

We have recently isolated an abundant cytosolic protein from human T-cells which specifically binds the immunosuppressive agent, FK-506. The FK-506-binding protein (FKBP) is a member of a novel class of proteins possessing peptidyl-prolyl cis-trans isomerase activity. These proteins are believed to play an important role in accelerating the rate at which proteins fold into their native conformations. In the present study, we demonstrate that FKBP is not a lymphoid-specific protein, but is widely distributed and phylogenically conserved. FKBP, purified from three sources (a human T-lymphocyte cell line JURKAT, bovine calf thymus, and Saccharomyces cerevisiae) exhibit identical molecular weights, immunological cross-reactivities, and a high degree of NH2-terminal amino acid sequence homology. In addition, FKBP from all sources possesses peptidyl-prolyl cis-trans isomerase activity which can be specifically inhibited by FK-506. We conclude that FKBP may serve an important biological function in all eukaryotic cells.     

Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes.

Although the immediate receptors (immunophilins) of the immunosuppressants cyclosporin A (CsA) and FK506 are distinct, their similar mechanisms of inhibition of cell signaling suggest that their associated immunophilin complexes interact with a common target. We report here that the complexes cyclophilin-CsA and FKBP-FK506 (but not cyclophilin, FKBP, FKBP-rapamycin, or FKBP-506BD) competitively bind to and inhibit the Ca(2+)- and calmodulin-dependent phosphatase calcineurin, although the binding and inhibition of calcineurin do not require calmodulin. These results suggest that calcineurin is involved in a common step associated with T cell receptor and IgE receptor signaling pathways and that cyclophilin and FKBP mediate the actions of CsA and FK506, respectively, by forming drug-dependent complexes with and altering the activity of calcineurin-calmodulin.     

Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands.

The peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin. With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506. Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex. By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions. Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes. Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo. These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin.     

Hsp90-mediated inhibition of FKBP38 regulates apoptosis in neuroblastoma cells

The FK506-binding protein 38 (FKBP38) is a pro-apoptotic regulator of Bcl-2 in neuroblastoma cells. Hsp90 inhibits the pro-apoptotic FKBP38/CaM/Ca(2+) complex and thus prevents interactions between FKBP38 and Bcl-2. Here we show that Hsp90 increases cell survival rates of neuroblastoma cells after apoptosis induction. Depletion of FKBP38 by short interference RNA significantly decreased the anti-apoptotic effect of Hsp90 expression. In addition, the influence of high cellular Hsp90 levels was only observed in post-stimulation apoptosis that is sensitive to selective FKBP38 active site inhibition. Similar anti-apoptotic effects in neuroblastoma cells were observed after stimulation of endogenous Hsp90 expression. Hence, the inhibition of FKBP38 by Hsp90 participates in programmed cell death control of neuroblastoma cells.