Ligand-switchable Substrates for a Ubiquitin-Proteasome System

Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.     

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.     

The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain

Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.     

A directed approach for engineering conditional protein stability using biologically silent small molecules

The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506- and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (DeltaDeltaG(U-F)) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.     

Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor.

FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2.     

A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules

Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.     

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.     

Ligands for FKBP12 Increase Ca2+ Influx and Protein Synthesis to Improve Skeletal Muscle Function

Rapamycin at high doses (2–10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 μg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca²⁺ influx to enhance refilling of sarcoplasmic reticulum Ca²⁺ stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.     

Mechanism of TGFbeta receptor inhibition by FKBP12.

Transforming growth factor-beta (TGFbeta) signaling requires phosphorylation of the type I receptor TbetaR-I by TbetaR-II. Although TGFbeta promotes the association of TbetaR-I with TbetaR-II, these receptor components have affinity for each other which can lead to their ligand-independent activation. The immunophilin FKBP12 binds to TbetaR-I and inhibits its signaling function. We investigated the mechanism and functional significance of this effect. FKBP12 binding to TbetaR-I involves the rapamycin/Leu-Pro binding pocket of FKBP12 and a Leu-Pro sequence located next to the activating phosphorylation sites in TbetaR-I. Mutations in the binding sites of FKBP12 or TbetaR-I abolish the interaction between these proteins, leading to receptor activation in the absence of added ligand. FKBP12 does not inhibit TbetaR-I association with TbetaR-II, but inhibits TbetaR-I phosphorylation by TbetaR-II. Rapamycin, which blocks FKBP12 binding to TbetaR-I, reverses the inhibitory effect of FKBP12 on TbetaR-I phosphorylation. By impeding the activation of TGFbeta receptor complexes formed in the absence of ligand, FKBP12 may provide a safeguard against leaky signaling resulting from the innate tendency of TbetaR-I and TbetaR-II to interact with each other.     

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.     

FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.     

Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo

The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes approximately 40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.     

Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor

The cardiac isoform of the ryanodine receptor (RyR2) from dog binds predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6), whereas RyR2 from other species binds both FKBP12.6 and the closely related isoform FKBP12. The role played by FKBP12.6 in modulating calcium release by RyR2 is unclear at present. We have used cryoelectron microscopy and three-dimensional (3D) reconstruction techniques to determine the binding position of FKBP12.6 on the surface of canine RyR2. Buffer conditions that should favor the "open" state of RyR2 were used. Quantitative comparison of 3D reconstructions of RyR2 in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds along the sides of the square-shaped cytoplasmic region of the receptor, adjacent to domain 9, which forms part of the four clamp (corner-forming) structures. The location of the FKBP12.6 binding site on "open" RyR2 appears similar, but slightly displaced (by 1-2 nm) from that found previously for FKBP12 binding to the skeletal muscle ryanodine receptor that was in the buffer that favors the "closed" state. The conformation of RyR2 containing bound FKBP12.6 differs considerably from that depleted of FKBP12.6, particularly in the transmembrane region and in the clamp structures. The x-ray structure of FKBP12.6 was docked into the region of the 3D reconstruction that is attributable to bound FKBP12.6, to show the relative orientations of amino acid residues (Gln-31, Asn-32, Phe-59) that have been implicated as being critical in interactions with RyR2. A thorough understanding of the structural basis of RyR2-FKBP12.6 interaction should aid in understanding the roles that have been proposed for FKBP12.6 in heart failure and in certain forms of sudden cardiac death.     

Energetic and structural analysis of the role of tryptophan 59 in FKBP12

Tryptophan 59 forms the seat of the hydrophobic ligand-binding site in the small immunophilin FKBP12. Mutating this residue to phenylalanine or leucine stabilizes the protein by 2.72 and 2.35 kcal mol(-1), respectively. Here we report the stability data and 1.7 A resolution crystal structures of both mutant proteins, complexed with the immunosuppressant rapamycin. Both structures show a relatively large response to mutation involving a helical bulge at the mutation site and the loss of a hydrogen bond that anchors a nearby loop. The increased stability of the mutants is probably due to a combination of improved packing and an entropic gain at the mutation site. The structures are almost identical to that of wild-type FKBP12.6, an isoform of FKBP12 that differs by 18 residues, including Trp59, in its sequence. Therefore, the structural difference between the two isoforms can be attributed almost entirely to the identity of residue 59. It is likely that in FKBP12-ligand complexes Trp59 provides added binding energy at the active site at the expense of protein stability, a characteristic common to other proteins. FKBP12 associates with the ryanodine receptor in skeletal muscle (RyR1), while FKBP12.6 selectively binds the ryanodine receptor in cardiac muscle (RyR2). The structural response to mutation suggests that residue 59 contributes to the specificity of binding between FKBP12 isoforms and ryanodine receptors.     

FKBP12 binding to RyR1 modulates excitation-contraction coupling in mouse skeletal myotubes

The skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel or ryanodine receptor (RyR1) binds four molecules of FKBP12, and the interaction of FKBP12 with RyR1 regulates both unitary and coupled gating of the channel. We have characterized the physiologic effects of previously identified mutations in RyR1 that disrupt FKBP12 binding (V2461G and V2461I) on excitation-contraction (EC) coupling and intracellular Ca2+ homeostasis following their expression in skeletal myotubes derived from RyR1-knockout (dyspedic) mice. Wild-type RyR1-, V246I-, and V2461G-expressing myotubes exhibited similar resting Ca2+ levels and maximal responses to caffeine (10 mm) and cyclopiazonic acid (30 microm). However, maximal voltage-gated Ca2+ release in V2461G-expressing myotubes was reduced by approximately 50% compared with that attributable to wild-type RyR1 (deltaF/Fmax = 1.6 +/- 0.2 and 3.1 +/- 0.4, respectively). Dyspedic myotubes expressing the V2461I mutant protein, that binds FKBP12.6 but not FKBP12, exhibited a comparable reduction in voltage-gated SR Ca2+ release (deltaF/Fmax = 1.0 +/- 0.1). However, voltage-gated Ca2+ release in V2461I-expressing myotubes was restored to a normal level (deltaF/Fmax = 2.9 +/- 0.6) following co-expression of FKBP12.6. None of the mutations that disrupted FKBP binding to RyR1 significantly affected RyR1-mediated enhancement of L-type Ca2+ channel activity (retrograde coupling). These data demonstrate that FKBP12 binding to RyR1 enhances the gain of skeletal muscle EC coupling.     

An Arabidopsis immunophilin, AtFKBP12, binds to AtFIP37 (FKBP interacting protein) in an interaction that is disrupted by FK506

AtFKBP12 is an Arabidopsis cDNA that encodes a protein similar to the mammalian immunophilin, FKBP12. AtFKBP12 was used as ‘bait’ in a yeast 2-hybrid system to screen for cDNAs in Arabidopsis encoding proteins that bind to FKBP12. Two partial cDNAs were recovered encoding the C-terminus of a protein we have called Arabidopsis thaliana FKBP12 interacting protein 37 (AtFIP37). AtFIP37 is similar to a mammalian protein, FAP48, that also binds to FKBP12. The interaction between AtFKBP12 and AtFIP37 in the 2-hybrid system, as assessed by histidine auxotrophy and β-galactosidase activity, was disrupted by FK506, but not by cyclosporin A, a drug that binds to cyclophilin A. AtFIP37 was also shown to bind in vitro to AtFKBP12 in GST-fusion protein binding assays. The binding was abolished by prior incubation of AtFKBP12 with FK506. These findings indicate that an Arabidopsis FKBP12 ortholog encodes a protein that binds FK506 and that the interaction between AtFKBP12 and AtFIP37 may involve the FK506 binding site of AtFKBP12. The interaction provides interesting new opportunities for controlling protein:protein interactions in vivo in plants.     

Re-evaluating the roles of proposed modulators of mammalian target of rapamycin complex 1 (mTORC1) signaling

Signaling through mammalian target of rapamycin complex 1 (mTORC1) is stimulated by amino acids and insulin. Insulin inactivates TSC1/2, the GTPase-activator complex for Rheb, and Rheb.GTP activates mTORC1. It is not clear how amino acids regulate mTORC1. FKBP38 (immunophilin FK506-binding protein, 38 kDa), was recently reported to exert a negative effect on mTORC1 function that is relieved by its binding to Rheb.GTP. We confirm that Rheb binds wild type FKBP38, but inactive Rheb mutants showed contrasting abilities to bind FKBP38. We were unable to observe any regulation of FKBP38/mTOR binding by amino acids or insulin. Furthermore, FKBP38 did not inhibit mTORC1 signaling. The translationally controlled tumor protein (TCTP) in Drosophila was recently reported to act as the guanine nucleotide-exchange factor for Rheb. We have studied the role of TCTP in mammalian TORC1 signaling and its control by amino acids. Reducing TCTP levels did not reproducibly affect mTORC1 signaling in amino acid-replete/insulin-stimulated cells. Moreover, overexpressing TCTP did not rescue mTORC1 signaling in amino acid-starved cells. In addition, we were unable to see any stable interaction between TCTP and Rheb or mTORC1. Accumulation of uncharged tRNA has been previously proposed to be involved in the inhibition of mTORC1 signaling during amino acid starvation. To test this hypothesis, we used a Chinese hamster ovary cell line containing a temperature-sensitive mutation in leucyl-tRNA synthetase. Leucine deprivation markedly inhibited mTORC1 signaling in these cells, but shifting the cells to the nonpermissive temperature for the synthetase did not. These data indicate that uncharged tRNA(Leu) does not switch off mTORC1 signaling and suggest that mTORC1 is controlled by a distinct pathway that senses the availability of amino acids. Our data also indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling.     

Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. Kube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. Wang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivastava, J. Virol. 72:1593-1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, M. C. Yoder, and A. Srivastava, J. Virol. 72:9835-9841, 1998). Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine- or threonine-phosphorylated FKBP52 caused approximately 40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.     

Tacrolimus (FK506) Prevents Early Stages of Ethanol Induced Hepatic Fibrosis by Targeting LARP6 Dependent Mechanism of Collagen Synthesis

Tacrolimus (FK506) is a widely used immunosuppressive drug. Its effects on hepatic fibrosis have been controversial and attributed to immunosuppression. We show that in vitro FK506, inhibited synthesis of type I collagen polypeptides, without affecting expression of collagen mRNAs. In vivo, administration of FK506 at a dose of 4 mg/kg completely prevented development of alcohol/carbon tetrachloride induced liver fibrosis in rats. Activation of hepatic stellate cells (HSCs) was absent in the FK506 treated livers and expression of collagen α2(I) mRNA was at normal levels. Collagen α1(I) mRNA was increased in the FK506 treated livers, but this mRNA was not translated into α1(I) polypeptide. No significant inflammation was associated with the fibrosis model used. FK506 binding protein 3 (FKBP3) is one of cellular proteins which binds FK506 with high affinity. We discovered that FKBP3 interacts with LARP6 and LARP6 is the major regulator of translation and stability of collagen mRNAs. In the presence of FK506 the interaction between FKBP3 and LARP6 is weakened and so is the pull down of collagen mRNAs with FKBP3. We postulate that FK506 inactivates FKBP3 and that lack of interaction of LARP6 and FKBP3 results in aberrant translation of collagen mRNAs and prevention of fibrosis. This is the first report of such activity of FK506 and may renew the interest in using this drug to alleviate hepatic fibrosis.