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.     

The plant nucleoplasmin AtFKBP43 needs its extended arms for histone interaction

The nucleoplasmin family of histone chaperones is a key player in governing the dynamic architecture of chromatin, thereby regulating various DNA-templated processes. The crystal structure of the N-terminal domain of Arabidopsis thaliana FKBP43 (AtFKBP43), an FK506-binding immunophilin protein, revealed a characteristic nucleoplasmin fold, thus confirming it to be a member of the FKBP nucleoplasmin class. Small-Angle X-ray Scattering (SAXS) analyses confirmed its pentameric nature in solution, and additional studies confirmed the nucleoplasmin fold to be highly stable. Unlike its homolog AtFKBP53, the AtFKBP43 nucleoplasmin core domain could not interact with histones and required the acidic arms, C-terminal to the core, for histone association. However, SAXS generated low-resolution envelope structure, ITC, and AUC results revealed that an AtFKBP43 pentamer with C-terminal extensions interacts with H2A/H2B dimer and H3/H4 tetramer in an equimolar ratio, like AtFKBP53. Put together, AtFKBP43 belongs to a hitherto unreported subclass of FKBP nucleoplasmins that requires the C-terminal acidic stretches emanating from the core domain for histone interaction.     

NMR assignments of the FKBP-type PPIase domain of FKBP42 from Arabidopsis thaliana

The Atfkbp42 gene is associated with reduced and disoriented growth of Arabidopsis thaliana. Resonance assignments are reported for the FKBP-type PPIase domain of AtFKBP42. Signal intensities reveal an additional structure element that is atypical for such FKBP domains.     

The immunophilin-interacting protein AtFIP37 from Arabidopsis is essential for plant development and is involved in trichome endoreduplication

The FKBP12 (FK506-binding protein 12 kD) immunophilin interacts with several protein partners in mammals and is a physiological regulator of the cell cycle. In Arabidopsis, only one specific partner of AtFKBP12, namely AtFIP37 (FKBP12 interacting protein 37 kD), has been identified but its function in plant development is not known. We present here the functional analysis of AtFIP37 in Arabidopsis. Knockout mutants of AtFIP37 show an embryo-lethal phenotype that is caused by a strong delay in endosperm development and embryo arrest. AtFIP37 promoter::beta-glucuronidase reporter gene constructs show that the gene is expressed during embryogenesis and throughout plant development, in undifferentiating cells such as meristem or embryonic cells as well as highly differentiating cells such as trichomes. A translational fusion with the enhanced yellow fluorescent protein indicates that AtFIP37 is a nuclear protein localized in multiple subnuclear foci that show a speckled distribution pattern. Overexpression of AtFIP37 in transgenic lines induces the formation of large trichome cells with up to six branches. These large trichomes have a DNA content up to 256C, implying that these cells have undergone extra rounds of endoreduplication. Altogether, these data show that AtFIP37 is critical for life in Arabidopsis and implies a role for AtFIP37 in the regulation of the cell cycle as shown for FKBP12 and TOR (target of rapamycin) in mammals.     

Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.     

Crystal structure of a plant immunophilin domain involved in regulation of MDR-type ABC transporters

We present the three-dimensional structure of the N-terminal FK506-binding protein (FKBP)-like domain of the immunophilin FKBP42 from Arabidopsis thaliana. The data provide the structural background for the explanation of key functional properties reported previously.     

Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-2

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.     

Wheat FKBP73 functions in vitro as a molecular chaperone independently of its peptidyl prolyl cis-trans isomerase activity

Peptidyl-prolyl cis-trans isomerases (PPIases) catalyse protein folding by accelerating the slow step of cis-trans isomerisation of peptidyl-prolyl bonds. Wheat (Triticum aestivum L.) FKBP73 (wFKBP73) is a peptidyl-prolyl cis-trans isomerase belonging to the FK506-binding protein (FKBP) family. It comprises three FKBP12-like domains, tetratricopeptide repeats and a calmodulin-binding domain (CaMbd). In vitro studies indicated that wFKBP73 possesses PPIase activity, binds calmodulin and forms a heterocomplex with mammalian p23 and wheat Hsp90 in wheat-germ lysate. To further study the role of wFKBP73 we have analysed its chaperone properties. Using the thermal unfolding and aggregation of citrate synthase (CS) as a model system, we have shown that the plant wFKBP73 exhibits chaperone activity, being able to suppress CS aggregation independently of its PPIase activity. The wFKBP73 interacts transiently with non-native CS and slows down its inactivation kinetics, whereas the mammalian homologue, hFKBP52 binds tightly to CS and does not affect its rate of inactivation. Hence, the first plant FKBP shown to function as a molecular chaperone has a mode of action different from that of the mammalian FKBP52.     

A novel role for the immunophilin FKBP52 in copper transport

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in (64)Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.     

Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor.hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.     

Association of immunophilins with mammalian TRPC channels

Drosophila photoreceptor channels TRP and TRPL are held in a large signalplex by the scaffolding protein, INAD. Immunophilin FKBP59, another member of the signalplex, binds to both INAD and TRPL. Mutation P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709), eliminates TRPL interaction with FKBP59. The first leucylprolyl (LP) dipeptide in this region is conserved in mammalian TRPC channel proteins. However, the second LP is changed to isoleucylprolyl (IP) in TRPC1, -C4, and -C5, and valylprolyl (VP) in TRPC3, -C6, and -C7. The purpose of the present study was to determine if mammalian FKBP12 or FKBP52 interact with TRPC channel proteins. Using TRPC-specific antibodies, immunoprecipitations from Sf9 cells individually co-expressing each of the TRPC proteins along with the immunophilins showed that TRPC3, -C6, and -C7 interact with FKBP12, whereas TRPC1, -C4, and -C5 interact with FKBP52. The binding of FKBP12 and FKBP52 was specific and could be displaced by the immunosuppressant drug FK506, at concentrations of 0.5 and 10 microm, respectively. To evaluate TRPC-immunophilin interactions in vivo, immunoprecipitations were performed using membrane lysates of rat cerebral cortex. FKBP12 co-immunoprecipitated with TRPC3, -C6, and -C7 from rat brain, whereas FKBP52 was found to associate with TRPC1, -C4, and -C5. The association of immunophilins with the TRPC channels in rat brain lysates could be displaced by FK506. Receptor-mediated activation of TRPC6, stably expressed in HEK cells, was significantly inhibited by FK506, which also disrupted interaction between TRPC6 and the endogenous immunophilin found in HEK cells. Pro to Gln mutations in the first LP dipeptide in the putative FKBP binding domain eliminated FKBP12 and FKBP52 interaction with TRPC3 and -C6, and TRPC1 and -C4, respectively. However, mutual swap of VP and IP in TRPC3 and TRPC5 did not alter the association or the selectivity of the channels for their respective immunophilin binding partner. These results suggest that immunophilins are TRPC channel accessory proteins that play an important role in the mechanism of channel activation following receptor stimulation.     

Molecular cloning and embryonic expression of dFKBP59, a novel Drosophila FK506-binding protein

A Drosophila cDNA encoding a structural homolog of mammalian FKBP59 (also identified as FKBP52), a member of the FK506-binding protein (FKBP) class of immunophilins, was isolated. The gene dFKBP59 corresponding to this cDNA has been characterized and mapped to the 30D3-4 region. The predicted amino acid sequence of this cDNA shows that the dFKBP59 protein contains one highly conserved FKBP12-like domain followed by two others with less conservation. Northern hybridization reveals that the dFKBP59 mRNA is expressed throughout the Drosophila life-cycle. In contrast to its mammalian homologs, in situ hybridization detected dFKBP59 expression in specific tissues: the lymph glands, Garland cells and oenocyte cells, which are all specialized tissues in which intensive exocytic/endocytic cycling takes place. Garland cells and oenocytes (also called Drosophila nephrocytes) function in taking up waste material from the hemolymph. Finally, I have mapped an enhancer trap element within the 5' region of dFKBP59 which may help in future studies to address the question of its function during Drosophila development.     

Novel structure of a high molecular weight FK506 binding protein fromArabidopsis thaliana

We have isolated clones of an Arabidopsis gene (ROF1, forrotamaseFKBP) encoding a high molecular weight member of the FK506 binding protein (FKBP) family. The deduced amino acid sequence of ROF1 predicts a 551-amino acid, 62 kDa polypeptide which is 44% identical to human FKBP59 — a 59 kDa FKBP which binds to the 90 kDa heat shock protein and is associated with inactive steroid hormone receptors. ROF1 contains three FKBP12-like domains in the N-terminal portion of the protein (in contrast to two domains in mammalian FKBP59), an internal repeat structure associated with protein-protein interactions (tetratricopeptide repeats), and a putative calmodulin binding domain near the C-terminal region of the protein. No sequences associated with protein translocation out of the cytosol were found in ROF1.ROF1 mRNA was found at equivalent low levels in light-grown roots, stems, and flowers and at slightly higher levels in leaves. The abundance ofROF1 mRNA increased several-fold under stress conditions such as wounding or exposure to elevated NaCl levels.     

Novel structure of a high molecular weight FK506 binding protein fromArabidopsis thaliana

We have isolated clones of an Arabidopsis gene (ROF1, forrotamaseFKBP) encoding a high molecular weight member of the FK506 binding protein (FKBP) family. The deduced amino acid sequence of ROF1 predicts a 551-amino acid, 62 kDa polypeptide which is 44% identical to human FKBP59 — a 59 kDa FKBP which binds to the 90 kDa heat shock protein and is associated with inactive steroid hormone receptors. ROF1 contains three FKBP12-like domains in the N-terminal portion of the protein (in contrast to two domains in mammalian FKBP59), an internal repeat structure associated with protein-protein interactions (tetratricopeptide repeats), and a putative calmodulin binding domain near the C-terminal region of the protein. No sequences associated with protein translocation out of the cytosol were found in ROF1.ROF1 mRNA was found at equivalent low levels in light-grown roots, stems, and flowers and at slightly higher levels in leaves. The abundance ofROF1 mRNA increased several-fold under stress conditions such as wounding or exposure to elevated NaCl levels.The nucleotide sequences in this paper have been submitted to the GenBank/EMBL Data bank with accession numbers U49453 and U57838     

Noncatalytic role of the FKBP52 peptidyl-prolyl isomerase domain in the regulation of steroid hormone signaling

Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity.     

Binding of hsp90-associated immunophilins to cytoplasmic dynein: direct binding and in vivo evidence that the peptidylprolyl isomerase domain is a dynein interaction domain

FKBP52 is a steroid receptor-associated immunophilin that binds via a tetratricopeptide repeat (TPR) domain to hsp90. FKBP52 has also been shown to interact either directly or indirectly via its peptidylprolyl isomerase (PPIase) domain with cytoplasmic dynein, a motor protein involved in retrograde transport of vesicles toward the nucleus. The functional role for the PPIase domain in receptor movement was demonstrated by showing that expression of the PPIase domain fragment of FKBP52 in 3T3 cells inhibits dexamethasone-dependent nuclear translocation of a green fluorescent protein-glucocorticoid receptor chimera. Here, we show that cytoplasmic dynein is co-immunoadsorbed with two other TPR domain proteins that bind hsp90 (the cyclophilin CyP-40 and the protein phosphatase PP5). Both proteins possess PPIase homology domains, and co-immunoadsorption of cytoplasmic dynein with each is blocked by the PPIase domain fragment of FKBP52. Using purified proteins, we show that FKBP52, PP5, and the PPIase domain fragment bind directly to the intermediate chain of cytoplasmic dynein. PP5 colocalizes with both cytoplasmic dynein and microtubules, and expression of the PPIase domain fragment of FKBP52 in 3T3 cells disrupts its cytoskeletal localization. We conclude that the PPIase domains of the hsp90-binding immunophilins interact directly with cytoplasmic dynein and that this interaction with the motor protein is responsible for the microtubular localization of PP5 in vivo.