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Published as doi: 10.1096/fj.06-7667com.
 (The FASEB Journal. 2007;21:2787-2797.)
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The immunophilin FKBP52 specifically binds to tubulin and prevents microtubule formation

Béatrice Chambraud*, Hamida Belabes*, Virginie Fontaine-Lenoir{dagger}, Arlette Fellous{dagger} and Etienne Emile Baulieu*,{dagger},1

* INSERM, Unité mixte de recherche 788, Université ParisXI, Kremlin Bicêtre, France; and

{dagger} MAPREG Company, Centre Hospitalier Universitaire de Bicêtre, Bâtiment Paul Langevin, Le Kremlin Bicêtre Cedex, France

1Correspondence: INSERM, Unité mixte de recherche 788, Université ParisXI, 80 rue du Général leclerc, Kremlin Bicêtre 94276, France. E-mail: baulieu{at}kb.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The FK506 binding protein FKBP52 belongs to the large family of immunophilins and is known as a steroid receptor-associated protein. Previous data suggest that FKBP52 is associated with the motor protein dynein and with the cytoskeleton during mitosis. Here we demonstrate a specific and direct interaction between FKBP52 and tubulin. The region of FKBP52 located between aa 267 and 400, which includes the tetratricopeptide repeat domain, is required for tubulin binding. We provide evidence that FKBP52 prevents tubulin polymerization and that an 84 residue sequence located in the C-terminal part of the molecule (aa 375–458) is necessary and sufficient for its microtubule depolymerization activity. In colocalization experiments in PC12 cells, FKBP52 is associated with tubulin in motile cellular compartments. Furthermore, we suggest that, by using siRNA, a decrease of FKBP52 expression in PC12 cells may lead to differentiated cell phenotype characterized by neurite extensions. Collectively, our data define an unexpected property of FKBP52 as a novel regulator of microtubule dynamics. The possible role of microtubule formation and tubulin binding of other immunophilins such as FKBP12 and FKBP51 is discussed.—Chambraud, B., Belabes, H., Fontaine-Lenoir, V., Fellous, A., Baulieu, E. E. The immunophilin FKBP52 specifically binds to tubulin and prevents microtubule formation.


Key Words: cytoskeleton • neurite differentiation • microtubule dynamics • FK506 binding protein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
FKBPS (FK506 BINDING PROTEINS) ARE A FAMILY of ubiquitously expressed intracellular receptors for immunosuppressant drugs such as FK506 and rapamycin, and therefore they belong to the large group of proteins known as immunophilins (1) .

Because of their ability to mediate the effects of immunosuppressant drugs (2) , FKBPs have been intensively studied. Initially found in the immune system, these proteins are in fact widely expressed (3 4) . Immunophilin levels are much higher in the brain than in the immune system (5) , and they participate in a variety of central nervous system functions (6 7 8) .

Ranging in size from 12 to 135 kDa (9) , FKBPs share a common domain for immunosuppressant ligand binding, but they differ in their affinity for FK506, the structure of other domains, and their subcellular localization, suggesting that each FKBP could have a distinct function.

FKBP52 was discovered as a component of steroid receptor heterooligomeric complexes. Analysis of cloned FKBP52 (10 11 12) and the definition of FKBP52's 3-dimensional structure (13) revealed a modular organization for this protein, including four individual and functional domains (14) . The FK506 binding site of FKBP52 (domain I) localized in the N-terminal part of the protein (aa 1 to 149) has peptidyl prolyl-isomerase (PPIase) activity (15) characteristic of all of the immunophilin protein family. While the second domain (aa 149 to 267) shares structural homology with domain I, its PPIase activity is residual and it does not bind FK506 (14 , 15) ; a noteworthy aspect of domain II is a consensus ATP-GTP binding sequence (16) . Domain III (aa 273 to 389) includes three tetratricopeptide repeats (TPR) that mediate the interaction with HSP90, which is also a component of steroid receptor complexes (17 18 19) . Beyond the final TPR motif, a short helical domain called domain IV contains a putative binding site for calmodulin (20) .

In the course of the search for the mechanistic contribution of FKBP52 to steroid receptor complexes, a role for FKBP52 in the nuclear translocation of steroid hormone receptors was suggested (21 22) , and it was found that FKBP52 is associated with the cytoskeleton and localized with the mitotic apparatus (23 24) . In addition, an interaction between the FKBP52 involving the PPI-ase domain and the motor protein dynein via dynamitin has been reported (25 26) .

Besides its effect on steroid receptor activity (27) , it has been reported that FKBP52 in the nervous system could mediate the neurotrophic and neuroprotective effects of FK506 (28 29) . It has also been postulated that FKBP52 by itself could increase the microtubule and microfilament tracks into axonal tips (30) . In addition, it has been speculated that FKBP52 could be involved in a retrograde signaling pathway from the growth cone to the neuronal cell body (31) , a possibility supported by the observation that FKBP52 binds to dynein.

Microtubules are noncovalent polymers composed of {alpha}/ß tubulin heterodimers and are found in all dividing cells and differentiated cells types (32 33) . They have diverse roles in eukaryotic cells ranging from mitotic spindle formation to membrane trafficking and organelle organization. They are major components of the neuronal cytoskeleton and play an essential role in the formation of axons and dendrites (34) . Tubulin assembly into microtubules is a reversible process that is highly regulated and known as dynamic instability (33 , 35) . This property allows both polymerizing and depolymerizing microtubules to exist in the same population and interconversion between these two states.

Here we report a specific association between FKBP52 and tubulin. We demonstrate that FKBP52 possesses microtubule depolymerization activity and is involved in microtubule network regulation of the PC12 clonal rat pheochromocytoma cell line. Taken together, our results suggest a novel function for the FKBP52 protein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Animals
Male adult Sprague-Dawley rats (body weight, 300 g) were obtained from Janvier (Le Genest-St.-Isle, France). Animals were killed by decapitation, and their entire brains were used immediately to prepare microtubules or tubulin.

Antibody
Anti-{alpha}-tubulin mAB (clone DM1A) and anti-ß-tubulin mAB (clone D66) were purchased from Sigma (St. Quentin Fallavier, France). Anti-ß-actin (clone mAbcam 8226) was purchased from Abcam (Cambridge, UK). Two peptides located at the N-terminal part (amino acids 19–33) and at the C-terminal part (amino acids 444–458) of rat FKBP52 were used to raise antisera in rabbits (Eurogentec, Herstal, Belgium). The specificity of these sera was tested by their ability to recognize the cognate protein by Western blot of the recombinant protein and of the rat brain cytosol.

Preparation of microtubules and tubulin
Microtubules were prepared from the brains of 50 adults rats by a temperature-dependent in vitro assembly-disassembly procedure as already described (36) . This preparation of MTs will be further referred to as "mixed tubulin."

Purified tubulin (without MAPs), referred to as tubulin, was isolated from the brain of 50 adults rats by two assembly-disasembly cycles in high-molarity buffer as described by Castoldi and Popov (37) , except that after the last centrifugation the microtubule pellets were resuspended at 16 mg/ml in buffer L x 2 (0.2 M Mes, 2 mM EGTA, 1 mM MgCl2, 2 mM EDTA, pH 6.4) to which 40% anhydrous glycerol and 0.5 mM GTP were added. The tubulin was frozen in 200 µl aliquots in liquid nitrogen.

Microtubule assembly
The assembly rate of tubulin and mixed tubulin was measured using a light scattering assay. Tubulin or mixed tubulin was used at a concentration of 2.5 mg/ml or 1 mg/ml, respectively. Defined protein amounts and drugs (taxol; Cytoskeleton, Inc., Tebu-bio, France) were incubated in 300 µl with mixed tubulin in a buffer A (buffer L complemented with 1 mM DTT/1 mM PMSF/1 mM GTP) or with tubulin in buffer A complemented with 6 mM MgCl2 and 0.5 mM GTP. Microtubule assembly was monitored by the increase of absorbance at 345 nm vs. time, with an uvicon spectrophotometer (Kontron, Montigny-le-bretonneux, France) equipped with an automatic six-sample charger thermostated at 37°C (15 or 30 min). The change of OD between time 0 and time 15 or 30 min reflects a variation of the medium's turbidity due to tubulin polymerization.

Expression vectors
All FKBP and mutant expression vectors, except those mentioned below, have been described (15 , 17) . The cDNA encoding human FKBP51 (a gift from S. Giraudier, Inserm U362 Villejuif France) was inserted into the XhoI restriction site of the pGEX4T3 vector (Amersham Biosciences, Uppsala, Sweden) to give pGST-FKBP51. The pGEX1{lambda}T plasmid, which contains the C-terminal part (CT) of FKBP52 from aa 374 to 458, was deleted from the sequence located between SmaI and BalI to give the FKBP52 mutant expressed from aa 374 to aa 405 as a fusion protein with GST and termed CT1. The mutant CT2 was obtained by cleaving the CT part of FKBP52 by BamH1 at the site located at aa 400 and the EcoRI site located at the cDNA end, and inserted into BamH1 and EcoRI restriction sites of pGEX5X2.

Overexpression of proteins (FKBP52 and deletion mutants, FKBP51 and FKBP12)
Affinity purification of full-length FKBPs and truncated FKBP52 was achieved as described (15) . Briefly, for microtubule polymerization assay, fusion proteins bound to glutathione-Sepharose beads were cleaved overnight at 4°C with 6 U of thrombin (Sigma) and dialyzed against buffer L with 10% glycerol and complemented to 1 mM GTP and 1 mM DTT before use. For GST pull-down experiments, an aliquot of fusion proteins was cleaved from beads by glutathione; after dialysis against buffer TG (20 mM Tris, 10% glycerol, pH 7.4), proteins concentrations were measured by BC assay (Perbio, France).

GST pull-down assay
Glutathione-Sepharose (100 µl) beads preloaded with 1 nmol of GST-FKBPs or truncated FKBP52 mutants were washed four times with 500 µl of buffer A, then resuspended in the same buffer containing 5 nmol purified tubulin. After rotating tubes for 30 min at 4°C, the GSH beads were washed five times with 1 ml of buffer A and twice with buffer TG, then eluted by GSH in a 50 µl final volume. Alternatively, the protein binding assay was carried out with microtubules polymerized at 37°C for 30 min (microtubule assembly was monitored by the increase of absorbance at 37°C). Subsequently, these microtubules were incubated at 37°C with beads containing GST, GST-FKBP52, or GST-FKBP51 for 30 min and eluted by GSH. The eluted proteins were subsequently analyzed for the presence of purified FKBPs and of truncated FKBP52 proteins by Coomassie staining, and for the presence of copurifying tubulin by SDS/PAGE Western blot analysis, using an mAb directed against {alpha}-tubulin.

Coimmunoprecipitation assay
Rat brain cytosol protein (500 mg) was incubated overnight in 200 µl of immunoprecipitation buffer [50 mM Tris (pH 7.5)/150 mM NaCl/2 mM MgCl2/0.1% Brij97 (Sigma)/10% glycerol/protease inhibitors] with 10 µl of affinity-purified anti-FKBP52 antibody or 10 µl of preimmune serum at 4°C. Subsequently, 10 µl protein A-Sepharose (Sigma) was added and incubated for 2 h at room temperature. The beads were washed four times with buffer A and subjected to SDS-PAGE (7% TAE NuPAGE [Novex]); proteins were detected with the indicated antibodies and visualized by enhanced chemiluminescence.

siRNA preparations
siRNA corresponding to rat FKBP52 were synthesized by Dharmacon (Perbio, France). They were designed as recommended by Elbaschir et al. (38) : siRNA1 was located in the first domain of rat FKBP52: 5'-AUUCUCCUUUGACCUGGGAdTdT-3' (sense) and 5'-UCCCAGGUCAAAGGAGAAUdTdT-3' (antisense); siRNA2 was located in the second domain of FKBP52:5'-AUGAUGGCGCUAUGGUAGAdTdT-3' (sense) and 5'-UCUACCAUAGCGCCAUCAUdTdT-3' (antisense); scrambled siRNA: 5'-AUCUUAGGCUGCUUACGCUdTdT-3' (sense) and 5'-AGCGUAAGCAGCCUAAGAUdTdT-3' (antisense). Annealing for duplex was performed according to the manufacturer's instructions. siRNA transfections were performed using lipofectamineTM (Invitrogen, Cergy Pontoise, France) in 6-well plates coated with 10 µg/ml poly(L)-lysine using 120 pmol of 21 bp duplex or sense strand oligonucleotides. The proteins were extracted at 24–72 h post-transfection and analyzed for the presence of FKBP52 by Western blot using the serum anti-FKBP52 (1/1000) and anti-ß-actin (1/1000). For immunocytochemistry, transfections were performed in 12-well plates using 60 pmol of siRNA.

Cell culture
PC12 cells were grown in DMEM containing 10% (v/v) horse serum and 5%(v/v) FBS (Invitrogen) at 37°c in 10% CO2. The differentiated neuronal phenotype of PC12 cells, grown on plastic dishes coated with poly(L)-lysine, was induced by adding 50 nM NGF (Invitrogen) for 5 days.

Immunocytochemistry
PC12 cells grown on glass coverslips precoated with 10 µg/ml poly(L)-lysine in 12-well tissue culture plates were fixed with methanol at –20°C for 6 min. After three washes in PBS, fixed cells were processed for immunocytochemical staining and incubated overnight with the affinity-purified primary anti-FKBP52 ("761," 1:1000) and anti-{alpha}-tubulin (1:2000). After three washes, cells were incubated for 1 h at room temperature with secondary antibodies [Alexa fluor 488 green-conjugated goat anti-rabbit IgG, 1/500 (Invitrogen); CyTM3 red-conjugated goat anti-mouse IgG, 1:2000 (GE Healthcare)]. After three washes, cells were mounted with glycergel mounting medium (DAKO, France) and examined using a Zeiss axioplan 2 microscope or confocal microscope (Zeiss, Thornwood, NY, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
FKBP52 binds directly tubulin
The fact that the immunophlin FKBP52 has been found to be associated with the cytoskeleton led us to undertake a study of this association. First, attention was focused on tubulin because it is the major component of the neuronal cytoskeleton, and protein binding assays were performed to investigate the possibility that FKBP52 interacts directly with tubulin. FKBP52, expressed and purified as fusion protein with GST, was immobilized on glutathione beads, then incubated with tubulin. We used either a tubulin preparation containing MAPs (microtubule-associated proteins) that copurifies with microtubule (MT), henceforth termed "mixed tubulin," or purified tubulin free of MAPs "tubulin." The experiments were carried out with depolymerized tubulin or mixed tubulin (4°C) and with polymerized mixed tubulin (37°C). The ability of GST-FKBP52 to interact directly with tubulin was revealed by immunoblotting using an anti-tubulin antibody. Tubulin or mixed tubulin, whether polymerized or not, was retained exclusively on GST-FKBP52 beads. The GST beads alone were unable to retain tubulin, confirming the specificity of this interaction (Fig. 1 A).


Figure 1
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  		Figure 1. Direct association between FKBP52 and tubulin. A) Immunoblot for tubulin showing the binding assay of soluble mixed tubulin, polymerized (37°C) or not (4°C), or of pure unpolymerized tubulin (4°C) incubated with GST-tagged FKBP52 or GST alone as negative control. The Coomassie-stained gel shows purified proteins used in this assay. B) Rat brain extract was subjected to immunoprecipitation (I.P.) with immunopurified antibody anti-FKBP52 or preimmune serum used as negative control. The supernatants (s) and precipitates (p) were immunoblotted (I.B.) with anti-{alpha}-tubulin and anti-ß tubulin antibody.

To confirm the specific interaction between FKBP52 and tubulin, coimmunoprecipitation assays using rat brain cytosol were carried out with an immunopurified anti-FKBP52 antibody (described above). As shown in Fig. 1B , immunoprecipitation of FKBP52 also brought down {alpha}-tubulin and ß-tubulin.

The interaction of FKBP52 with tubulin involves several binding sites
Protein binding assays were used to determine which part of FKBP52 binds tubulin. The ability of several FKBP52 mutants (Fig. 2 B) to bind tubulin was measured using GST pull-down assays. We show that the NH2-terminal part of the protein [domain I+II (aa 1–267)] does not bind tubulin, unlike the C-terminal part of FKBP52 [domain III+IV (aa 267–458)] (Fig. 2C ). Of the different truncated mutants tested, including I+II+III (aa 1–400), I+II+III{Delta} (aa 1–375), III+IV (aa 267–458), III (aa 267–400), and the C-terminal part of FKBP52 (CT aa 375–458), all bind tubulin, but the CT2 mutant with residues 400 to 458 and the CT1 mutant (aa 375–400) expressed as fusion proteins with GST could not bind tubulin (Fig. 2C ). These results suggest that several anchoring sequences, including the TPR domain of FKBP52, are involved in the binding with tubulin, which means that in the C-terminal part of FKBP52, the segment expressed from aa 375 to 405 and that from 400 to 458 are necessary but not individually sufficient for FKBP52 binding to tubulin.


Figure 2
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  		Figure 2. In vitro binding assay for FKBP52 truncated mutants with depolymerized tubulin. A) The 3-dimensional structure of FKBP52 (aa 1–427) showing the main structural domains. The cartoon was produced from the PDB coordinates (accessions 1Q1C and 1PSQ) using Swiss-PdbViewe v3.5 (56) . B) The structure of FKBP52 is shown schematically with a diagram of the various FKBP52 mutants used for the binding tubulin assay and MT assembly assay. Amino acid positions are indicated. C) GST pull-down assay was carried out by incubation of full-length or truncated FKBP52 mutants expressed as fusion proteins with GST, or GST alone, with tubulin at 4°C. The name of different mutants used in these experiments is indicated. The presence of tubulin was revealed by immunoblotting using an anti-{alpha}-tubulin antibody (1/1000). Coomassie-stained gels show purified proteins used in these assays.

FKBP52 affects microtubule stability in vitro
To determine whether FKBP52 is involved in MT formation, we measured the assembly rate of tubulin using a light scattering assay.

Control mixed tubulin purified from rat brain started to polymerize after a time lag of {approx}120 s, with a steep initial slope for the first 5 min followed by a smooth increase up to a variation of OD between 0.12 and 0.15 after 15 min or 0.2 after 30 min. Recombinant soluble FKBP52 added to mixed tubulin was able to inhibit MT polymerization in a dose-dependent manner (Fig. 3 A), unlike GST, which was used as negative control (Fig. 3B ). Seventy-eight percent (±4) inhibition of polymerization could be observed after the addition of 3.5 µM FKBP52 (molar ratio of FKBP52 to tubulin dimer was ~1:6). To investigate whether FKBP52 is able to induce depolymerization of assembled MT, 3.5 µM FKBP52 was added to the reaction mixture after 15 min of polymerization at 37°C. FKBP52 induced a rapid and dramatic disassembly of MT (Fig. 3C ). Similar experiments were performed in the presence of taxol to assess whether FKBP52 could antagonize the MT-stabilizing effect of the drug. For this, 4 µM taxol was used because at this concentration it stabilizes MT formation without increasing tubulin polymerization, which required 5 or 10 µM taxol (data not shown). Figure 3D shows that 3.5 µM FKBP52 was able to induce a partial disassembly of 4 µM taxol-stabilized MT.


Figure 3
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  		Figure 3. Effect of FKBP52 on the mixed tubulin polymerization. Assembly of MT was performed by switching the samples from 4°C to 37°C and the change in turbidity was monitored at 345 nm for 15 min. A) Dose-dependent inhibition of MT assembly by FKBP52. Microtubule proteins purified from rat brain cytosol (1mg/ml) were incubated in the absence ({diamond}) or presence of 3.5 ({blacksquare}), 1.75 ({blacktriangleup}), 0.7 ({square}), and 0.35 µM ({diamondsuit}) FKBP52. The polymerization was induced by changing the temperature to 37°C at time 0. Each curve was performed in triplicate with three different preparations of FKBP52. The inset on the right shows the percentage of polymerization inhibition as a function of total FKBP52 concentrations. B) The same assay was carried out for 30 min in the absence ({diamond}) or presence of FKBP52 ({blacksquare}) or GST ({blacktriangleup}). C) Effect of FKBP52 on MT disassembly. Samples containing mixed tubulin were allowed to polymerize for 15 min at 37°C, then 3.5 µM FKBP52 ({blacksquare}) or buffer was added as control ({diamond}) and measurements were continued for another 15 min. D) Effect of FKBP52 on taxol-stabilized MT. MT proteins were incubated without ({diamond}) or with 4 µM taxol ({blacksquare}); 3.5 µM FKBP52 was added alone to MT ({square}) or concomitant with taxol ({blacktriangleup}).

To gain a better understanding of how FKBP52 prevents the formation of microtubules in vitro, we checked the effect of FKBP52 on purified tubulin (without MAP) polymerization. Under these experimental conditions, no effect of FKBP52 on MT formation could be observed (data not shown).

C-terminal part of FKBP52 is required to prevent tubulin polymerization
To map the region within FKBP52 that is involved in the inhibition of MT formation, the assembly rate of mixed tubulin was determined in the presence of different truncated FKBP52 mutants (Fig. 2B ). When FKBP52 is deleted in the C-terminal region (mutant I+II or I+II+III containing residues 1–267 and 1–400, respectively), we did not observe any effect on MT formation, contrary to the effect observed with 3.5 µM NH2-terminal truncated mutant (mutant III+IV with residues 267 to 458) (Fig. 4 A). These results led us to check the effects of mutant CT of FKBP52 (residues 375 to 458) on the assembly of microtubules. MT formation was partially prevented by the addition of 3.5 µM of mutant CT; however, 75% (±7) inhibition of polymerization could be observed when 10 µM of mutant CT was added to mixed tubulin (Fig. 4A ). Domain III (3.5 µM and 7 µM), with residues 267 to 400 (including the TPR region), did not have an inhibitory effect on polymerization but was able to inhibit the depolymerization activity observed with full-length FKBP52 (Fig. 4B ). This last observation suggests that domain III, devoid of MT depolymerizing activity itself, competes with the full-length FKBP52 for binding to tubulin and thus could impede the latter's depolymerizing activity.


Figure 4
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  		Figure 4. The C-terminal part of FKBP52 prevents MT assembly. A) MT assembly assays were carried out with FKBP52 or different FKBP52 mutants. MT formation is expressed as percentage inhibition of polymerization at 15 min. For each protein tested, mixed tubulin polymerization was carried out without added protein and represents the control, which is considered to be 0% inhibition, or after addition of protein, as indicated on the graph. Each assay was performed at least three times. B) Effect of domain III (aa 267–400) on MT formation with or without FKBP52. MT assembly assay was done without ({diamond}) or with 3.5 µM ({blacksquare}), 7 µM ({blacktriangleup}) of domain III or 3.5 µM of FKBP52 full-length ({blacksquare}), or with 3.5 µM full-length FKBP52 in the presence of 3.5 µM ({blacktriangleup}) or 7 µM (•) domain III.

Colocalization of FKBP52 and tubulin in PC12 cells
PC12 cells were used to further investigate the subcellular distribution of FKBP52 and the potential relationship between tubulin and FKBP52. We chose this cell line, of neuronal crest origin, because it is an experimental model largely used to study the early stages of neuronal differentiation. When exposed in culture to nerve growth factor (NGF), PC12 cells cease to divide, grow, and undergo neuronal differentiation, characterized by elongation of neuritic processes.

The FKBP52 subcellular distribution in PC12 cells was examined by immunofluorescence confocal microscopy. For this, we generated a polyclonal antibody directed against two peptides located in the N and C terminals of the protein. The specificity of this antibody (Abs 761) was tested by Western blot analysis. Cytosolic protein isolated from rat brain as well as the recombinant FKBP52 protein, unlike recombinant FKBP51, is recognized by Abs 761, and the signal obtained with this antibody was inhibited by the antigenic peptides (Fig. 5 A). In PC12 cells, affinity-purified anti-FKBP52 antibody shows a predominantly diffuse cytoplasmic staining that is inhibited by the antigenic peptides (Fig. 5B ). Finally, FKBP52 and tubulin staining revealed a preferential colocalization of these two proteins in the growth cones of outgrowing neurites and at the nascent neurites of PC12 cells treated with NGF (Fig. 5C ).


Figure 5
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  		Figure 5. Coimmunolocalization of FKBP52 and tubulin. Characterization of anti-FKBP52 antiserum 761. A) 1 µg recombinant FKBP51 (R-FKBP51), recombinant FKBP52 (R-FKBP52), or 50 µg rat brain cytosolic extracts were analyzed by immunoblotting using affinity-purified anti-FKBP52 antibody 761 (1/1000) preincubated or not with the peptides. B) PC12 cells treated with 50 nM NGF for 4 days were fixed with methanol and stained with affinity-purified anti-FKBP52 antibody 761 (1/1000) preincubated (761+peptides) or not (761) with peptides and analyzed by confocal microscopy. Scale bar: 10 µm. C) PC12 cells treated with 50 nM NGF for 4 days were double stained after methanol fixation with affinity-purified anti-FKBP52 antibody 761 (1/000) and anti-{alpha}-tubulin (1/2000), then analyzed by confocal microscopy. The boxed area in the merged panel is shown at higher magnification on the right. Arrows indicate preferential colocalization of FKBP52 and tubulin. Scale bar: 10 µm.

FKBP52 siRNA induces a differentiated phenotype of PC12 cells
We examined the effect of depletion of FKBP52 in PC12 cultured cells by introducing two different small, interfering RNA (siRNA) duplexes specific for rat FKBP52, named RNAi 1 and RNAi 2. The sense sequence of siRNAs and an oligonucleotide duplex with a scrambled sequence corresponding to RNAi 1 were used as negative control. In these experiments, the level of FKBP52 analyzed by Western blot became substantially reduced after 48 h (not shown) and remained low 72 h post-transfection (Fig. 6 ). Tubulin and FKBP52 staining was performed 72 h post transfections. As expected in cells transfected with RNAi 1 or 2, FKBP52 staining was significantly lower than that observed in control cells, and tubulin staining revealed a change in the PC12 cell phenotype—in particular, the loss of FKBP52 in PC12 cells results in these cells forming extensions (Fig. 7 ). Therefore, these cells acquired a differentiated phenotype that could be compared with PC12 cells treated with NGF. No significant modification could be observed in cells transfected with controls.


Figure 6
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  		Figure 6. Validation of siRNA targeting of FKBP52. Depletion of FKBP52 levels on SiRNA transfection. PC12 cells either were not (Nt) or were transfected with lipofectamine alone (Lp), with two different FKBP52 siRNA (RNAi 1), (RNAi 2), or with scrambled RNAi oligonucleotide duplexes (RNAi sc) or sense oligonucleotides corresponding to RNAi 1, RNAi 2, and RNAi sc and called respectively control S1, control S2, and control Ssc. 72 h post-transfection, cells were harvested and cell lysates were analyzed by immunoblotting using FKBP52-specific antibody (761) (1/1000) and a ß-actin antibody as a control for gel loading.


Figure 7
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  		Figure 7. SiRNA targeting of FKBP52 leads to a modification of PC 12 MT networks. PC12 cells were transfected with two different FKBP52 siRNA (RNAi 1), (RNAi 2), or scrambled RNAi oligonucleotide duplexes (RNAi sc) or sense oligonucleotides corresponding to RNAi 1, RNAi 2 called control S1, control S2, respectively. PC12 cells were fixed 72 h post-transfection and analyzed by fluorescence microscopy using anti-{alpha}-tubulin (1/2000) and affinity-purified anti-FKBP52 antibody (761) (1/1000).

FKBP51, but not FKBP12, affects partially the MT formation
Of the FKBP family, the main cytoplasmic FKBP isoform is FKBP12, and FKBP51 could be regarded as FKBP52's twin (39) . FKBP51 has {approx}75% sequence similarity with FKBP52, and they share similar crystallographic structures (13 , 40) . A specific common feature of these proteins is the TPR domain lacking from FKBP12. Therefore, the effects of FKBP12 and FKBP51 on the assembly and disassembly of MT were investigated. FKBP51 has a residual activity with respect to MT polymerization compared to that observed with FKBP52: 26% (±6) inhibition of polymerization was obtained after the addition of 3.5 µM FKBP51 vs. 78% (±4) inhibition after addition of 3.5 µM FKBP52; 37% (±8) inhibition of polymerization was observed with 7 µM FKBP51 (Fig. 8 A).


Figure 8
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  		Figure 8. Effects of FKBP51 and FKBP12 on tubulin polymerization. A) MT polymerization assays were performed without proteins ({diamond}) or in the presence of 3.5 µM ({blacktriangleup}), 7 µM ({blacksquare}) FKBP51, or 3.5 µM ({square}) FKBP52. B) MT polymerization assays were performed without proteins ({diamond}) or in the presence of 13 µM ({blacksquare}) FKBP12 or 3.5 µM ({square}) FKBP52. C) Protein binding assays were carried out with FKBP51, FKBP12, FKBP52, or GST alone used as negative control and pure depolymerized tubulin (4°C) or mixed polymerized tubulin (37°C), as indicated. The presence of tubulin was revealed by immunoblotting using an anti-{alpha}-tubulin antibody (1/1000). Coomassie-stained gels show the purified proteins used in this assay.

No effect of FKBP12 on the mixed tubulin polymerization could be observed, and the addition of FKBP12 of up to 13 µM could not prevent or inhibit MT polymerization (Fig. 8B ).

The ability of FKBP51 to associate directly with tubulin was checked by a protein binding assay. Tubulin is specifically retained on GST-FKBP51 beads. However, when the protein binding assay was performed at 37°C on polymerized mixed tubulin, no tubulin binding on GST-FKBP51 beads was observed. No tubulin binding was observed with FKBP12 (Fig. 8C ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Biochemical and functional interactions between tubulin and FKBP52
Dynamic instability of microtubules is an intrinsic property of microtubules that allows them to switch between phases of elongation and rapid shortening. These processes are complex and highly regulated. In addition to the post-translational modifications such as phosphorylation or acetylation (41) involved in these processes, two main categories of proteins are required to regulate microtubule dynamic instability: some stabilize microtubules such as the structural microtubule-associated proteins (MAPs), and other proteins such as stathmin (42 , 43) or members of the kinesin subfamily (such as XKCM1) (44) act as potent destabilizers of microtubules. They destabilize MT by sequestering tubulin dimers or by promoting catastrophe at MT plus ends. MTs can also be destabilized by the action of severing proteins such as katanin, which cuts MTs at internal sites generating an increased number of free ends (45) .

The results presented in this report document a specific interaction between FKBP52 and tubulin. First, the immunostaining carried out on NGF differentiated PC12 cells revealed a preferential colocalization of tubulin and FKBP52 in the growth cones and at the nascent neurites, suggesting that FKBP52 may be able to modulate MT dynamics in these highly motile cellular compartments. Indeed, experiments using purified tubulin or mixed tubulin and purified FKBP52 demonstrate the ability of FKBP52 to interact directly with tubulin whether polymerized or not. In addition, coimmunoprecipitation assays confirmed the existence of a complex between FKBP52 and tubulin (alpha or beta isoforms). These cytological and biochemical observations are strong evidence for a FKBP52-tubulin complex. Second, we provide evidence that the consequence of the FKBP52/tubulin interaction is to prevent or inhibit MT formation; however, no effect of FKBP52 on tubulin polymerization could be observed when experiments were carried out with pure tubulin. From this last result, we can rule out the possibility that FKBP52, unlike stathmin (42 43) , acts as a sequestering protein.

On the other hand, PC12 cells were used to check the ability of FKBP52 to inhibit MT formation ex vivo. When stimulated by NGF, PC12 cells differentiate into neuronal-like cells and neuritic processes elongate; the resulting extensions are filled with microtubules (46) . There is a direct correlation between the increase in neurite length and the rate of microtubule assembly (47) . In this work we show that the decreased level of FKBP52 using siRNA induces a differentiated phenotype of PC12 cells characterized by elongation of neuritic processes, as observed in PC12 cells treated with NGF. These results suggest that FKBP52 can prevent the differentiation process in PC12 cells by acting as a blocker of tubulin polymerization. Collectively, our results suggest that by its ability to interact with tubulin, FKBP52 could be involved in the process of MT dynamic instability. According to these results, we can define the immunophilin FKBP52 as a new regulator of MT that inhibits and prevents tubulin polymerization

Mechanism of FKBP52 action
To address the question of which mechanism was responsible for FKBP52-mediated regulation of the MT system, identification of the FKBP52 subsequences in tubulin binding and in MT formation was undertaken.

The effects of several FKBP52 mutants on MT formation and tubulin binding were examined. Deletion of the C-terminal part of FKBP52 (mutant I+II+III) abolished its ability to prevent MT formation, unlike deletion of the N-terminal part of the FKBP52 (mutant III+IV), which mimics effects of the full-length protein at the same concentration; we show that the sequence of 84 aa of the C-terminal part (CT mutant) of FKBP52 is the minimal sequence to cause a loss of microtubule polymer. However, it is necessary to add 3-fold more (10 µM vs. 3.5 µM) of this sequence than of full-length protein or domain III+IV to obtain the same inhibition of MT formation. This decreasing efficiency of the CT mutant on MT formation could be explained by the observation that FKBP52 has several anchoring sites on tubulin and that they all participate in stabilizing the association of FKBP52 with tubulin. These observations are supported by protein binding assay experiments that allowed mapping of the FKBP52 sequences involved in tubulin binding. Indeed, domains I+II+III{Delta} and CT mutant, which do not share any common sequence, both bind tubulin. In addition, we have shown that domains I+II+III, III+IV, and III specifically bind with tubulin, unlike domains I+II, CT1, or CT2 mutants. Based on these results, one might conclude that the tubulin binding sites are restricted to the sequence from aa 267 to 400, which includes the three TPRs of FKBP52. Furthermore, these data lead to delineation of a minimal sequence of 28 aa (aa 375 to 400) in the C-terminal part of FKBP52, necessary but not sufficient for tubulin binding. Considering that the TPR domain is included in the FKBP52 tubulin binding sites and that this part of FKBP52 does not present any MT depolymerization activity contrary to the CT mutant, we can conclude that the tubulin binding activity is not superimposable on the MT depolymerization activity of FKBP52. In addition, competition experiments carried out with domain III and FKBP52 in MT formation assay show that domain III inhibits the tubulin depolymerization obtained with FKBP52.

All data indicated that FKBP52 could neither act as a sequestering protein nor compete for tubulin binding with other proteins that could antagonize the effect of FKBP52; nor could it induce structural rearrangements of tubulin (such as bending of MT) (48) , preventing tubulin polymerization. Rather, the data suggest that post-translational modifications of FKBP52, by compound(s) present in the so-called "mixed tubulin" could explain the loss of tubulin depolymerization activity obtained with pure tubulin, contrary to the results with mixed tubulin. Alternatively, the FKBP52 C-terminal domain may anchor a molecule with depolymerization activity. The C-terminal domain of FKBP52 includes a calmodulin binding site, and calmodulin has been described as affecting tubulin polymerization (49) . However, FKBP52 is a Ca2+-dependent calmodulin binding protein (20) , and all MT polymerization assays were carried out in the presence of EGTA, a Ca2+ chelator, thus eliminating calmodulin as the candidate molecule.

It is noteworthy that the PPIase domain of FKBP52 is not involved in the association of FKBP52 and tubulin. However, it has been reported that FKBP52 interacts with the motor protein dynein by its PPIase domain (25 , 26) , and it is tempting to speculate that tubulin, FKBP52, and dynein form a heterooligomeric complex whose function remains to be elucidated.

Three recent papers that refer to FKBP52 KO mice phenotypes (50 51 52) indicate specific differences unrelated to microtubule disassembly function. However, the MT dynamic process is highly regulated and the absence of FKBP52 could be compensated for by other factors, as already described for this process. For example, MAP2 KO mice developed without any apparent abnormalities, and only double KO for MAP2 and MAP1, lead to observed abnormalities (53) .

FKBP51 and tubulin polymerization
Despite their overall similarities, FKBP52 and FKBP51 do not have the same effect in vitro on MT polymerization. The low MT depolymerization activity of FKBP51 compared with FKBP52 could be explained in part by the difference in their tubulin binding activity. Although FKBP51 binds depolymerizated tubulin, as does FKBP52, we observed no association of FKBP51 with polymerized tubulin under our experimental conditions, contrary to FKBP52. These observations recall that the two immunophilins, FKBP51 and FKBP52, associate differently with steroid receptors (54) . TPR domain chimera and truncation experiments have shown that C-terminal sequences outside the TPR domain of FKBP51 and FKBP52, which are involved in binding to HSP90, are different (55) . The recent definition of the 3-dimensional structure of FKBP52 shows key structural differences between FKBP52 and FKBP51, including relative orientations of the four constitutive domains and some important residue substitutions that could account for the differential functions of these FKBPs (13) . Additional experiments ex vivo and with chimera and truncation mutants of these two FKBPs would be necessary to identify which sequence differences between the two FKBPs are responsible for their differential tubulin polymerization activity.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
We report here a novel function of FKBP52 that is involved in the process of MT dynamics and therefore places the protein in the large family of cytoskeleton-modulating proteins. Our demonstration that FKBP52 binds and regulates tubulin polymerization suggests that FKBP52 could contribute to the neuronal differentiation via regulation of microtubule dynamics, and confirms and extends the suggestion that immunophilins are proteins that have additional functions not yet explored.


   ACKNOWLEDGMENTS
 
We are grateful to Pr. J. Y. Lallemand (ISCN-CNRS, Gif sur Yvette, France) for providing support to H.B, Dr. A. Sobel (INSERM U706, Paris) for thoughtful discussions and important comments on the manuscript, and Dr. K. Rajkowski (INSERM U788, Kremlin-Bicetre, France) for a critical reading. We thank Dr. S. Giraudier for the gift of FKBP51 plasmid (INSERM U362, Villejuif, France), B. Eychenne (INSERM U788, Kremlin-Bicetre, France) and S. Lachkar (INSERM U706, Paris) for their contributions to this work, S. David for his assistance in illustrating, and P. Leclerc (IFR 93, Kremlin-Bicetre, France) for confocal photography. This work was funded in part by the Association pour la Recherche sur le Cancer (Villejuif, France) [contract no. 3232], the Fondation National de Gérontologie (Paris, France), and the Collège de France.

Received for publication November 6, 2006. Accepted for publication March 15, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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D. L. Riggs, M. B. Cox, H. L. Tardif, M. Hessling, J. Buchner, and D. F. Smith
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