Effects of FK506-binding Protein 12 and FK506 on Autophosphorylation of Epidermal Growth Factor Receptor*

Marco Lopez-Ilasaca, Cordelia SchieneDagger , Gerhard KüllertzDagger , Thomas TradlerDagger , Gunter FischerDagger , and Reinhard Wetzker§

From the Research Unit, Molecular Cell Biology, Drackendorfer Strabeta e 1, D-07747 Jena, Germany and Dagger  Max Planck Research Unit, Enzymology of Protein Folding, Kurt-Mothes Str. 3, D-06120 Halle, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

FK506-binding proteins and cyclophilins are intracellular proteins that express peptidylproline cis-trans-isomerase (PPIase) activity. The effects of FK506-binding protein 12 (FKBP12) and the cyclophilins 18 and 23 on autophosphorylation of the epidermal growth factor (EGF) receptor prepared from plasma membranes of the human epidermoid cell line A431 have been investigated. Whereas FKBP12 inhibited EGF receptor tyrosine kinase activity in a concentration-dependent manner, the cyclophilins did not affect autophos- phorylation. In contrast to the wild-type enzyme, several variants of FKBP12 with greatly reduced PPIase activity were unable to suppress EGF receptor tyrosine kinase significantly. Pervanadate an inhibitor of protein tyrosine phosphatases, abolished the effect of FKBP12 on EGF receptor autophosphorylation. Finally, FK506 and rapamycin, which are known to block the PPIase activity of FKBP12, induced a significant stimulation of EGF receptor autophosphorylation in intact A431 cells suggesting suppression of EGF receptor autophosphorylation by intracellular FKBP12 in vivo. Taken together the data point to an inhibitory function of FKBP12 in EGF receptor signaling, possibly induced by stimulation of a protein tyrosine phosphatase coupled to the EGF receptor. Both PPIase activity and substrate specificity of FKBP12 seem to be indispensable for this effect.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Peptidylproline cis-trans-isomerases (PPIases1; EC number 5.2.1.8) are ubiquitous and abundant enzymes conserved from prokaryotes to eukaryotes. They accelerate the slow cis/trans isomerization of peptidylprolyl bonds in peptides and unfolded proteins (1-3). Until today three distinct PPIase families have been defined on the basis of primary structure differences: cyclophilins (Cyp), FK506-binding proteins (FKBP), and parvulins. The FKBP group may include the ribosomal trigger factors of bacteria as a subfamily that is not able to sequester the peptidomacrolide FK506 in the active site (4, 5). The occurrence of at least one member of PPIases in virtually all cell types or organisms along with pronounced amino acid sequence conservation suggests that these enzymes may play an important role in a range of biological processes.

In human T cells the cytosolic proteins Cyp18 and FKBP12, which are the archetypal PPIases, were shown to represent the primary targets of the immunosuppressive drugs cyclosporin A (CsA), rapamycin, and FK506. These microbial products were identified as tightly binding inhibitors of the enzymatic activity of their respective family of PPIases (6-8). However, it was demonstrated that inhibition of the catalyzed prolyl isomerization did not cause immunosuppression. According to the current knowledge, suppression of clonal expansion of T cells by FK506 or CsA is attributed to the inhibitory effect of the FK506·FKBP12 (or CsA·Cyp18) complex at the active site of the Ser/Thr phosphatase calcineurin (9) leading to an increase in the phosphorylation state of the transcription factor NF-AT (10, 11).

In addition to its effect on T cell proliferation, FK506 modulates certain signaling events in other cellular systems. Thus different effects of the drug on intracellular protein phosphorylation have been observed (12-18) but their molecular basis remains obscure. Obviously multiple effects of the immunosuppressive drugs on their respective PPIases, such as inhibition of PPIase activity and disruption of heteroligomeric PPIase complexes, make it difficult to achieve information about molecular details of FK506-dependent reactions. Correspondingly investigation of direct effects of FKBP12 or other PPIases allows to circumvent the complexity of reactions induced by FK506 or CsA.

To a large extent PPIases have yet unknown physiological functions. There are just a few examples reported of how PPIases affect cellular events in the absence of the inhibitory drugs at the molecular level (19-21). Recent evidence points to an involvement of the parvulin-like nuclear PPIase Pin1 in the regulation of proliferation and differentiation. Thus it has been shown that PPIase inactive Pin1 variants, in contrast to the wild-type enzyme, were unable to complement yeast mutants lacking the yeast Pin1 homologue ESS1/PTF1 (22). Similarly the PPIase activity of Cyp18 was shown to be critical for the functional expression of homoligomeric neuronal nicotinic and type 3 serotonin receptors (23). In complex biological model systems, such as liver regeneration (24) or neutrophil chemotaxis (25) it was shown that FKBP12 causes considerable influence on signaling proteins. Thus FKBP binds to and modulates the function of calcium release channels (26, 27) and has also been found complexed to the type I receptors of transforming growth factor-beta (28). These pieces of evidence point to a more widespread role of FKBPs in intracellular signal transduction.

This study is aimed at investigating the effects of FKBP12 on epidermal growth factor (EGF) receptor function in the human epidermoid cell line A431. In these cells, EGF receptor function has been studied in detail (29). We show that FKBP12 inhibits EGF receptor autophosphorylation in vitro and present evidences for a similar effect in vivo. In contrast to wild-type FKBP12 several variants of the protein with largely reduced PPIase activity were unable to inhibit EGF receptor tyrosine kinase. Suppression of EGF receptor autophosphorylation by FKBP12 could be abolished by FK506 and pervanadate. The results suggest that inhibition of EGF receptor tyrosine kinase may involve a FKBP12 dependent activation of a phosphotyrosine phosphatase associated to the receptor and that PPIase activity is important for this function.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
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References

Materials-- Recombinant human FKBP12 was produced using the plasmid pQE60 (Qiagen) in Escherichia coli strain K12 M15/pREP4. FK506 was a gift from A. Lawen, CsA from Arzneimittelwerk Dresden GmbH, and rapamycin was a gift from Dr. H. Fliri. Human recombinant Cyp18 was from Boehringer Mannheim. Authentic mature mitochondrial Cyp23 from rat liver was a gift of M. Kramer (Halle). [gamma -32P]ATP was purchased from Amersham Pharmacia Biotech (Braunschweig). Recombinant FKBP12 and variants of the protein have been produced as described recently (30). All other reagents were obtained from Sigma (Deisenhofen).

Cell Culture-- A431 cells (CRL 1555, obtained from the American Type Culture Collection (Rockville, MD)) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with L-glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 IU/ml) and 7.5% fetal calf serum and maintained at 37 °C and 5% CO2. Cellular DNA synthesis was measured by assaying [3H]thymidine incorporation as described (31).

Analysis of EGF Receptor Phosphorylation in Intact A431 Cells-- A431 cells were grown in 6-well plates in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal bovine serum. The cells were deprived overnight in serum-free Dulbecco's modified Eagle's medium in the presence or absence of 10 µM CsA or 10 µM FK506. The medium was removed, and the cells were incubated with 100 ng/ml EGF for 10 min. The cells were washed twice with phosphate-buffered saline containing leupeptin (1 mg/ml), pepstatin A (1 mg/ml), orthovanadate (1 mM), and phenylmethanesulfonyl fluoride (1 mM), solubilized with Laemmli buffer, and boiled for 5 min. After electrophoresis and transfer to polyvinylidene difluoride membranes (Millipore) the blots were developed with antiphosphotyrosine antibodies (RC20-peroxidase conjugate, Affiniti, Nottingham) at 1:2,500 dilution. Bound peroxidase was visualized by enhanced chemiluminescence using the ECL kit from Amersham Pharmacia Biotech.

EGF Receptor Preparation from A431 Membranes-- A431 cells were washed twice with ice-cold buffer Tris-maleate 20 mM, 100 mM NaCl, pH 7.2, and once with buffer 20 mM PIPES, pH 7.2, and scraped. The cells were collected by centrifugation at 1200 rpm and 4 °C and homogenized by sonication in a Branson sonifier (1 min, level 3) at 4 °C in the same buffer containing leupeptin (1 mg/ml), pepstatin A (1 mg/ml), chymostatin (1 mg/ml), phenylmethanesulfonyl fluoride (1 mM), EGTA (1 mM), and dithiothreitol (10 mM). The homogenate was centrifuged at 25,000 × g for 40 min at 4 °C in a Sorvall SS34 rotor. The pellet was resuspended in 20 mM PIPES buffer, pH 7.2, and used as a membrane fraction. EGF receptor was prepared from these membranes by a procedure described by Akiyama et al. (32). Briefly, this protocol includes solubilization of the membranes with Triton X-100, affinity chromatography on wheat germ agglutinin, and tyrosine-Sepharose chromatography.

EGF Receptor Autophosphorylation-- Assay of EGF receptor autophosphorylation was carried out in a final volume of 30 µl. EGF receptor preparation from A431 cells (15.5 µg of protein per assay) was incubated in the presence of 1.2 µg/ml EGF, 50 mM HEPES (pH 7.5), and 3 mM MnCl2 (final concentrations) for 30 min on ice. Immunophilins were added 30 min before treatment with the growth factor. In some experiments, sodium pervanadate, prepared as described by Pumiglia et al. (33), was added together with FKBP12. Phosphorylation was initiated by the admixture of [gamma -32P]ATP (3-5 µCi, final concentration 2 µM) and terminated after 10 min by addition of 30 µl of 2-fold concentrated SDS-polyacrylamide gel electrophoresis buffer. The samples were subjected to SDS-polyacrylamide gel electrophoresis using 10% acrylamide gels and to autoradiographic analysis. Radioactivity incorporated into the EGF receptor band was quantified using a GS250 Molecular Imager (Bio-Rad). Determination of ATP concentration in the assay after phosphorylation was performed by evaluating the peak area of the isolated ATP peak at a migration time of 7.10 min at 200 nm in capillary electrophoresis (Applied Biosystems 270 A-HAT. 25 kV, 100 mM phosphate buffer, pH 6.6).

    RESULTS
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Results
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References

FKBP12 Inhibits Autophosphorylation of EGF Receptor-- The effect of different PPIases on EGF receptor tyrosine kinase prepared from A431 cells has been investigated. Recombinant human FKBP12 or the cyclophilins 18 and 23 were mixed with the EGF receptor and autophosphorylation was assayed. As shown in Fig. 1A FKBP12 significantly suppressed EGF receptor phosphorylation, whereas pig kidney cytosolic cyclophilin (Cyp18) or mature rat liver mitochondrial cyclophilin (Cyp23) were ineffective. No concomitant phosphorylation of the added FKBP12 was observed (results not shown). ATP depletion of the assay as a basis for the observed effect could be excluded as well. Changing the ATP concentration from 1 to 100 µM did not affect the ratio of the EGF receptor autophosphorylation in the absence of to that in the presence of 1.5 µM FKBP12. In an experiment using 2 µM ATP, no significant decrease in the peak area of the ATP peak (±5%) during phosphorylation reaction could be determined by capillary electrophoresis (data not shown). The effect of FKBP12 was found to be dose- and time-dependent, being most prominent at high protein concentrations and long incubation times (Fig. 1, B and C). After 10 min of incubation, half-maximal inhibition of autophosphorylation was achieved at about 1.2 µM FKBP12.


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Fig. 1.   Effect of PPIases on EGF receptor autophosphorylation. A, effect of recombinant human Cyp18, rat mitochondrial Cyp23, and human recombinant FKBP12 on the autophosphorylation of the EGF receptor isolated from A431 cells. EGF receptor preparation was preincubated for 30 min with 4 µM of the indicated PPIases and stimulated with 1.2 µg/ml EGF; autophosphorylation was assayed as described under "Experimental Procedures." B, dose response of the FKBP12 effect on EGF receptor autophosphorylation. The indicated amounts of FKBP12 correspond to 0.3, 0.6, 1.2, 2.5, 5, and 10 µM final concentrations. C, time course of EGF receptor autophosphorylation in the presence of 0.3 µM FKBP12.

Several approaches were utilized to elucidate the mechanism of this inhibitory effect on EGF receptor autophosphorylation. Using FKBP12 variants, we investigated the role of PPIase activity on the autophosphorylation of purified EGF receptor preparations. As shown in Fig. 2, FKBP12 variants bearing D37L or F99Y mutations expressed greatly reduced inhibitory effects. In comparison to wild-type FKBP, both protein variants are known to be unaffected in the secondary structure (30). They both exhibit 3-6% residual activity of the wild-type enzyme when assayed with tetrapeptide standard substrates for PPIase activity but have an altered specificity for amino acid residues flanking proline in substrates (30). To further explore the specificity of the inhibitory effect of FKBP12, we tested the FKBP-like core of the trigger factor. The trigger factor is a fully active FKBP-like PPIase but structurally distinct from FKBP12 by active site construction (30). Compared with the nearly complete inhibitory effect induced by wild-type FKBP12, the trigger factor (TF) caused only about 30% reduction of autophosphorylation (Fig. 2). Thus, both PPIase activity and substrate specificity of FKBP12 seem to be essential for the suppression of EGF receptor autophosphorylation.


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Fig. 2.   Effect of FKBP12 variants with reduced PPIase activity and of trigger factor (TF) on EGF receptor autophosphorylation. EGF receptor preparation from A431 membranes was treated with 1.5 µM (final concentration) of the indicated PPIase species and stimulated with 1.2 µg/ml EGF; autophosphorylation of the EGF receptor was assayed as described under "Experimental procedures".

We next elucidated the effect of the peptidomacrolide FK506 on the inhibition of autophosphorylation of the EGF receptor induced by FKBP12. FK506 is known to suppress PPIase activity of FKBP12. As shown in Fig. 3A FK506 at a 1.5 molar ratio to FKBP12 completely reversed the inhibitory effect of the PPIase. Direct stimulation of the EGF receptor tyrosine kinase by FK506 can be excluded because in the absence of FKBP12 the macrolide induced a weak inhibition of autophosphorylation (about 10% inhibition at 1.5 µM FK506). Rapamycin, which is another effective inhibitor of FKBP12 activity (34), likewise abolished the suppression of EGF receptor tyrosine phosphorylation by the PPIase when applied stoichiometrically (data not shown).


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Fig. 3.   Suppression of the inhibitory effect of FKBP12 on EGF receptor autophosphorylation by FK506 and pervanadate. A, effect of increasing concentrations of FK506 on the inhibition of EGF receptor phosphorylation by FKBP12. EGF receptor preparation was treated with the indicated amounts of FK506 and FKBP12 and stimulated with EGF; autophosphorylation of the EGF receptor was assayed by immunoblotting with antiphosphotyrosine antibodies. B, effects of pervanadate and FKBP12 on EGF receptor autophosphorylation. EGF receptor preparations were treated with 1.5 µM FKBP12, 0.1 mM pervanadate, and 1.2 µg/ml EGF as indicated, and EGF receptor autophosphorylation was assayed.

The autophosphorylated EGF receptor in A431 cell membranes is rapidly dephosphorylated by endogenous protein tyrosine phosphatase (35). Recently association of SH2 domain-containing phosphotyrosine phosphatases SHP1 and SHP2 to the EGF receptor in A431 cells has been demonstrated (35). SHP1 acts as an opponent of EGF receptor tyrosine kinase and catalyzes dephosphorylation of the receptor phosphotyrosine residues. To resolve a possible involvement of tyrosine phosphatase in the observed FKBP12 effects we studied the influence of pervanadate, which acts as a specific inhibitor of protein tyrosine phosphatases. Fig. 3B shows that treatment of A431 membranes with 100 µM pervanadate nearly abolishes the inhibitory effect of FKBP12 on EGF receptor autophosphorylation. The basal level of autophosphorylation, which is slightly decreased by FKBP12, has been proved to be insensitive toward pervanadate.

Effect of FK506 on the Growth of A431 Cells and EGF Receptor Autophosphorylation-- To study the interaction of FKBP12 and EGF receptor in vivo, we investigated the effect of FK506 on the proliferation of A431 cells. It has been recently shown that EGF is able to inhibit the growth of this cell line but the mechanism of this effect is unknown (13). One could expect that FK506, which abolished the inhibitory effect of FKBP12 on EGF receptor autophosphorylation, would produce an inhibition of A431 cell growth via an indirect stimulation of EGF receptor tyrosine kinase activity. As shown in Fig. 4A, a corresponding effect could indeed be detected. These data are similar to a recent observation by Richter et al. (13).


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Fig. 4.   Effects of FK506 on proliferation of A431 cells and EGF receptor autophosphorylation. A, inhibition of A431 cell growth by FK506. A431 cells were treated with the indicated concentrations of FK506 and [3H]thymidine incorporation into cellular DNA was assayed. B, effects of FK506 and CsA on EGF receptor autophosphorylation in A431 cells. Cells were pretreated with 10 µM FK506 or CsA as indicated and stimulated with 100 ng/ml EGF for 10 min. EGF receptor autophosphorylation was assayed by immunoblotting with antiphosphotyrosine antibodies. EGF receptor expression was investigated in parallel by immunoblotting using anti-EGF receptor antibodies.

Finally we investigated the effects of FK506 and CsA on EGF receptor autophosphorylation in intact A431 cells. As shown in Fig. 4B FK506 induced a significant increase of EGF receptor autophosphorylation in vivo, whereas CsA showed no gross effect in comparison to the control. Most probably the in vivo effect of FK506 should be due to a direct interaction of the macrolide with endogenous FKBP12. The expression of EGF receptor protein in the cells was unaltered excluding possible contribution of protein synthesis to the effect of FK506. Taken together the effects observed after treatment of A431 cells with FK506 are in agreement with the results on the inhibition of EGF receptor autophosphorylation induced by FKBP12 in vitro.

    DISCUSSION
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Discussion
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The sequential signaling events induced by binding of EGF to the EGF receptor include receptor dimerization, stimulation of its intrinsic tyrosine kinase activity, and finally autophosphorylation of defined tyrosine residues of the receptor protein. Negative regulation of the autophosphorylation is mediated by the protein tyrosine phosphatase SHP1, which has been shown to associate tightly with the phosphorylated EGF receptor (35).

According to this schema, the inhibitory effect of FKBP12 on EGF receptor autophosphorylation could be due to either suppression of the receptor tyrosine kinase activity or to a stimulation of associated protein tyrosine phosphatase. The partial elimination of the FKBP12 effect induced by the tyrosine phosphatase inhibitor, pervanadate, points to the latter explanation. The role of FKBP12 in generating the inhibitory effect on EGF receptor autophosphorylation might be related to its enzymatic function as a PPIase. In line with this interpretation, two site-directed FKBP12 variants, D37L and F99Y, with reduced PPIase activity exhibited reduced ability to suppress EGF receptor tyrosine phosphorylation (30). It is tempting to speculate that PPIase activity of FKBP12 could facilitate conformational changes during the proposed interaction of the protein tyrosine phosphatase with the phosphorylated EGF receptor thus accelerating dephosphorylation of this protein substrate.

Alternatively or in addition to a PPIase effect, specific binding of FKBP12 to the EGF receptor or the associated phosphatase may be involved in the inhibitory effect on EGF receptor autophosphorylation. Thus the failure of the trigger factor to efficiently affect EGF receptor autophosphorylation demonstrates that PPIase catalysis does not suffice for the effect. The trigger factor domain is more active than FKBP12 toward most tetrapeptides used in the routine PPIase assay. However, distinct active sites were indicated by the lack of inhibition by FK506 on the trigger factor PPIase activity (4, 30). In a similar manner the absence of inhibitory effects of Cyp18 and Cyp23 could be explained.

In our experiments, retardation of EGF receptor autophosphorylation was abolished in response to the FKBP12 inhibitors FK506 and rapamycin. Since stoichiometric amounts of the inhibitors were found to be required for complete reversal of the FKBP12 effect, any function of the FKBP12·drug complexes reminiscent of inhibition of calcineurin could be ruled out. Indeed, the in vivo effects of FK506 on the autophosphorylation of the EGF receptor and the proliferation of A431 correspond to the results obtained in vitro. Assuming endogenous FKBP12 as the intracellular target for FK506, the increased autophosphorylation of the EGF receptor can be interpreted on the basis of an inhibition of the enzymatic activity of the PPIases by the peptidomacrolide. Suppression of PPIase activity could induce a stimulation of EGF receptor tyrosine kinase via inhibition of an associated protein tyrosine phosphatase. The inhibitory effect of FK506 on the proliferation of A431 cells can be explained on the same basis. In contrast to the growth stimulation of epithelial cells, which is commonly observed after treatment with EGF, A431 cells are known to be inhibited by admixture of EGF and following activation of the EGF receptor (37). Correspondingly, stimulation of EGF receptor tyrosine kinase by FK506 should induce growth inhibition of the cell line.

Enhanced phosphorylation associated with FK506 treatment was already obtained for neuronal cell lines transfected with nitric oxide synthase (14). Another link of PPIases to neuronal protein phosphorylation was established by the growth-associated protein 43, whose phosphorylation was found to be augmented by both FK506 and CsA (17). The strong stimulation by FK506 of neurite outgrowth was thought to be associated with its effects on phosphorylation. Thus the intracellular protein phosphorylation machinery seems to represent an important target for immunophilins.

Taken together we could demonstrate inhibition of EGF receptor autophosphorylation by FKBP12 in vitro and in vivo. Our data suggest that the inhibitory effect of FKBP12 is due to stimulation of a protein tyrosine phosphatase associated with the EGF receptor, and PPIase activity of FKBP12 could be involved in this effect. Considering the cellular concentration of FKBP12, which approaches 20 µM in Jurkat cells (38), its observed inhibitory function for EGF receptor autophosphorylation may be of biological relevance.

After submission of this paper, an inhibition of the transphosphorylation of the transforming growth factor-beta receptor subunits I and II by FKBP12 was demonstrated (39). Rapamycin, which blocks binding of FKBP12 to the transforming growth factor-beta receptor has been shown to reverse the inhibitory effect of FKBP12 on the receptor phosphorylation. Together with our data these findings point to a more general role of FKBP12 in the regulation of receptor-induced phosphorylation events.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 49-3641-304-460; Fax: 49-3641-304-462; E-mail: i5rewe{at}rz.uni-jena.de.

1 The abbreviations used are: PPIase, peptidylproline cis-trans-isomerase; FKBP, FK506-binding protein; EGF, epidermal growth factor; CsA, cyclosporin A; Cyp, cyclophilin; PIPES, 1,4-piperazinediethanesulfonic acid.

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References

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