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[Cancer Research 62, 1559-1566, March 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK), a Novel Hsp90-Client Tyrosine Kinase: Down-Regulation of NPM-ALK Expression and Tyrosine Phosphorylation in ALK+ CD30+ Lymphoma Cells by the Hsp90 Antagonist 17-Allylamino,17-demethoxygeldanamycin1

Paolo Bonvini2, Tamara Gastaldi, Brunangelo Falini and Angelo Rosolen

Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova, 35128 Padua, Italy [P. B., T. G., A. R.]; Dipartimento di Medicina Clinica Sperimentale, Università di Perugia, 06122 Perugia, Italy [B. F.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anaplastic large cell lymphomas (ALCL) are characterized by the expression of a chimeric protein, NPM-ALK, which originates from fusion of the nucleophosmin (NPM) and the membrane receptor anaplastic lymphoma kinase (ALK) genes. The NPM-ALK kinase, on dimerization, shows phosphotransferase activity and, through its interaction with various ALK-adapter proteins, induces cell transformation and increases cell proliferation in vitro. The chaperones heat shock proteins 90 (Hsp90) and 70 (Hsp70) play a critical role in the folding and maturation of several oncogenic protein kinases, and perturbation of Hsp90 structure affects the stability and degradation of Hsp90- and Hsp70-bound substrates. This process is triggered by benzoquinone ansamycin antibiotics, Hsp90-binding small molecules. We have studied the effect of 17-allylamino,17-demethoxygeldanamycin (17-AAG), a benzoquinone ansamycin, on NPM-ALK steady-state level in ALCL cells. Treatment with 17-AAG decreased NPM-ALK expression and phosphorylation, thus impairing its association with phospholipase C-{gamma}, Src homology 2 domain-containing protein (Shc), growth factor receptor-bound protein 2 (Grb2), and insulin receptor substrate-1 (IRS-1). We also observed that NPM-ALK associates with Hsp90, and incubation with 17-AAG disrupts this complex without affecting Hsp90 expression. As shown previously for other Hsp90 client proteins, destabilization of the Hsp90/NPM-ALK complex induced by 17-AAG resulted in increased binding of the chimeric protein to Hsp70, which is known to affect protein degradation. Hsp/NPM-ALK complex formation appears to be independent of NPM sequences, because we were unable to coimmunoprecipitate NPM with either Hsp90 or Hsp70. Similar to NPM-ALK, the exogenously expressed variant fusion protein TPR-ALK showed decreased expression and phosphorylation after 17-AAG treatment, suggesting that the effect of 17-AAG on ALK chimeric proteins depends on the ALK portion and not on the partner protein moiety. Our data demonstrate that NPM-ALK cell content is determined by its interaction with Hsp90 and Hsp70, and suggest that the alteration of such associations can interfere with NPM-ALK function in ALCL cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALCLs3 represent a well-recognized subgroup of non-Hodgkin lymphomas, characterized by the expression of the membrane receptor CD30/Ki-1, a member of the tumor necrosis factor receptor family (1) . About 50–60% of ALCLs possesses a reciprocal chromosomal translocation, t(2;5)(p23;q35), that fuses the NH2-terminal portion of the ubiquitously expressed NPM to the entire cytoplasmic portion of the insulin-like receptor ALK tyrosine kinase, thus originating a soluble Mr 80,000 chimeric protein, NPM-ALK (2, 3, 4, 5, 6) . Because of an oligomerization motif conserved in the NPM coding sequence, the fusion protein can homodimerize or associate with wild-type NPM, localizing both in the cytoplasm and nucleus (7 , 8) . Cytoplasmic, dimerized NPM-ALK was found to interact with and phosphorylate the Shc, IRS-1, and Grb2 ALK-adapter proteins as well as PLC{gamma}1, contributing to the dysregulated growth of the t(2;5)-harboring cells (2 , 9 , 10) . NPM-ALK-transfected cells show increased cell proliferation and up-regulation of G1 cyclins and several immediate early gene products involved in signal transduction (11 , 12) . Mutational analysis has revealed the important role of PLC{gamma}1 in regulating such mitogenic stimuli (13) , whereas no biological function of Shc or IRS-1 has been found to modulate NPM-ALK oncogenicity (11) .

Similar to many other RTKs, dimerization is strictly required to activate the catalytic domain of NPM-ALK (14) . However, although RTK ligand-dependent dimerization requires proper membrane localization, NPM-ALK is constitutively active, although it does not localize to the plasma membrane (11 , 15) . For several RTKs, plasma membrane localization depends on chaperone activity. Chaperones represent a class of molecules that participate in folding, intracellular trafficking, and in degradation of a subset of proteins that includes receptor or nonreceptor tyrosine, and serine/threonine kinases, steroid hormone receptors, and transcription factors (16 , 17) . In particular, the chaperones Hsp70 and Hsp90, together with several accessory proteins, cooperate in the folding and maturation of several oncogenic protein kinases (18, 19, 20) . Hsp90/Hsp70 folding activity is achieved through conversion of the Hsp90 heterocomplex from an ADP-bound to an ATP-bound state (20, 21, 22) . The Hsp90-substrate association may be disrupted by benzoquinone ansamycin antibiotics (23, 24, 25, 26) . In particular, the ansamycin GA has been shown to prevent Hsp90 activation, thereby resulting in the acceleration of proteosomal degradation of several Hsp90-client proteins (27, 28, 29, 30) .

Here, we examined the ability of ansamycins to regulate stability and kinase activity of the NPM-ALK tyrosine kinase. To assess this effect, we used the GA analogue 17-AAG, currently in Phase I clinical trial in the United States and United Kingdom, which has been shown to retain potent antitumor activity in vitro and reduced toxicity in vivo compared with GA (31, 32, 33, 34) . We found that the functional chimeric protein, NPM-ALK, displays 17-AAG sensitivity in anaplastic lymphoma cell lines and that a 17-AAG-dependent decrease in the phosphorylation of ALK-adapter proteins reflects NPM-ALK steady-state down-regulation. Furthermore, we observed that NPM-ALK binds to both Hsp90 and Hsp70 molecular chaperones. However, whereas 17-AAG prevented Hsp90/NPM-ALK complex formation, the association of Hsp70 with NPM-ALK markedly increased after drug treatment, suggesting an involvement of the latter chaperone in NPM-ALK degradation. Taken together, these observations underline the importance of the Hsps in regulating NPM-ALK stability and function, and suggest a rationale for the assessment of the antineoplastic activity of 17-AAG in ALCL cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The human ALCL cell lines, Karpas299 and SR786, harboring the t(2;5)(p23;q35) translocation, were maintained in RPMI 1640 containing 15% heat-inactivate FCS, 2 mmol/liter glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin under standard tissue-culture conditions. COS-7 monkey renal epithelial cells were cultivated in DMEM, supplemented with 10% heat-inactivated FCS, glutamine, penicillin, and streptomycin, as described above.

Reagents and Antibodies.
17-AAG was obtained from Dr. L. Neckers (National Cancer Institute, NIH), dissolved in DMSO, and stored at -80°C. Anti-PLC-{gamma}-1, anti-Shc, anti-Grb2, and anti-IRS-1 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), as well as antiphosphotyrosine (PY99) and anti-HSP70 monoclonal antibodies. Anti-HSP90{alpha} rat monoclonal antibody was purchased from StressGen (StressGen Biotechnologies Corp., Victoria, BC, Canada). Anti-ALK (ALKc) and anti-NPM (NPM-376) monoclonal antibodies were described previously (5) . Anti-Cyt-c and anti-HDCA1 rabbit polyclonal antibodies were from Santa Cruz Biotechnology. Rabbit antimouse IgG1 was obtained from CAPPEL (ICN Biomedicals Inc.). Horseradish peroxidase-conjugated sheep antimouse and donkey antirabbit antibodies were purchased from Amersham (Amersham Pharmacia Biotech Inc.), as well as Protein A-Sepharose beads and Protein G-Sepharose Fast-Flow beads. Horseradish peroxidase-conjugated rabbit antirat was from StressGen. Bicinchoninic acid protein assay reagents, NE-PER Nuclear and Cytoplasmic Extraction Reagents, and Western Blot chemiluminescence reagents were all obtained from PIERCE (Pierce Chemical Co.). Protran nitrocellulose membranes were from Schleicher & Schuell. All of the other chemicals used in this study were purchased from Sigma Chemical Co.

Immunoprecipitation and Immunoblotting.
Cells were washed twice in ice-cold 1x PBS and incubated on ice for 20 min with lysis buffer [10 mM Tris-HCl (pH 7.5), 130 mM NaCl, 5 mM EDTA, 1 mg/ml BSA, 20 mM sodium phosphate (pH 7.5), 10 mM sodium PPi (pH 7.0), 25 mM glycerophosphate; 1 mM sodium orthovanadate, 10 mM sodium molybdate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 20 µg/ml aprotinin] containing 0.05–0.5% Triton X-100 for p80NPM/ALK coprecipitation analysis, 1% Triton X-100 to precipitate p80NPM-ALK, or tyrosine phosphorylated p80NPM/ALK. Cell lysates were clarified by centrifugation (at 4°C) at 14000 x g for 30 min. Protein concentrations were determined by the bicinchoninic acid assay using BSA as a standard. Protein extracts were then incubated for 2–12 h with 1–2 µg of antibody at 4°C as indicated. Immunocomplexes were precipitated with 20 µl of Protein G-Sepharose beads or by using a rabbit antimouse IgG1 antibody, bound previously to 150 µl Protein A-Sepharose beads, for 2 h at 4°C. The immunoabsorbed pellets were washed four times with 1% Triton X-100 lysis buffer and heated at 95°C in 1x reducing Laemmli loading buffer. Aliquots of cell lysates, diluted in 5x reducing Laemmli loading buffer [2% SDS, 100 mM DTT, 60 mM Tris (pH 6.8), 0.01% bromophenyl blue, and 10% glycerol], and immunoprecipitates were fractionated by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. Proteins were visualized by chemiluminescence using a commercial kit (Pierce). Films (Hyperfilm; Amersham) were scanned and analyzed by using image analysis software (NIH Image). To coprecipitate p80NPM-ALK with both Hsp70 and Hsp90 molecular chaperones under heat stress, exponentially growing SR786 cells were maintained at 42°C under standard tissue culture conditions for 60 min, whereas control cells were maintained for the same time interval at 37°C. After the heat shock, half of the cells were left to recover at 37°C for an additional hour. Cells were lysed in ice-cold 0.1% Triton X-100 lysis buffer, and the heterocomplexes were obtained and detected as described above.

Preparation of Cytoplasmic and Nuclear Fractions.
To extract both cytoplasmic and nuclear p80NPM/ALK, a NE-PER extraction kit (Pierce) was used. In brief, ALCL cells were washed in 1x PBS twice, pelleted by low-speed centrifugation, and incubated with ice-cold Cytoplasmic Extraction Reagent I (Pierce) lysis buffer for 10 min, followed by addition of an aliquot of ice-cold Cytoplasmic Extraction Reagent II (Pierce) lysis buffer. Samples were then centrifuged (at 4°C) at 14,000 x g for 5 min, and supernatants (cytoplasmic fraction) were transferred into a fresh tube. Pellet fractions, representing intact nuclei, washed twice in ice-cold 1x PBS, were incubated on ice with Nuclear Extraction Reagent (Pierce) lysis buffer for 40 min, and soluble nuclear proteins were recovered by high-speed centrifugation. Lysates from each fraction were quantified, electrophoresed, and Western blotted as described previously. To demonstrate the integrity of the two fractions, Cyt-c and HDCA1 were respectively used as markers of the cytoplasmic and nuclear fraction, and detected by immunoblotting.

Transient Transfection.
To determine the effect of 17-AAG on the steady-state level and tyrosine phosphorylation of ectopically expressed NPM-ALK and TPR-ALK, 3 µg of NPM-ALK- and TPR-ALK-pSR{alpha}MSVtkneo retroviral vector, a generous gift of Prof. S. W. Morris (St. Jude Children’s Research Hospital, Memphis, TN), were transiently transfected into 1 x 106 COS-7 cells using the Multicomponent lipid-base transfection reagent FuGENE6, according to manufacturer’s instructions (Roche Diagnostic Corp.). After 24 h, cells were maintained in the presence or in the absence of 17-AAG (0.5 µM) for an additional 24 h. COS-7 cells were then incubated with ice-cold 1% Triton X-100 lysis buffer and scraped after 20 min. Lysates were clarified, immunoprecipitated, and resolved by SDS-PAGE as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
p80NPM/ALK Expression Is Down-Regulated in Anaplastic Lymphoma Cells by the Benzoquinone Ansamycin 17-AAG.
To study the effect of 17-AAG on the expression of p80NPM/ALK, two ALCL cell lines characterized previously for the presence of the t(2;5) translocation, Karpas299 and SR786, were chosen as experimental models (35) . Karpas299 and SR786 cells were maintained in log phase growth and treated with different concentrations of 17-AAG for 24 h (Fig. 1A)Citation . Cells were lysed in Triton X-100 lysis buffer, and p80NPM/ALK was detected by using an antibody (ALKc) specific for the cytoplasmic region of the mature ALK receptor (5) . As shown in Fig. 1Citation (Lanes 1–5), p80NPM/ALK steady-state decreased proportionally to the increase of 17-AAG concentration, reaching 15% of the control in cells exposed to 0.5 µM 17-AAG. As the cytotoxicity, in the experimental conditions used, viability was >=90% in all of the experiments, being SR786 more sensitive to 17-AAG than Karpas299 cells, and this correlated with the p80NPM-ALK down-regulation described above. Interestingly, higher concentrations of the drug were unable to further down-regulate the cell content of the fusion protein in both cell lines (Fig. 1ACitation , Lanes 8–10). This raised the question of whether the ability of p80NPM/ALK to form homo- or heterodimers, or its subcellular localization, might render the protein insensitive to the drug. To rule out the latter possibility, Karpas299 cells were treated with 17-AAG up to 0.5 µM, and nuclear soluble p80NPM/ALK was separated from its cytoplasmic counterpart and analyzed. We observed that 17-AAG negatively regulated both p80NPM/ALK pools to a similar extent (Fig. 1B)Citation , whereas it did not affect the steady-state level of either Cyt-c and HDCA1 proteins, which were chosen to verify the integrity of the cytoplasmic and nuclear fraction, respectively. We then analyzed the kinetics of the effect of AAG treatment on the steady-state level of p80NPM/ALK in both Karpas299 and SR786 ALCL cells. Because 0.5 µM 17-AAG was the lowest most effective dose, ALCL cells were exposed to this concentration of drug for various time intervals. The p80NPM/ALK protein expression declined significantly between 4 and 8 h of treatment, and it became almost undetectable at 48 h (Fig. 1C)Citation . Sensitivity of p80NPM/ALK to 17-AAG was compared with that one of the ALK-adapter protein Shc, and unlike NPM-ALK, Shc levels remained unaltered.



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  		Fig. 1. A, exponentially growing Karpas299 and SR786 ALCL cells were cultured for 24 h in the presence of different concentrations of 17-AAG, as indicated. Cells were lysed, and 30 µg of protein lysate were resolved by reducing 10% SDS-PAGE. Nitrocellulose blots were probed with an anti-ALK antibody (ALKc). B, analysis of cytosolic and nuclear p80NPM/ALK in Karpas299 cells treated with 17-AAG for 24 h as in Lanes 1–5 of panel A. Cell fractions were obtained as described in "Materials and Methods," and 30 µg of protein lysate from each fraction were resolved and analyzed by Western blotting for p80NPM/ALK. Band densities were quantified using image analysis software (NIH image) and were expressed as percentage of control. To verify the integrity of the two fractions, blots were reprobed with anti-Cyt-c and anti-HDCA1 antibody. C, time-dependent 17-AAG effect on both p80NPM/ALK and Shc steady-state was also measured by exposing Karpas299 and SR786 cells to 17-AAG (0.5 µM) for increasing time periods, as shown. p80NPM/ALK was visualized as described in A, whereas 60 µg of cell lysates were resolved to detect Shc by immunoblotting.

 
Inhibition of p80NPM/ALK Signaling by 17-AAG.
The recruitment and phosphorylation of the downstream effector proteins requires p80NPM-ALK dimerization and autophosphorylation (7) . Thus, we measured the level of tyrosine-phosphorylated p80NPM/ALK in SR786 cells exposed for 24 h to 0.5 µM 17-AAG. Tyrosine-phosphorylated p80NPM/ALK was readily detectable in ALCL cells; however after exposure to 17-AAG the total phosphotyrosine level of p80NPM/ALK declined markedly, as shown in Fig. 2ACitation (compare Lanes 3 and 4). Furthermore, the phosphorylation of an Mr 80,000 protein detected in Shc immunoprecipitates was completely abrogated by the drug treatment as well (Fig. 2BCitation , Lanes 1 and 2, arrowhead). This protein is most likely p80NPM/ALK, because we found that 17-AAG inhibited the complex formation between Shc and p80NPM/ALK (Fig. 2BCitation , Lane 6 and 7), thereby causing the inhibition of Shc phosphorylation (Fig. 2BCitation , Lanes 1 and 2). To confirm these results we immunoprecipitated PLC{gamma}-1, Grb2, and IRS-1 in addition to Shc, and after SDS-PAGE of immunoprecipitates, immunoblots were probed for p80NPM/ALK (Fig. 3, A and B)Citation . Unlike p80NPM/ALK, the total level of these proteins remained unchanged after 17-AAG treatment (small panels). Nevertheless, we found that p80NPM/ALK could no longer be coimmunoprecipitated with PLC{gamma}-1 and Shc (Fig. 3ACitation , Lanes 3 and 4, and 9 and 10), or with Grb2 and IRS-1 (Fig. 3BCitation , Lanes 3 and 4, and 5 and 6). To our surprise, p80NPM/ALK bound to IRS-1 was particularly difficult to detect, even when SR786 cells were exposed to the phosphatase inhibitor orthovanadate. Once again, we detected a major Mr 80,000 protein when Grb2 immunoprecipitates were probed with an antiphosphotyrosine antibody, which completely disappeared when cells were treated, as in Fig. 3BCitation , with 17-AAG (data not shown).



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  		Fig. 2. A, to detect tyrosine-phosphorylated p80NPM/ALK, 1 mg of cell lysate was precipitated with 10 µl of anti-ALKc antibody from the detergent-soluble fraction of SR786 cells, treated (+) with 17-AAG (0.5 µM) for 24 h, or untreated (-). Immunoprecipitates were probed with an antiphosphotyrosine antibody, whereas as negative controls, lysates were immunoprecipitated with 0.5 µg of normal mouse IgG. Total protein lysates (30 µg) were also included (Lane 5 and 6). B, to measure the phosphorylation status of Shc after exposing SR786 cells to AAG as above, 1 mg of cell lysates was precipitated by using 1 µg of anti-Shc antibody (Lanes 1–5). Immunoabsorbed phospho-Shc was then detected with an antiphosphotyrosine antibody. Negative controls (IgG) and total lysates (Lanes 4–5) are also shown. To detect coimmunoprecipitation of p80NPM/ALK and Shc, 1 mg of ALKc immunoprecipitates were Western blotted with an antibody recognizing Shc (Lanes 6–7); normal mouse IgG immunoprecipitates and aliquots of total lysates are shown in Lanes 8 and 9–10, respectively.

 


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  		Fig. 3. ALCL SR786 cells were treated with 0.5 µM 17-AAG (+) for 24 h or left untreated (-). A, PLC{gamma}-1- and Shc-bound p80NPM/ALK were detected by precipitating 1 mg of protein lysates with 1 µg of anti-PLC{gamma}-1 or an anti-Shc antibody (Lanes 3–4 and 9–10, respectively). Immunoprecipitates and aliquots of total lysates (Lanes 5–6 or 11–12), were then probed with anti-ALKc antibody. Steady-state levels of PLC{gamma}-1 and Shc were also analyzed (smaller panels). B, similarly, to detect p80NPM/ALK complexed to Grb2 or IRS-1, 1 mg of total protein was precipitated with 1 µg of an antibody recognizing Grb2 (Lanes 3 and 4) or IRS-1 (Lanes 5 and 6), and Western blotted for p80NPM/ALK. Aliquots of total lysates are shown in Lanes 7 and 8. Steady-state levels of Grb2 and IRS-1 are also shown (smaller panels). Uncoupled Protein A-Sepharose beads (PAS) were used as a negative control for the immunoprecipitation (A, Lanes 1–2 and 7–8; B, Lanes 1–2).

 
The Constitutive Association of p80NPM/ALK with Hsp90 Is Impaired in 17-AAG-treated ALCL Cells.
Because 17-AAG leads to destabilization of p80NPM/ALK, we determined whether this effect might be the result of the ability of the drug to prevent the association of the molecular chaperone Hsp90 with the oncoprotein p80NPM/ALK, as shown previously with other Hsp90 client proteins (36, 37, 38, 39, 40) . To rule out an overall decrease of p80NPM/ALK protein after 24 h of AAG treatment, ALCL cells SR786 were exposed to 0.5 µM 17-AAG for either 4 or 24 h. p80NPM/ALK was immunoprecipitated from AAG-treated and untreated SR786 cells, and p80NPM/ALK-bound Hsp90 was visualized with an antibody recognizing both free and complexed Hsp90 (Fig. 4ACitation , Lanes 1–7). As hypothesized, we found that AAG rapidly caused the loss of association of Hsp90 with p80NPM/ALK (Fig. 4ACitation , Lanes 3–6), without perturbing the Hsp90 protein level (Fig. 4ACitation , Lanes 15–18). In addition, we carried out reverse coimmunoprecipitation, and confirmed that p80NPM/ALK constitutively binds to Hsp90, whereas the association was totally prevented in the presence of 17-AAG (Fig. 4ACitation , Lanes 10–13), although the amount of p80NPM/ALK was not significantly changed at the shorter exposure time (Fig. 4ACitation , Lanes 19 and 20). Interestingly, we observed that although NPM was complexed with p80NPM/ALK and that this interaction was sensitive only to prolonged AAG exposures, NPM did not coprecipitate with Hsp90 (Fig. 4B)Citation . These data raise the possibility that heterodimerization with NPM may render p80NPM/ALK insensitive to 17-AAG.



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  		Fig. 4. SR786 ALCL cells were cultivated for both 4 and 24 h in the presence of 17-AAG (0.5 µM). A, to detect the association between p80NPM/ALK and Hsp90, cell lysates (1 mg) were immunoprecipitated with an anti-ALKc (Lanes 1–7) or anti-Hsp90 (Lanes 8–14) antibody, whereas 1 µg of normal mouse IgG, conjugated to Protein G –Sepharose beads, was used as negative control. The association between the two proteins was demonstrated by the anti-Hsp90 and anti-p80NPM/ALK immunoblotting of samples 1–7 and 8–14, respectively. Total Hsp90 (Lanes 15–18) and p80NPM/ALK (lanes 19–22) level was measured by Western blotting of SR786 cell lysates prepared from cells exposed to 17-AAG for both 4 and 24 h, or left unexposed. B, duplicate samples of p80NPM/ALK and Hsp90 immunoprecipitates (Lanes 1–7 and 8–14, respectively) were also assayed for NPM by using an antibody (NPM-376) recognizing the NH2-terminal portion of the Mr 38,000 NPM protein. As shown in the figure, NPM coprecipitated with NPM-ALK, and this association was relatively sensitive to 17-AAG (compare Lanes 3–4 with 5–6), whereas NPM was undetectable in Hsp90 immunoprecipitation (Lanes 10–13).

 
17-AAG-sensistive Association of p80NPM/ALK with Hsp70: Role of Hsp70 in p80NPM/ALK Destabilization.
In the case of several Hsp90 client proteins, the destabilization induced by GA is preceded by an increased association with the chaperone Hsp70 (41) , which has been related to the ubiquitination and proteosomal degradation of some of these proteins (42, 43, 44) . To examine whether endogenous Hsp70 was associated with p80NPM/ALK, SR786 cells were exposed to 0.5 µM 17-AAG for 4 and 24 h, and the heterocomplex was measured by precipitating both p80NPM/ALK and Hsp70. As expected, 17-AAG increased the amount of Hsp70 that coprecipitated with p80NPM/ALK in a time-dependent manner (Fig. 5ACitation , compare 4 h, Lanes 1 and 2, to 24 h, Lanes 7 and 8). Accordingly, p80NPM/ALK markedly complexed with Hsp70 after AAG treatment (Fig. 5B)Citation , although a time-dependent increase in the association of the two proteins was less evident than in Fig. 5ACitation . Again, we did not find NPM coprecipitate with Hsp70, neither after 4 nor 24 h of AAG exposure (Fig. 5BCitation , Lanes 11 and 12, and 15 and 16), supporting the idea that the interaction of p80NPM/ALK with Hsp90 or Hsp70 would occur independently of NPM, both in SR786 (Figs. 4Citation and 5Citation ) and Karpas299 cells (data not shown). To confirm these observations, the association of p80NPM/ALK with the two molecular chaperones was measured after heat stress. Transcription of Hsp70, as well as of several other chaperones, is strongly induced under heat-stress conditions to avoid general protein misfolding and protect cells from stress-induced apoptosis (45 , 46) . However, it remains unclear whether Hsp70-mediated protection occurs independently of Hsp90. Thus, SR786 cells were heat-shocked at 42°C for 60 min, and Hsp90 or p80NPM/ALK immunoprecipitates were respectively probed for p80NPM/ALK and Hsp70 (Fig. 5C)Citation . Perfectly correlating with what is described above, heat shock resulted in the down-regulation of the Hsp90/p80NPM/ALK association, whereas it up-regulated the level of Hsp70 complexed to p80NPM/ALK, which further increased if the cells were allowed to grow at 37°C for an additional hour after the heat shock.



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  		Fig. 5. SR786 cells were treated with 17-AAG as described in Fig. 4Citation . A, to detect p80NPM/ALK-bound Hsp70, cell lysates of both 4 h- (Lanes 1 and 2) and 24 h-treated (Lanes 7 and 8) cells were precipitated with an anti-ALK antibody and probed for Hsp70. The p80NPM/ALK/Hsp70 complex is demonstrated by Hsp70 immunoblotting, as shown in Lanes 5–6 and 9–10. IgG represents the negative control used. B, to demonstrate that p80NPM/ALK binds to Hsp70 and that complex formation is independent of NPM, anti-Hsp70 immunoprecipitates and aliquots of cell lysates were resolved by 10% SDS-PAGE, electrotransferred to nitrocellulose membranes, and probed for both p80NPM/ALK (Lanes 1–10) or NPM (Lanes 11–20). C, SR786 cells were heat shocked for 1 h at 42°C. Half of the shocked cells were incubated at 37°C for an additional hour, as described in "Materials and Methods." p80NPM/ALK was then coprecipitated with the two molecular chaperones Hsp90 and Hsp70, as indicated, and band densities of p80NPM/ALK and Hsp70 proteins, bound respectively to Hsp90 and p80NPM/ALK, were quantified and expressed in histograms as folds of control using NIH image software. Steady-state level of p80NPM/ALK, of both untreated and treated SR786 cells, is also shown (small panel).

 
Inhibition of p80NPM/ALK Synthesis Blocks p80NPM/ALK/Hsp70 Complex Formation.
Hsp70 is a molecular chaperone primarily involved in the folding of newly synthesized proteins at the membrane bound ribosomes (47 , 48) . To determine whether Hsp70 was bound to newly synthesized p80NPM/ALK, ALCL cells, Karpas299, were grown in the presence of the protein synthesis inhibitor CHX for various time intervals, and p80NPM/ALK was immunoprecipitated. As seen in Fig. 6ACitation (top panel), CHX induced the down-regulation of the mature, detergent-soluble p80NPM/ALK although it proceeded slowly after the first 4 h of treatment, probably because of the low degradation rate of the protein. When 17-AAG was added to CHX to the culture medium, expression of the mature protein declined more rapidly (Fig. 6ACitation , bottom panel), in agreement with the even faster decline of its phosphorylated form (Fig. 6CCitation , compare Lanes 4 and 5 to 9 and 10). Thus, p80NPM/ALK immunoprecipitates were analyzed by Western blot with an anti-Hsp70 antibody. In cells cultivated in the presence of CHX alone, the complex was difficult to detect, whereas, consistent with our previous data, 17-AAG stimulated the p80NPM/ALK/Hsp70 complex formation in a time-dependent manner (Fig. 6ACitation , middle panel). Interestingly, blocking p80NPM/ALK synthesis resulted in the complete inhibition of the AAG-induced association between the two proteins (Fig. 6ACitation , bottom panel) although the mature p80NPM/ALK kinase was still easily detectable, and no down-regulation of the Hsp70 steady-state by CHX was measured (Fig. 6B)Citation . Taken together these data suggest the Hsp70/p80NPM-ALK complex is strictly dependent on p80NPM-ALK synthesis.



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  		Fig. 6. Exponentially growing Karpas299 cells were treated with 0.5 µM 17-AAG for various time intervals, as indicated. To block protein synthesis, AAG-treated and untreated cells were cultured in the presence of 10 µg/ml CHX for the times shown. A, p80NPM/ALK was precipitated from 0.5 mg protein extracts of CHX-, AAG-, or CHX+AAG-treated Karpas299 cells, and immunoprecipitates were then analyzed for both p80NPM/ALK and Hsp70. B, Hsp70 steady-state was also measured in cells treated with CHX or 17-AAG alone. C, to detect tyrosine phosphorylated p80NPM/ALK, 1 mg of protein extracts from CHX-treated ALCL cells, cultivated in the presence (17-AAG) or in the absence of the drug (-), were precipitated with 10 µl of anti-ALKc antibody and immunoblotted with an antiphosphotyrosine antibody. Tyrosine-phosphorylated NPM-ALK measured in the presence of CHX alone or in combination with 17-AAG is shown in Lanes 1–5 and 6–10, respectively.

 
17-AAG Sensitivity of p80NPM/ALK Does Not Depend on the Aminoterminal Sequences Fused to ALK.
Because wild-type NPM protein failed to be coimmunoprecipitated with both Hsp90 and Hsp70 chaperones, we hypothesized that p80NPM/ALK sensitivity to 17-AAG did not depend on the ALK-fused partner sequences. Thus, we examined the effect of 17-AAG on the status of a previously described engineered ALK-fusion protein, TPR-ALK, in which the two leucine zipper domains of the nuclear pore protein, TPR, mediate dimerization and activation of the ALK catalytic domain (7 , 10) . We transiently transfected TPR-ALK cDNA (Fig. 7A)Citation into COS-7 epithelial cells, and the expression of the fusion protein was determined both in untreated and in 0.5 µM AAG-treated cells. TPR-ALK was immunoprecipitated by using anti-ALKc antibody, because the ALK portion of TPR-ALK and p80NPM/ALK is identical. As illustrated in Fig. 7BCitation , TPR-ALK was readily detectable and highly tyrosine phosphorylated in AAG-untreated, transfected COS-7 cells (Fig. 7BCitation , Lanes 5 and 12, respectively). However when 17-AAG was added, the expression of the fusion protein was markedly reduced, and tyrosine-phosphorylated TPR-ALK became almost undetectable (Fig. 7BCitation , Lanes 6 and 13). In addition, we ectopically expressed p80NPM/ALK (NPM-ALK) in the same model system. Similar to TPR-ALK, and in accordance to our previous findings, 17-AAG negatively regulated the total amount of the exogenous NPM-ALK, as well as its tyrosine-phosphorylated form (Fig. 7BCitation , Lanes 3 and 4, and 10 and 11, respectively), whereas it had no effect on the steady-state of endogenous NPM (Fig. 7BCitation , Lanes 14–19).



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  		Fig. 7. A, schematic representation of NPM/ALK and TPR/ALK cDNA. Indicated motifs are: putative metal binding domain (MB); leucine zippers regions (LZ); and tyrosine kinase catalytic domain (TKc). B, exponentially growing COS-7 cells were transiently transfected with 3 µg of NPM-ALK or TPR-ALK expression vectors, and treated with 0.5 µM 17-AAG, as described in "Materials and Methods." To measure expression as well as tyrosine phosphorylation of the two ectopically expressed proteins, 0.5 mg of COS-7 protein extracts were immunoprecipitated and Western blotted with anti-ALKc (Lanes 1–6) or antiphosphotyrosine (Lanes 8–13) antibody. COS-7 cells transfected with 3 µg of an empty vector were used as negative control (Lanes 1–2, 8–9, and 14–15), whereas Karpas299 cells were used as positive control (Lane 7). Position of ectopic NPM-ALK or TPR-ALK is indicated by (>). Steady-state level of endogenous NPM protein, of both non-transfected or transfected COS-7 cells, is shown in Lanes 14–19.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Abnormalities at chromosome 2p23 that result in fusion of the intracellular domain of the insulin-like receptor ALK tyrosine kinase with dimerizable partners have been reported in more than half of ALCLs (5 , 8 , 49) . The majority of the ALK-positive ALCLs carry the t(2;5)(p23;q35) reciprocal translocation, generating the oncogenic tyrosine kinase NPM-ALK fusion protein. The resulting Mr 80,000 hybrid protein lacks the extracellular ligand-binding domain necessary for signaling activation of the wild-type ALK transmembrane receptor (2 , 3 , 50) . However, because of a putative dimerization motif in the NPM portion, NPM-ALK is constitutively activated in ALCL cells and is capable of causing malignant transformation through phosphorylation of downstream PTB- or SH2-containing molecules (7 , 11 , 13) . Although a correlation between cyclin A and D1 transcriptional up-regulation, and NPM-ALK ectopic expression has been observed in rat-1 fibroblasts (12) , the details of the NPM-ALK signal-transducing cascade remains largely unknown. The oncogenic potential of NPM-ALK depends on its phosphotransferase activity, ectopic expression of wild-type NPM-ALK accelerates cell cycle entry, and promotes soft-agar anchorage-independent growth of murine cells in vitro, as well as lymphoid malignancies in vivo (51, 52, 53) . In contrast, either kinase-defective mutants or NPM-ALK deletion mutants that are unable to oligomerize fail to induce transformation in vitro (7 , 11) . Surprisingly, Simonitsch et al. (54) have shown recently that NPM-ALK homodimerization is necessary but not sufficient to transform rat embryonic cells. However, additional proof of the critical role of NPM-ALK in lymphomagenesis is the recent observation that phosphorylation of the phosphatidylinositol 3'-kinase regulatory subunit p85, by NPM-ALK, leads to activation of the Akt serine/threonine kinase and thereby inhibits Bad-induced apoptosis in Bad-expressing COS-1 cells (55 , 56) .

In this study we demonstrated that NPM-ALK is a novel Hsp90 client protein, and exposure to the Hsp90 inhibitor, 17-AAG, disrupted NPM-ALK/Hsp90 complex formation, thereby resulting in destabilization and degradation of both the cytosolic- and nuclear-localized fusion protein, in a dose- and time-dependent manner. In addition, our analysis of the tyrosine kinase activity in ALCL cells revealed that phosphorylation of NPM-ALK was strongly inhibited in AAG-treated cells, and in accordance with the failure of the PTB-containing Shc protein and NPM-ALK to coprecipitate in the presence of 17-AAG, we also demonstrated that tyrosine phosphorylation of the ALK-adapter protein, Shc, became undetectable after 17-AAG, although its expression level remained unchanged. Furthermore, immunoprecipitation of the ALK-downstream effector proteins, PLC{gamma}-1, Grb-2, and IRS-1, failed to detect NPM-ALK in AAG-treated samples, whereas, similar to Shc, the steady-state level of these proteins was unaffected by the drug.

Both GA and its derivative 17-AAG bind to the ATP-binding pocket found in the NH2-terminal domain of the Hsp90 molecular chaperone and thereby inhibit the Hsp90-mediated holding/folding activity (26 , 57 , 58) . As a consequence, many signal-transducing Hsp90 client proteins, including the oncogenic kinases Raf-1, v-Src, erbB2, and c-Met, are rapidly degraded in the presence of GA (27 , 36 , 38 , 59, 60, 61) . Therefore, to determine whether the down-regulation of NPM-ALK expression level was achieved through disruption of Hsp90 chaperoning, we carried out a series of coimmunoprecipitation experiments and demonstrated that NPM-ALK constitutively associates with Hsp90, whereas 17-AAG treatment disrupts this association, before any effect on the NPM-ALK protein level. Furthermore, our data have shown that 17-AAG-induced down-regulation of NPM-ALK correlates with and is preceded by an up-regulation of its binding to Hsp70 in a time-dependent manner. Indeed, blocking of NPM-ALK synthesis resulted in an immediate inhibition of the 17-AAG-induced NPM-ALK/Hsp70 association. Interestingly, we were unable to coprecipitate NPM with either Hsp90 or Hsp70 in both 17-AAG-treated or untreated cells, suggesting that NPM-ALK binds to both chaperones independently of and before heterodimerization with wild-type NPM.

As reported recently for the CFTR, immature CFTR and its mutant pathological counterpart, CFTR{Delta}508, bind strongly to Hsp70, becoming ubiquitinated and proteasome-degraded (62) . Furthermore, mutation analysis revealed that synthesis and stability of a mature insulin receptor is finely regulated by its kinase domain integrity, because amino acid Asp1179 and Leu1193 substitution in the catalytic domain produces receptors that are rapidly recognized by Hsp70 and degraded through the 26S proteasome (63, 64, 65) . Our results showed that under normal conditions NPM-ALK is only partially complexed with Hsp70, whereas most of the mature fusion protein binds more strongly to Hsp90. 17-AAG, by binding to and inhibiting Hsp90 function, would force NPM-ALK to associate with Hsp70, thereby causing NPM-ALK to be degraded. However, under these circumstances we cannot exclude the involvement of Hsp90 in NPM-ALK degradation as well, and, as hypothesized for another oncogenic fusion tyrosine kinase, Bcr-Abl, which is also dependent on Hsp90 association for its stability (41 , 66) , the apparent loss of Hsp90 after 17-AAG treatment might be because of the increased hydrophobicity and detergent sensitivity of Hsp90 compared with its ATP-bound form.

The interaction of Hsp90 with several protein kinases has been shown to specifically involve the catalytic domain of the kinase (67) . Xu et al. (68) reported recently that mature ErbB2 tyrosine kinase is recruited by Hsp90 through its catalytic domain. Kinase domain-deleted ErbB2 mutants lose GA sensitivity, whereas kinase-deficient mutants do not. This implies that the ALK kinase domain might interact with Hsp90 and, therefore, all of the other known ALK-fused variants should be as sensitive to 17-AAG as NPM-ALK. To test this hypothesis, we analyzed the ALK-fused protein TPR-ALK, of which the oncogenicity has been demonstrated in vitro (10) . We transiently transfected a TPR-ALK expression vector into COS-7 epithelial cells, and found that 17-AAG-dependent reduction of TPR-ALK total protein content, as well as depletion of tyrosine-phosphorylated TPR-ALK, was similar to what we observed with both endogenous and exogenous NPM-ALK tyrosine kinase, suggesting that these phenomena depend on the ALK protein moiety rather than on its fused NH2-terminal sequences. In conclusion, our data demonstrate that NPM-ALK expression is strictly dependent on its interaction with Hsp90 and Hsp70, and that alteration of these molecular complexes results in NPM-ALK-mediated signal transduction down-regulation.


   ACKNOWLEDGMENTS
 
We thank Dr. L. Neckers (NCI, NIH, Bethesda, MD) for providing 17-AAG drug; Dr. S. W. Morris (SJCRH, Memphis, TN) for pSR{alpha}MSVtkneo-NPM-ALK and pSR{alpha}MSVtkneo-TPR-ALK expression plasmids; Dr. L. Neckers and E. G. Mimnaugh for critical review of the manuscript; and E. Tosato for technical support.


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

1 Supported by Fondazione Citta della Speranza; P. B. is recipient of a fellowship by Associazione Italiana contro le Leucemie. Additional support was provided by Consiglio Nazionale delle Ricerche and Ministero dell’Università e Ricerca Scientifica e Tecnologica. Back

2 To whom requests for reprints should be addressed, at Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova, via Giustiniani 3, 35128 Padova, Italy. Phone: 39-049-8215678; Fax: 39-049-8215679; E-mail: paolobonvini{at}hotmail.com. Back

3 The abbreviations used are: ALCL, anaplastic large cell lymphoma; RTK, receptor tyrosine kinase; ALK, anaplastic lymphoma kinase; Hsp, heat shock protein; NPM, nucleophosmin; TPR, translocated promoter region protein; HDCA1, histone deacetylase 1; Cyt-c, cytochrome c; PLC{gamma}-1, phospholipase C-{gamma}; IRS-1, insulin receptor substrate-1; Grb2, growth factor receptor-bound protein 2; Shc, Src homology 2 domain-containing protein; 17-AAG, 17-allylamino-17-demethoxygeldanamycin; GA, geldanamycin; CHX, cycloheximide; CFTR, cystic fibrosis transmembrane regulator. Back

Received 9/26/01. Accepted 1/ 4/02.


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 RESULTS
 DISCUSSION
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