• Paolo Bonvini, Tamara Gastaldi, Brunangelo Falini, and Angelo Rosolen.
• Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova, 35128 Padua, Italy. paolobonvini@hotmail.com
• Cancer Res. 2002 Mar 1; 62 (5): 1559-66.

AbstractAnaplastic 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.

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