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-demethoxygeldanamycin.
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.
Bonvini
Paolo
P
Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova, 35128 Padua, Italy. paolobonvini@hotmail.com
Gastaldi
Tamara
T
Falini
Brunangelo
B
Rosolen
Angelo
A
D000077548:Anaplastic Lymphoma Kinase; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D016227:Benzoquinones; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D006801:Humans; D017730:Ki-1 Antigen / Q000032:analysis*; D047029:Lactams, Macrocyclic; D016403:Lymphoma, Large B-Cell, Diffuse / Q000201:enzymology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000502:physiology*; D020794:Receptor Protein-Tyrosine Kinases; D017828:Rifabutin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D017730:Ki-1 Antigen; D047029:Lactams, Macrocyclic; D017828:Rifabutin; D014443:Tyrosine; C112765:tanespimycin; C107023:p80(NPM-ALK) protein; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases