Abstract Background. Among the posttranslational modifications occurring in eukaryotic cells, phosphorylation is the most critical for proteins, as phosphate-mediated conformational switching of catalytic and non-catalytic motifs impacts expression and activity through changes in structure stability. Activation by phosphorylation is the rule for protein kinases, including oncogenic protein tyrosine kinase NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) in Anaplastic Large Cell Lymphomas, though it renders the kinase prone to degradation. For several of them, however, the maintenance of functional stability is provided by the molecular chaperone Hsp90, through the recognition of specific motifs within the catalytic domain. Objectives. Herein, we determined whether NPM-ALK kinase activity facilitates Hsp90 binding, and whether structural motifs within NPM-ALK catalytic domain favor or impair Hsp90 interaction. Design/Methods. To test this hypothesis we prepared a series of N- and C-terminal deletion mutants of wild-type NPM-ALK, as well as inactive point mutants by site-directed mutagenesis of the NPM-ALK ATP-binding site and activation loop. Results. Using NPM-ALK constructs in COS7 and 293T cells, we showed that Hsp90 strongly interacts with wild-type NPM-ALK, but fails to co-adsorb with inactive point mutants and with deletion mutants lacking the entire kinase domain. By comparison, binding to Hsp90 family members Hsc70 and Hsp70 is independent of NPM-ALK activity, suggesting a greater dependence of active NPM-ALK on Hsp90 function. Accordingly, phosphorylated NPM-ALK associated with Hsp90 and co-chaperone cdc37, and pharmacological inhibition of NPM-ALK prevented the interaction of NPM-ALK with both proteins. Of note, mutants unmasking the αC-helix motif in the N-terminal lobe of the catalytic domain maintained the ability to bind Hsp90, whereas mutants lacking αC-helix did not. Conclusions. Our findings, therefore, suggest a model in which, in addition to a role in folding nascent NPM-ALK, Hsp90 provides an “ongoing folding support” to activated NPM-ALK kinase, by protecting self-phosphorylated NPM-ALK from ubiquitination through recognition of specific unstable motifs within its catalytic domain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3034. doi:1538-7445.AM2012-3034