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J. Biol. Chem., Vol. 283, Issue 7, 3743-3750, February 15, 2008

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Characterization of Some Molecular Mechanisms Governing Autoactivation of the Catalytic Domain of the Anaplastic Lymphoma Kinase^*

Carmen J. Tartari¹, Rosalind H. Gunby, Addolorata M. L. Coluccia, Roberta Sottocornola, Barbara Cimbro, Leonardo Scapozza^¶, Arianna Donella-Deana^||, Lorenzo A. Pinna^||, and Carlo Gambacorti-Passerini^**

From the Department of Clinical and Prevention Medicine, University of Milano-Bicocca, via Cadore 48, Monza 20052, Italy, the Department of Experimental Oncology, National Cancer Institute Foundation, 20133 Milan, the ^¶School of Pharmaceutical Sciences, University of Lausanne, University of Geneva, CH-1211 Geneva, Switzerland, the ^||Department of Biological Chemistry, University of Padua, and Venetian Institute for Molecular Medicine, 35122 Padua, Italy, and the ^**Department of Internal Medicine, McGill University, Montreal H3A2T5, Canada

NPM/ALK is an oncogenic fusion protein expressed in 50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr Phe) three potential autophosphorylation sites contained in the "YXXXYY" motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.

Received for publication, July 24, 2007 , and in revised form, November 27, 2007.

^* This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Min. San. Ricerca Finalizzata (2003); the CNRS and Ministry of Education, University, and Research-COFIN and PRIN programs (2003, 2004, and 2005); Fondazione Cariplo; the European Union (Prokinase Network 503467); the Canadian Fund for Innovation; and the National Cancer Institute of Canada. 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 To whom correspondence should be addressed: Tel.: 39-02-6448-8362; Fax: 39-02-6448-8363; E-mail: carmen.tartari{at}unimib.it.