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

Barbara Cimbro,Carmen J. Tartari,Leonardo Scapozza,Addolorata M.L. Coluccia,Lorenzo A. Pinna,Carlo Gambacorti-Passerini,Roberta Sottocornola,Rosalind H. Gunby,Arianna Donella-Deana

doi:10.1074/jbc.m706067200

Abstract

NPM/ALK is an oncogenic fusion protein expressed in approximately 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.

Highlights

The most commonly expressed anaplastic lymphoma kinase (ALK) fusion protein in anaplastic large cell lymphoma is NPM/ALK, which derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the kinase domain of ALK to the dimerization domain of nucleophosmin (NPM) [5]
In Vitro Kinase Activity of NPM/ALK Activation Loop Mutants—We systematically mutated tyrosine residues to phenylalanine in the YXXXYY motif of the NPM/ALK activation loop (A-loop) and determined the effect of these mutations on ALK activity
The absence of all three tyrosine residues in the A-loop appears to cause the destabilization of the inactive conformation leading to a partially active conformation, whose activity does not require the phosphorylation of the first tyrosine

Summary

EXPERIMENTAL PROCEDURES

Full-length human NPM/ALK cDNA, subcloned in the expression vector pcDNA3, was kindly provided by Dr S. We centrifugation, washed once with ice-cold phosphate-buffered observed that purified ALK kinase domain preferentially phos- saline, and lysed for 30 min in lysis buffer These in 1 mM NaVO4, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl vitro results suggest that the mechanism of autoregulation of fluoride, 10 ␮g/ml of Pepstatin-A, leupeptin, and aprotinin). Mutations were verified by reverse transcription-PCR and DNA sequencing

RESULTS

DISCUSSION

Pinna and Carlo Gambacorti-Passerini