Characterization of some molecular mechanisms governing autoactivation of the catalytic domain of the anaplastic lymphoma kinase.
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.
Tartari
Carmen J
CJ
Department of Clinical and Prevention Medicine, University of Milano-Bicocca, via Cadore 48, Monza 20052, Italy. carmen.tartari@unimib.it
Gunby
Rosalind H
RH
Coluccia
Addolorata M L
AM
Sottocornola
Roberta
R
Cimbro
Barbara
B
Scapozza
Leonardo
L
Donella-Deana
Arianna
A
Pinna
Lorenzo A
LA
Gambacorti-Passerini
Carlo
C
D000595:Amino Acid Sequence; D000077548:Anaplastic Lymphoma Kinase; D015153:Blotting, Western; D020134:Catalytic Domain; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases