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

Abstract

NPM/ALK is an oncogenic fusion protein expressed in ϳ50% 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 3 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. The anaplastic lymphoma kinase (ALK)2 is a receptor tyrosine kinase that belongs to the insulin receptor (IR) kinase subfamily of receptor tyrosine kinases (1). ALK is normally expressed in both the central and peripheral nervous systems during embryogenesis (2, 3). Expression of ALK is down-regulated before birth and in the adult ALK expression is restricted to specific cells in the central and peripheral nervous system (4). However, ALK is also aberrantly expressed in cancer as a result of chromosomal rearrangements involving the alk gene located on the 2p23 chromosome. ALK fusion proteins have been detected in anaplastic large cell lymphoma, diffuse large B-cell lymphoma, and in inflammatory myofibroblastic tumors (5-7).The most commonly expressed 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). Dimerization of NPM/ALK stimulates autophosphorylation and activation of the ALK kinase domain (8, 9) and, subsequently, phosphorylation of other tyrosine residues within NPM/ALK that are important for the activation of oncogenic signaling pathways. Phosphorylated tyrosine residues act as docking sites for SH2 and PTB domains contained within signaling molecules or adap...

Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells

“…The following primers were used: rat MYCN (forward 5 0 -TGCCAACAGTGGCGAGCA-3 0 ; reverse autoactivation of the ALK kinase domain and the transformation ability of NPM-ALK. 40 In response to treatment with crizotinib tyrosine phosphorylation of Y1278 was clearly abrogated (Figures 3a-c, lane 2). We observed identical results when employing NVP-TAE684 as inhibitor (data not shown).…”

The R1275Q Neuroblastoma Mutant and Certain ATP-competitive Inhibitors Stabilize Alternative Activation Loop Conformations of Anaplastic Lymphoma Kinase

“…Interactions of the A-loop ␣-helix with both the N-terminal and C-terminal lobes of the kinase and a hydrogen bond between Tyr 1278 and Cys 1097 from the N-terminal ␤-turn motif serve to stabilize the observed conformation. The fact that Tyr 1278 is phosphorylated upon formation of fully activated ALK underscores the inactive nature of the observed structures (40,41). The fully activated ALK kinase is expected to resemble the activated form of the insulin receptor kinase (IRK), the structure of which has been reported previously using the Tris-phosphorylated IRK kinase domain crystallized with a substrate peptide and an ATP analog (42).…”

“…The fact that Tyr 1278 makes no specific hydrogen bonding interactions in this structure and is adjacent to the beginning of the disordered region of the A-loop is clearly suggestive of more facile autophosphorylation of this residue. As previous studies have shown that Tyr 1278 , the first residue in the activation loop YXXXYY motif, is a key driver of ALK activation, the R1275Q structure provides a structural rationale of the activating nature of this mutant (40,41).…”

Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry

“…It is well established that inappropriate activation of various tyrosine kinases represents one of the important mechanisms underlying tumorigenesis (1,2). NPM-ALK, an oncogenic fusion protein found exclusively in a subset of ALK-positive anaplastic large cell lymphoma, is believed to promote tumorigenesis via its constitutively active tyrosine kinase (3).…”