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Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity

Donella-Deana, Arianna
Marin, Oriano
Cesaro, Luca
Gunby, Rosalind H.
Ferrarese, Anna
Coluccia, Addolorata M. L.
Tartari, Carmen J.
Mologni, Luca
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Published in Biochemistry. 2005, vol. 44, no. 23, p. 8533-8542
Abstract The anaplastic lymphoma kinase (ALK), whose constitutively active fusion proteins are responsible for 5-10% of non-Hodgkin's lymphomas, shares with the other members of the insulin receptor kinase (IRK) subfamily an activation loop (A-loop) with the triple tyrosine motif Y-x-x-x-Y-Y. However, the amino acid sequence of the ALK A-loop differs significantly from the sequences of both the IRK A-loop and the consensus A-loop for this kinase subfamily. A major difference is the presence of a unique "RAS" triplet between the first and second tyrosines of the ALK A-loop, which in IRK is replaced by "ETD". Here we show that a peptide reproducing the A-loop of ALK is readily phosphorylated by ALK, while a homologous IRK A-loop peptide is not unless its "ETD" triplet is substituted by "RAS". Phosphorylation occurs almost exclusively at the first tyrosine of the Y-x-x-x-Y-Y motif, as judged by Edman analysis of the phosphoradiolabeled product. Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY). A number of substitutions in the YFF peptide outlined the importance of Ile and Arg at positions n - 1 and n + 6 in addition to the central triplet, to ensure efficient phosphorylation by ALK. Such a peculiar substrate specificity allows the specific monitoring of ALK activity in crude extracts of NPM-ALK positive cells, using the YFF peptide, which is only marginally phosphorylated by a number of other tyrosine kinases.
Keywords Amino Acid SequenceAmino Acid SubstitutionCell LineTumorEnzyme ActivationHumansLymphomaLarge-CellAnaplastic/enzymologyMolecular Sequence DataOligopeptides/chemical synthesisPeptides/chemical synthesisPhosphorylationProtein StructureTertiaryProtein-Tyrosine Kinases/chemistry/metabolismReceptor Protein-Tyrosine Kinases/chemistry/metabolismSubstrate SpecificityTyrosine/metabolism
PMID: 15938644
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DONELLA-DEANA, Arianna et al. Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity. In: Biochemistry, 2005, vol. 44, n° 23, p. 8533-8542. doi: 10.1021/bi0472954 https://archive-ouverte.unige.ch/unige:81203

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Deposited on : 2016-03-02

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