Unique Substrate Specificity of Anaplastic Lymphoma Kinase (ALK): Development of Phosphoacceptor Peptides for the Assay of ALK Activity †

Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.
Biochemistry (Impact Factor: 3.02). 07/2005; 44(23):8533-42. DOI: 10.1021/bi0472954
Source: PubMed

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 Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY).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.

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Available from: Luca Mologni, Sep 26, 2015
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    • "Tyr kinases substrates are depicted in blue and those for S/T kinases in red. A table of kinases and their K M values (Donella-Deana et al., 2005; Fan et al., 2005; Sarno et al., 1996; Ubersax et al., 2003). (C) Distribution of modified lysine residues over the protein sequence (intervals [À10;À5], [À5;0], [0;5], [5;10]) compared to random occurrences (dashed lines) plotted separately for S, T, and Y. "
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    Cell Reports 08/2014; 8(5). DOI:10.1016/j.celrep.2014.07.036 · 8.36 Impact Factor
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