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

Biochemistry. 2005 Jun 14;44(23):8533-42. doi: 10.1021/bi0472954.


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 Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY).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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Anaplastic Lymphoma Kinase
  • Cell Line, Tumor
  • Enzyme Activation
  • Humans
  • Lymphoma, Large-Cell, Anaplastic / enzymology
  • Molecular Sequence Data
  • Oligopeptides / chemical synthesis
  • Peptides / chemical synthesis*
  • Phosphorylation
  • Protein Structure, Tertiary
  • Protein-Tyrosine Kinases / chemistry*
  • Protein-Tyrosine Kinases / metabolism*
  • Receptor Protein-Tyrosine Kinases / chemistry
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Substrate Specificity
  • Tyrosine / metabolism


  • Oligopeptides
  • Peptides
  • Tyrosine
  • ALK protein, human
  • Anaplastic Lymphoma Kinase
  • Protein-Tyrosine Kinases
  • Receptor Protein-Tyrosine Kinases