Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity.
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 Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY).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.
Donella-Deana
Arianna
A
Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.
Marin
Oriano
O
Cesaro
Luca
L
Gunby
Rosalind H
RH
Ferrarese
Anna
A
Coluccia
Addolorata M L
AM
Tartari
Carmen J
CJ
Mologni
Luca
L
Scapozza
Leonardo
L
Gambacorti-Passerini
Carlo
C
Pinna
Lorenzo A
LA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000077548:Anaplastic Lymphoma Kinase; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D006801:Humans; D017728:Lymphoma, Large-Cell, Anaplastic / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010455:Peptides / Q000138:chemical synthesis*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism
D009842:Oligopeptides; D010455:Peptides; D014443:Tyrosine; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases