Biochemistry, 2005, 44(23):8533-42
Donella-Deana A, Marin O, Cesaro L, Gunby RH, Ferrarese A, Tartari C,
Mologni L, Scapozza L, Gambacorti-Passerini C, Pinna L.
Department of Biochemistry, University of
Padova, Viale G. Colombo 3, 35121 Padova, Italy.
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had been replaced by phenylalanine (FYY) was almost unaffected by ALK.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contrast, a peptide in which the second and third tyrosines had been replaced by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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