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 Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY).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 He 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. © 2005 American Chemical Society.

Donella deana, A., Marin, O., Cesaro, L., Gunby, R., Ferrarese, A., Tartari, C., et al. (2005). Unique substrate specificity of Anaplastic Lymphoma Kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity. BIOCHEMISTRY, 44(23), 8533-8544 [10.1021/bi0472954].

Unique substrate specificity of Anaplastic Lymphoma Kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity

MOLOGNI, LUCA;GAMBACORTI PASSERINI, CARLO;
2005

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 He 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. © 2005 American Chemical Society.
Articolo in rivista - Articolo scientifico
Scientifica
alk, anaplastic lymphoma
English
Donella deana, A., Marin, O., Cesaro, L., Gunby, R., Ferrarese, A., Tartari, C., et al. (2005). Unique substrate specificity of Anaplastic Lymphoma Kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity. BIOCHEMISTRY, 44(23), 8533-8544 [10.1021/bi0472954].
Donella deana, A; Marin, O; Cesaro, L; Gunby, R; Ferrarese, A; Tartari, C; Mologni, L; Scapozza, L; GAMBACORTI PASSERINI, C; Pinna, L
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/15010
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