Article

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 Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY).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

0 Followers
 · 
199 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.
    Cell Reports 08/2014; 8(5). DOI:10.1016/j.celrep.2014.07.036 · 7.21 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1,596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples—at three hot spots and 13 minor sites—and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from nononcogenic mutations and allowed robust computational prediction of their effects. The mutated variants also showed differential in vitro crizotinib sensitivities. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma and will allow tailoring of ALK-targeted therapy to specific mutations.
    Cancer Cell 11/2014; 26(5):682-694. DOI:10.1016/j.ccell.2014.09.019 · 23.89 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: Anaplastic lymphoma kinase (ALK), a tyrosine kinase receptor, has been initially identified through its involvement in chromosomal translocations associated with anaplastic large cell lymphoma. However, recent evidence that aberrant ALK activity is also involved in an expanding number of tumor types, such as other lymphomas, inflammatory myofibroblastic tumor, neuroblastomas and some carcinomas, including non-small cell lung carcinomas, is boosting research progress in ALK-targeted therapies. Areas covered: The first aim of this review is to describe current understandings about the ALK tyrosine kinase and its implication in the oncogenesis of human cancers as a fusion protein or through mutations. The second goal is to discuss its interest as a therapeutic target and to provide a review of the literature regarding ALK inhibitors. Mechanisms of acquired resistance are also reviewed. Expert opinion: Several ALK inhibitors have recently been developed, offering new treatment options in tumors driven by abnormal ALK signaling. However, as observed with other tyrosine kinase inhibitors, resistance has emerged in patients treated with these agents. The complexity of mechanisms of acquired resistance recently described suggests that other therapeutic options, including combination of ALK and other kinases targeted drugs, will be required in the future.
    Expert Opinion on Therapeutic Targets 10/2012; 16(11). DOI:10.1517/14728222.2012.719498 · 4.90 Impact Factor

Full-text

Download
71 Downloads
Available from
May 20, 2014