ABSTRACT
Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor originally identified as part of the chimeric nucleophosmin-ALK protein in the t(2;5) chromosomal rearrangement associated with anaplastic large cell lymphoma. We recently demonstrated that the ALK kinase is constitutively activated by gene amplification at the ALK locus in several neuroblastoma cell lines. Forming a stable complex with hyperphosphorylated ShcC, activated ALK modifies the responsiveness of the mitogen-activated protein kinase pathway to growth factors. In the present study, the biological role of activated ALK was examined by suppressing the expression of ALK kinase in neuroblastoma cell lines using an RNA interference technique. The suppression of activated ALK in neuroblastoma cells by RNA interference significantly reduced the phosphorylation of ShcC, mitogen-activated protein kinases, and Akt, inducing rapid apoptosis in the cells. By immunohistochemical analysis, the cytoplasmic expression of ALK was detected in most of the samples of neuroblastoma tissues regardless of the stage of the tumor, whereas significant amplification of ALK was observed in only 1 of 85 cases of human neuroblastoma samples. These data demonstrate the limited frequency of ALK activation in the real progression of neuroblastoma.
Subject(s)
Neuroblastoma/enzymology , Neuroblastoma/genetics , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Anaplastic Lymphoma Kinase , Apoptosis/physiology , Blotting, Southern , Cell Line, Tumor , Child, Preschool , Enzyme Activation/physiology , Gene Amplification , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Infant , Mitogen-Activated Protein Kinases/metabolism , Neuropeptides/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , RNA Interference , Receptor Protein-Tyrosine Kinases , Reverse Transcriptase Polymerase Chain Reaction , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 3ABSTRACT
ShcC is a family member of the Shc docking proteins that possess two different phosphotyrosine-binding motifs and conduct signals as Grb2-binding substrates of various receptor tyrosine kinases. We have recently shown that some neuroblastoma cell lines, such as NB-39-nu cells, express a protein complex of hyperphosphorylated ShcC and anaplastic lymphoma kinase (ALK), which is self-activated by gene amplification. Here, we demonstrate that the expression of a mutant ShcC lacking Grb2-binding sites, 3YF-ShcC, significantly impaired the survival, differentiation and motility of NB-39-nu cells by blocking the ERK and Akt pathways. On the other hand, cells overexpressing ShcC or 3YF-ShcC, but not a mutant ShcC that lacks SH2, showed decreased anchorage independency and in vivo tumorigenicity, suggesting a novel ShcC-specific suppressive effect through its SH2 domain on cell transformation. Notably, overexpression of ShcC suppressed the sustained phosphorylation of Src family kinase after cell detachment, which might be independent of phosphorylation of Grb2-binding site. It was indicated that the Src/Fyn-Cas pathway is modulated as a target of these suppressive effects by ShcC. Reciprocal change of ShcC expression and phosphorylation observed in malignant neuroblastoma cell lines might be explained by these phosphotyrosine-dependent and -independent functions of ShcC.
Subject(s)
Neuroblastoma/metabolism , Neuropeptides/physiology , Agar/chemistry , Animals , Apoptosis , Cell Adhesion , Cell Differentiation , Cell Line, Tumor , Cell Movement , Cell Proliferation , Genes, Dominant , Humans , Immunoblotting , Immunohistochemistry , In Situ Nick-End Labeling , Mice , Mice, Nude , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mutation , Neoplasm Invasiveness , Neoplasm Transplantation , Neuropeptides/metabolism , Phosphorylation , Phosphotyrosine/chemistry , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 3 , Thymidine/chemistry , Time Factors , Transfection , Tretinoin/metabolism , Wound Healing , src Homology DomainsABSTRACT
Shc family of docking proteins, ShcA, ShcB and ShcC, play roles in cellular signal transduction by binding to phosphotyrosine residues of various activated receptor tyrosine kinases. Both ShcB and ShcC proteins are selectively expressed in the neural system of adult mouse tissues. In most of neuroblastoma cells, obvious tyrosine phosphorylation of ShcC was observed, whereas expression of ShcB was considerably low. Phosphoproteins associated with hyperphosphorylated ShcC were purified from neuroblastoma cell lines, and identified by mass-spectrometry. Anaplastic lymphoma kinase (ALK), which turned out to be one of these phosphoproteins, was constitutively activated and associated with the PTB domain of ShcC in three neuroblastoma cells. In vitro kinase assay revealed that ShcC is a potent substrate of the activated ALK kinase. The ALK gene locus was significantly amplified in both of these cell lines, suggesting that gene amplification leads to constitutive activation of the ALK kinase, which results in hyperphosphorylation of ShcC. Constitutive activation of ALK appeared to interfere with signals from other receptor tyrosine kinases. ALK-ShcC signal activation, possibly caused by co-amplification with the N-myc gene, might give additional effects on malignant tumor progression of neuroblastoma.
