pervanadate and Neuroblastoma

The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.

Topics: Cell Differentiation; Cell Line, Tumor; Colforsin; Golgi Apparatus; Guanine Nucleotide-Releasing Factor 2; HEK293 Cells; Humans; Mutant Proteins; Neurites; Neuroblastoma; Phosphorylation; Phosphotyrosine; Protein Binding; Protein Structure, Tertiary; Protein Transport; Protein Tyrosine Phosphatase, Non-Receptor Type 2; src-Family Kinases; Structure-Activity Relationship; Vanadates

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimer's disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimer's disease.

Topics: Aged, 80 and over; Alzheimer Disease; Amino Acid Sequence; Amino Acid Substitution; Animals; Brain; Brain Chemistry; Cell Line, Tumor; Chlorocebus aethiops; CHO Cells; COS Cells; Cricetinae; Cricetulus; Female; Fetal Proteins; Humans; Mass Spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Nerve Tissue Proteins; Neuroblastoma; Neurofibrillary Tangles; Neurons; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-abl; Proto-Oncogene Proteins c-fyn; src-Family Kinases; tau Proteins; Transfection; Vanadates

beta-Amyloid precursor protein (APP) is a widely expressed transmembrane protein of unknown function that is involved in the pathogenesis of Alzheimer's disease. The cytoplasmic tail of APP interacts with phosphotyrosine binding (PTB) domain containing proteins (Fe65, X11, mDab-1, and JIP-1) and may modulate gene expression and apoptosis. We now identify Shc A and Shc C, PTB-containing adapter proteins that signal to cellular differentiation and survival pathways, as novel APP-interacting proteins. The APP cytoplasmic tail contains a PTB-binding motif (Y(682)ENPTY(687)) that, when phosphorylated on Tyr(682), precipitated the PTB domain of Shc A and Shc C, as well as endogenous full-length Shc A. APP and Shc C were physically associated in adult mouse brain homogenates. Increase in phosphorylation of APP by overexpression of the nerve growth factor receptor Trk A in 293T cells promoted the interaction of transfected APP and endogenous Shc A. Pervanadate treatment of N2a neuroblastoma cells resulted in tyrosine phosphorylation and association of endogenous APP and Shc A. Thus, APP and Shc proteins interact in vitro, in cells, and in the mouse brain. Tyrosine phosphorylation of APP may promote the interaction with Shc proteins.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Amino Acid Motifs; Amyloid beta-Protein Precursor; Animals; Apoptosis; Binding Sites; Brain; Cell Differentiation; Cell Line; Cell Membrane; COS Cells; Cytoplasm; Enzyme Inhibitors; Glutathione Transferase; Humans; Immunoblotting; Mice; Mice, Inbred BALB C; Models, Biological; Nerve Tissue Proteins; Neuroblastoma; Neuropeptides; Phosphorylation; Precipitin Tests; Protein Binding; Protein Biosynthesis; Protein Structure, Tertiary; Proteins; Receptor, trkA; Recombinant Fusion Proteins; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Src Homology 2 Domain-Containing, Transforming Protein 3; Transcription, Genetic; Tyrosine; Vanadates

The ectodomain of certain transmembrane molecules can be released by proteolysis, and the solubilized antigens often exert important biological functions. We demonstrated before that the L1 adhesion molecule is shed from the cell surface. Here we show that L1 release in AR breast carcinoma cells is mediated by a member of the disintegrin metalloproteinase (ADAM) family of proteinases. Up-regulation of L1 shedding by phorbol ester or pervanadate involved distinct mechanisms. Pervanadate induced shedding and rounding-up of cells from the substrate, which was blocked by the Src kinase inhibitor PP2. Tyr phosphorylation of the L1 cytoplasmic tail and the Src kinase Fyn was observed following pervanadate treatment. Up-regulation of L1 release and activation of Fyn occurred also when cells were detached by EDTA suggesting that the regulation of L1 shedding by this pathway was linked to cell morphology and adhesion. The phorbol 12-myristate 13-acetate-induced shedding was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway. Soluble L1 binds to the proteoglycan neurocan and in bound form could support integrin-mediated cell adhesion and migration. We propose that the release of cell-associated adhesion molecules such as L1 may be relevant to promote cell migration.

Topics: Animals; Antigens, Surface; Breast Neoplasms; Cell Size; CHO Cells; Cricetinae; Disintegrins; Enzyme Inhibitors; Female; Flavonoids; Humans; Leukocyte L1 Antigen Complex; Membrane Glycoproteins; Metalloendopeptidases; Mice; Neural Cell Adhesion Molecules; Neuroblastoma; Phosphorylation; Protease Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Recombinant Proteins; Signal Transduction; src-Family Kinases; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured; Vanadates