guanosine-triphosphate and staurosporine-aglycone

Dendritic arborization of neurons is regulated by brain-derived neurotrophic factor (BDNF) together with its receptor, TrkB. Endocytosis is required for dendritic branching and regulates TrkB signaling, but how postendocytic trafficking determines the neuronal response to BDNF is not well understood. The monomeric GTPase Rab11 regulates the dynamics of recycling endosomes and local delivery of receptors to specific dendritic compartments. We investigated whether Rab11-dependent trafficking of TrkB in dendrites regulates BDNF-induced dendritic branching in rat hippocampal neurons. We report that TrkB in dendrites is a cargo for Rab11 endosomes and that both Rab11 and its effector, MyoVb, are required for BDNF/TrkB-induced dendritic branching. In addition, BDNF induces the accumulation of Rab11-positive endosomes and GTP-bound Rab11 in dendrites and the expression of a constitutively active mutant of Rab11 is sufficient to increase dendritic branching by increasing TrkB localization in dendrites and enhancing sensitization to endogenous BDNF. We propose that Rab11-dependent dendritic recycling provides a mechanism to retain TrkB in dendrites and to increase local signaling to regulate arborization.

Topics: Analysis of Variance; Animals; Antibodies; Brain-Derived Neurotrophic Factor; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cells, Cultured; Dendrites; Embryo, Mammalian; Endocytosis; Endosomes; Enzyme Inhibitors; Female; Gene Expression Regulation; Green Fluorescent Proteins; GTP-Binding Proteins; Guanosine Triphosphate; Hippocampus; Indole Alkaloids; Male; Microscopy, Confocal; Microtubule-Associated Proteins; Mutation; Myosins; Neurons; Rats; Receptor, trkB; RNA, Small Interfering; Thiazolidines; Transfection

Extracellular adenosine triphosphate (ATPo) has been suggested to play a role in lymphocyte effector functions. Recently, it has been suggested that MgATP2- may be the molecular species which is involved in modulating the lytic interaction between cytotoxic T-lymphocytes (CTL) and their target cells. In this study, we provide evidence that ATPo mediates the phosphorylation of extracellular proteins on T-lymphocytes through the action of ectoprotein kinases. The ectophosphorylation is temperature-dependent, supported by Mg2+ and Mn2+, and both ATP and GTP, whereas kinase activity and/or substrates were removed by pretreatment of intact lymphocytes with trypsin. We show the presence of extracellular ATP/GTP-binding sites, indicating the presence of ectoenzymes on intact lymphocytes. The major ectoprotein kinase was identified as a casein kinase II-like protein kinase and could be inhibited by heparin, whereas its activity was enhanced by spermine. The ectoprotein kinase showed remarkable substrate specificity, phosphorylating the serum protein vitronectin, but not fibronectin. In experiments with the cell-impermeable protein kinase inhibitor K-252b, we demonstrate the possible functional importance of ectoprotein kinase in CTL-mediated cytotoxicity, i.e., target cell death was completely blocked by K-252b without affecting intracellular phosphorylation. These results suggest that ectoprotein phosphorylation may possibly be an important event in immunologically relevant cell-cell interactions.

Topics: Adenosine Triphosphate; Animals; Carbazoles; Casein Kinase II; Cations; Cell Membrane; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Enzyme Inhibitors; Guanosine Triphosphate; Indole Alkaloids; Membrane Proteins; Mice; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Substrate Specificity; T-Lymphocytes, Cytotoxic

The ras gene product (p21) is thought to transduce signals from various growth and differentiation factors. p21 is a GTP-binding protein, and its activity is regulated by the bound GDP/GTP ratio. We analysed p21-bound nucleotides in cell lysates of rat pheochromocytoma cell line PC12 cells stimulated with various factors. Nerve growth factors (NGF) rapidly increased the relative amount of active p21-GTP complex to as much as 20% of the total amount of p21 within 2 min. The amount of p21-GTP then declined to 8% after 10 min, and this level was sustained for at least 2 h. Epidermal growth factor (EGF) also stimulated a rapid accumulation of p21-GTP to the same extent as seen with NGF, but the amount of p21-GTP declined to 5% after 10 min and gradually returned to the basal level within 60 min. In contrast, basic fibroblast growth factor, interleukin 6 and dibutyryl cAMP, which induce neuronal differentiation of PC12 cells, did not stimulate the accumulation of p21-GTP at any time point examined. Phorbol 12-myristate 13-acetate also had no effect. Interestingly, the protein kinase inhibitor K-252a specifically suppressed the NGF-induced accumulation of p21-GTP, but did not suppress the EGF-induced response. These results strongly suggest that an active p21-GTP complex transduces the differentiation signal from NGF. It may also be suggested that the process of activating p21 is mediated by a K-252a-sensitive protein kinase(s).

Topics: Animals; Carbazoles; Epidermal Growth Factor; GTP-Binding Proteins; Guanosine Triphosphate; In Vitro Techniques; Indole Alkaloids; Nerve Growth Factors; PC12 Cells; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); Rats; Signal Transduction

Activation of p21ras, demonstrated directly as an increase in p21ras-associated GTP, was induced rapidly but transiently by both nerve growth factor (NGF) and epidermal growth factor (EGF) in PC12 cells. The factors activate p21ras to equal extents and with virtually identical time courses. Growth factor-induced p21ras activation and tyrosine phosphorylation have similar time courses and sensitivities to genistein inhibition, indicating that p21ras activation is a result of tyrosine kinase activity. Furthermore, PC12 mutants lacking the Trk NGF receptor tyrosine kinase also lack NGF-inducible p21ras activation. The protein kinase inhibitor K252a and the methyltransferase inhibitor MTA abolish NGF-induced, but not EGF-induced, p21ras activation--effects correlated with inhibition only of NGF-induced tyrosine phosphorylation. In spite of differences in sensitivity to genistein, MTA, and K252a, EGF- and NGF-stimulated p21ras activation are not additive, implying that they do share at least one step in common.

Topics: Animals; Carbazoles; Epidermal Growth Factor; Genistein; Guanosine Diphosphate; Guanosine Triphosphate; Indole Alkaloids; Isoflavones; Kinetics; Nerve Growth Factors; PC12 Cells; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Tyrosine