calyculin-a and Neuroblastoma

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity.

Topics: Alzheimer Disease; Animals; Cell Line, Tumor; Cyclin-Dependent Kinase 5; Cytoskeleton; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Marine Toxins; Mice; Naphthoquinones; Nerve Degeneration; Neuroblastoma; NIMA-Interacting Peptidylprolyl Isomerase; Oxazoles; Peptidylprolyl Isomerase; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 2; Purines; Rats; Receptors, GABA-A; Roscovitine; tau Proteins; Tauopathies

Nerve growth factor, retinoic acid, dibutyryl cAMP, ganglioside G(Q1b), and botulinum C3 exoenzyme were evaluated for their neural differentiating potential on human neuroblastoma GOTO cells. C3 exoenzyme is an ADP-ribosyltransferase inactivating Rho protein (a small GTP-binding protein). C3 exoenzyme caused the fastest differentiation of GOTO cells into neural cells and induced the strongest network of the cells. Fasudil, an inhibitor of Rho-kinase, induced outgrowth of the neurites in the GOTO cells. Calyculin A, an inhibitor of phosphatases including myosin phosphatase, counteracted C3 exoenzyme-induced neurite outgrowth of the cells. These findings suggest that differentiation of GOTO cells triggered by C3 exoenzyme is attained via inactivation of Rho protein, inhibition of Rho-kinase, and activation of myosin phosphatase. Because of the strong differentiating potential of C3 exoenzyme, the transduction pathway consisting of Rho protein, Rho-kinase, and myosin phosphatase seems to be main stream in the neural differentiation of GOTO cells. A single GOTO cell was observed continuously after treatment with C3 exoenzyme. The cell started to change shape from its original morphology only 15 min after treatment with C3 exoenzyme, and it was completely spherical within 60 min. Neurites appeared on the membrane of the cell 2 hr after the treatment and then gradual outgrowth began. These observations are fundamental information in elucidating the mechanism of neural differentiation, especially at an early stage.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; ADP Ribose Transferases; Botulinum Toxins; Cell Differentiation; Cell Line, Tumor; Humans; Intracellular Signaling Peptides and Proteins; Marine Toxins; Neuroblastoma; Oxazoles; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction

Hyperphosphorylation of cytoskeletal proteins seen in Alzheimer's disease is most probably the result of an imbalanced regulation in protein kinases and protein phosphatases (PP) in the affected neurons. Previous studies have revealed that PP-2A and PP-1 play important roles in the pathogenesis. Employing human neuroblastoma cells, we found that 10 nM calyculin A (CA), a selective inhibitor of PP-2A and PP-1, significantly increased phosphorylation and accumulation of neurofilament (NF) in the cells. Levels of NF-M (middle chain) and NF-L (light chain) mRNA decreased after CA treatment. Additionally, CA led to a decreased cell viability determined by MTT and crystal violet assay. Melatonin efficiently protects the cell from CA-induced alterations in NF hyperphosphorylation and accumulation, suppressed NF gene expression as well as decreased cell viability. It is concluded that inhibition of PP-2A/PP-1 by CA induces abnormalities in NF metabolism and cell survival, and melatonin efficiently arrests the lesions.

Topics: Cell Survival; Enzyme Inhibitors; Humans; Marine Toxins; Melatonin; Neuroblastoma; Neurofilament Proteins; Neuroprotective Agents; Neurotoxicity Syndromes; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Tumor Cells, Cultured

Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.

Topics: Animals; Bradykinin; Calcium; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glioma; Hybrid Cells; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxazoles; Phosphoric Monoester Hydrolases; Phosphorylation; Signal Transduction; Thapsigargin; Type C Phospholipases