Journal of neurochemistry
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Journal of neurochemistry · Mar 1999
Gö 6976 is a potent inhibitor of neurotrophin-receptor intrinsic tyrosine kinase.
We report here that addition of the protein kinase C inhibitor Gö 6976 blocked neurotrophin-induced signaling and autophosphorylation of neurotrophin-specific tyrosine kinase (Trk) receptors, either Trk B in cortical neurons or Trk A in GT1-1-trk9 cells. The effect of Gö 6976 on Trk autophosphorylation was not inhibited by 100 microM orthovanadate, suggesting that the block was not due to the activation of tyrosine phosphatases. ⋯ Gö 6976 also blocked the ability of brain-derived neurotrophic factor to promote cortical neuronal survival and the ability of nerve growth factor to promote PC12 cell survival and differentiation. These results suggest that Gö 6976, besides its known inhibitory effects on lipid- and calcium-dependent isoforms of protein kinase C, can also inhibit neurotrophin signaling by directly inhibiting the intrinsic Trk.
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Journal of neurochemistry · Feb 1999
Differential phosphorylation of syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) isoforms.
The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (alpha-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). ⋯ The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca2+ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or alpha-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.
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Journal of neurochemistry · Dec 1998
GABA synthesis in astrocytes after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase 65 or 67.
Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA. GAD vector infection resulted in isoform-specific expression of GAD as determined by western blotting and immunohistochemistry. Astrocytes infected with a beta-galactosidase vector or uninfected expressed no GAD and contained no detectable GABA. ⋯ Vector infection did not alter levels of other intracellular amino acids. GABA was tonically released from astrocytes infected with the GAD67 vector, but no increase in release could be detected after treatment of the cells with K+, veratridine, glutamate, or bradykinin. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurologic disorders associated with hyperexcitable or diminished inhibitory activity.
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Journal of neurochemistry · Nov 1998
Regulation of sensory neuron precursor proliferation by cyclic GMP-dependent protein kinase.
Cyclic GMP (cGMP) is a molecular messenger involved in diverse cellular processes. Recently, cGMP-dependent protein kinase (cGK) type II was determined to be a regulator of endochondral ossification and bone growth, identifying a role for cGMP in the regulation of cellular proliferation. Here, we demonstrate the presence of cGK type I (cGKI) in cells of the developing trigeminal ganglia. cGKI occurs in some proliferating precursors as evidenced by double labeling with an antibody to cGKI and 5-bromo-2'-deoxyuridine(BrdU) incorporation. ⋯ The cGKI inhibitor Rp-8-pCPT-cGMPS decreases proliferation and BrdU incorporation in transfected PC12 cells but has no effect on control cells. The PC12 cells do not express NOS, indicating that this effect is also independent of NOS. Thus, cGKI regulates the proliferation of sensory neurons as a result of activation of a NOS-independent pathway, representing a novel pathway by which the number of sensory neurons is regulated.
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Journal of neurochemistry · Aug 1998
p75 and TrkA receptor signaling independently regulate amyloid precursor protein mRNA expression, isoform composition, and protein secretion in PC12 cells.
The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. ⋯ These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors--in contrast to p75NTR-associated signaling--stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.