Anthony W. M. Cheng

ATP acts via P2Y1 receptors to stimulate acetylcholinesterase and acetylcholine receptor expression: transduction and transcription control.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter–reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

ATP induces post-synaptic gene expressions in vertebrate skeletal neuromuscular junctions

In vertebrate neuromuscular junctions (nmjs), adenosine 5′-triphosphate (ATP) is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Several lines of evidence suggest that the synaptic ATP can act as a synapse-organizing factor at the nmjs, mediated by metabotropic P2Y1 receptors. P2Y1 receptor mRNAs in chicken and rat muscles are low in embryo but increases markedly in the adult, and decreased after denervation. The P2Y1 receptor protein is restricted to the nmjs and co-localized with AChRs in adult muscles. The activation of P2Y1 receptor by adenine nucleotides in cultured chick myotubes stimulated the accumulation of inositol phosphates, intracellular Ca2+ mobilization, protein kinase C activity and phosphorylation of extracellular signal-regulated kinases. The receptor activation led to an increase in the expression of transcripts encoding AChE catalytic subunit and AChR subunits. The ATP-induced post-synaptic gene expression is possibly mediated by the activation of signaling cascades of mitogen-activated protein kinase. Therefore, a model is being proposed here that the synaptic ATP has a role of synergy with other regulatory signals, such as neuregulin, which act via their post-synaptic receptors to activate second signaling molecules locally to enhance the transcription of AChR/AChE genes specifically in the adjacent sub-synaptic nuclei during the formation and, especially, the maintenance of post-synaptic specializations at the nmjs.

B-raf is critical for mapk activation during mitosis and is regulated in an M-phase dependent manner in xenopus egg extracts

Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. Here we purified and characterized the MEK kinase activity present in Xenopus M phase-arrested egg extracts. Our results show that B-Raf was the critical MEK kinase required for M phase activation of the MAPK pathway. Consistent with this, B-Raf was activated and underwent hyperphosphorylation in an M phase-dependent manner. Interestingly B-Raf hyperphosphorylation at mitosis occurred, at least in part, as a consequence of a feedback loop involving MAPK-mediated phosphorylation within a conserved C-terminal SPKTP motif. The kinase activity of a B-Raf mutant defective at both phosphorylation sites was substantially greater than its wild type counterpart when incubated in Xenopus M phase egg extracts. Furthermore suppression of MAPK feedback at mitosis enhanced B-Raf activity, whereas constitutive activation of MAPK at mitosis strongly suppressed B-Raf activity. These results suggest that feedback phosphorylation by MAPK negatively regulates B-Raf activity at mitosis. Collectively our data demonstrate for the first time a role for B-Raf at mitosis and provide new insight into understanding the regulation and function of B-Raf during cell proliferation.

Three N-Glycosylation Sites of Human Acetylcholinesterase Shares Similar Glycan Composition

Acetylcholinesterase (AChE; EC 3.1.1.7) is a glycoprotein possessing three conserved N-linked glycosylation sites in mammalian species, locating at 296, 381, and 495 residues of the human sequence. Several lines of evidence demonstrated that N-glycosylation of AChE affected the enzymatic activity, as well as its biosynthesis. In order to determine the role of three N-glycosylation sites in AChE activity and glycan composition, the site-directed mutagenesis of N-glycosylation sites in wild-type human AChET sequence was employed to generate the single-site mutants (i.e., AChETN296Q, AChETN381Q, and AChETN495Q) and all site mutant (i.e., AChET3N→3Q). The mutation did not affect AChE protein expression in the transfected cells. The mutants, AChET3N→3Q and AChETN381Q, showed very minimal enzymatic activity, while the other mutants showed reduced activity. By binding to lectins, Con A, and SNA, the glycosylation profile was revealed in those mutated AChE. The binding affinity with lectins showed no significant difference between various N-glycosylation mutants, which suggested that similar glycan composition should be resulted from different N-glycosylation sites. Although the three glycosylation sites within AChE sequence have different extent in affecting the enzymatic activity, their glycan compositions are very similar.

Transcriptional activity of acetylcholinesterase gene is regulated by DNA methylation during C2C12 myogenesis.

The expression of acetylcholinesterase (AChE), an enzyme hydrolyzes neurotransmitter acetylcholine at vertebrate neuromuscular junction, is regulated during myogenesis, indicating the significance of muscle intrinsic factors in controlling the enzyme expression. DNA methylation is essential for temporal control of myogenic gene expression during myogenesis; however, its role in AChE regulation is not known. The promoter of vertebrate ACHE gene carries highly conserved CG-rich regions, implying its likeliness to be methylated for epigenetic regulation. A DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), was applied onto C2C12 cells throughout the myotube formation. When DNA methylation was inhibited, the promoter activity, transcript expression and enzymatic activity of AChE were markedly increased after day 3 of differentiation, which indicated the putative role of DNA methylation. By bisulfite pyrosequencing, the overall methylation rate was found to peak at day 3 during C2C12 cell differentiation; a SP1 site located at -1826bp upstream of mouse ACHE gene was revealed to be heavily methylated. The involvement of transcriptional factor SP1 in epigenetic regulation of AChE was illustrated here: (i) the SP1-driven transcriptional activity was increased in 5-Aza-treated C2C12 culture; (ii) the binding of SP1 onto the SP1 site of ACHE gene was fully blocked by the DNA methylation; and (iii) the sequence flanking SP1 sites of ACHE gene was precipitated by chromatin immuno-precipitation assay. The findings suggested the role of DNA methylation on AChE transcriptional regulation and provided insight in elucidating the DNA methylation-mediated regulatory mechanism on AChE expression during muscle differentiation.