c-Src protein tyrosine kinase activity is required for muscarinic receptor-mediated DNA synthesis and neurogenesis via ERK1/2 and c-AMP-responsive element-binding protein signaling in neural precursor cells.
The G protein-coupled muscarinic acetylcholine receptor (mAChR) isoforms have been identified in neural stem/progenitor (or precursor) cells. In previous studies, activation of these receptors induced elevations in intracellular Ca(2+) signals and mitogen-activated protein (MAP) kinase activity that led to enhanced DNA synthesis along with neurogenesis in neural precursor cells. Here we report that the nonreceptor protein tyrosine kinase c-src activity is required for the muscarinic receptor-activated MAP kinase and cAMP-responsive element-binding protein (CREB). Stimulation of neural precursor cells dissociated from embryonic day 13 rat cortical neuroepithelium with the muscarinic receptor agonist carbachol (CCh) induced phosphorylations of c-src that were detected by antibodies raised against phospho-Tyr416 (Ptyr416), phospho-Tyr527 (Ptyr527), and phospho-Tyr215 (Ptyr215) of the kinase. Although an increase in Ptyr416 suggested direct activation of c-src, Ptyr215 may serve as an alternative mechanism underlying activation of c-src without dephosphorylation of Ptyr-527. Both extracellular signal-regulated kinase (Erk1/2) and CREB were significantly activated after CCh treatment indicated by increases in phosphorylation of these two proteins. The c-Src inhibitor PP1 abolished the CCh-induced activation of Erk1/2 and CREB in a dose-dependent manner. Moreover, CCh stimulated expression of the neuronal specific marker MAP2, which was inhibited by PP1. Cell proliferation assays and immunocytochemistry revealed that PP1 inhibited the CCh-induced DNA synthesis and MAP2(+) production. These results suggest that c-src activity is essential for the muscarinic receptor-mediated proliferation and neurogenesis in neural precursor cells via Erk1/2 and CREB signaling pathways.
Zhao
Wei-Qin
WQ
Blanchette Rockefeller Neurosciences Institute, Rockville, Maryland 20850, USA. zhaow@brni-jhu.org
Alkon
Daniel L
DL
Ma
Wu
W
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000254:growth & development; D018679:Cholinergic Agonists / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism
D018679:Cholinergic Agonists; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D011976:Receptors, Muscarinic; D002217:Carbachol; D004247:DNA; D016392:Proto-Oncogene Proteins pp60(c-src); D020928:Mitogen-Activated Protein Kinases