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. © 2003 Wiley‐Liss, Inc.