Stimulation of M-3 muscarinic receptors induces phosphorylation of the Cdc42 effecter activated Cdc42Hs-associated kinase-1 via a Fyn tyrosine kinasesignaling pathway

The tyrosine kinase, activated Cdc42Hs-associated kinase-1 (ACR-1), is a sp ecific effector of the Rho family GTPase Cdc42. GTP-bound Cdc42 has been sh own to facilitate neurite outgrowth elicited by activation of muscarinic ch olinergic receptors (mAChRs). Because tyrosine kinase activity is a require ment for neuritogenesis in several cell systems, we investigated whether en dogenous mAChRs (principally of the M-3 subtype) expressed in human SN-SY5Y neuroblastoma cells would signal to ACK-1. Incubation of cells with the ch olinergic agonist oxotremorine-M (Oxo-M) induced an approximately 6-fold in crease in the tyrosine phosphorylation of ACK-1 which was inhibited by atro pine. ACK-1 phosphorylation was blocked by Clostridium difficile toxin B, a n inhibitor of Rho family GTPases. In contrast, disruption of the actin cyt oskeleton with cytochalasin D stimulated ACK-1 phosphorylation, and moreove r, addition of Oxo-M 60 cells preincubated with this agent elicited a furth er increase in phosphorylation, indicating that an intact cytoskeleton is n ot required for mAChR signaling to ACH-1. Although stimulation of M-3 mAChR s induces bath an increase in intracellular Ca2+ and activation of protein kinase C (PKC), neither of these second messenger pathways was required for receptor-stimulated ACK-1 phosphorylation. Instead, inhibition of PKC resu lted in a 2-fold increase in Oxo-M-stimulated ACK-1 phosphorylation, wherea s acute activation of PRC with phorbol ester decreased ACK-1 phosphorylatio n. The agonist-induced tyrosine phosphorylation of ACK-1 was blocked by inh ibitors of Src family kinases, and ACK-1 was coprecipitated with Fyn (but n ot Src) in an agonist-dependent manner. Finally, scrape loading cells with glutathione S-transferase fusion proteins of either the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR-stimulated ACK-1 tyrosine phosphoryla tion, The data are the first to shaw phosphorylation of ACK-1 after stimula tion of a receptor coupled to neurite outgrowth and indicate that a Rho fam ily GTPase (i.e, Cdc42) and Fyn are essential upstream elements of this sig naling pathway.