J. Neurochem. (2012) 120, 684–698.

cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di‐butyric cAMP (db‐cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db‐cAMP up‐ and down‐regulates GTP‐RhoA levels in PC12 cells in a time‐dependent manner. Tat‐C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)‐RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db‐cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db‐cAMP increases GTP‐Rap1 levels, and dominant negative (DN)‐Rap1 and DN‐Rap‐dependent RhoGAP (ARAP3) block neurite outgrowth induced by db‐cAMP. DN‐p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c‐Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db‐cAMP.