The N-terminal domain of NogoA, called amino-Nogo, inhibits axonal outgrowth and cell spreading via a largely unknown mechanism. In the present study, we show that amino-Nogo decreases Rac1 activity and inhibits fibroblast spreading. 12-OTetradecanoylphorbol-13-acetate-type tumor promoters, such as phorbol 12-myristate 13-acetate (PMA) and teleocidin, increase Rac1 activity and overcome the amino-Nogo-induced inhibition of cell spreading. The stimulating effect of tumor promoters on cell spreading requires activation of protein kinase D and the subsequent activation of Akt1. Furthermore, we identified Akt1 as a new signaling component of the amino-Nogo pathway. Akt1 phosphorylation is decreased by amino-Nogo. Activation of Akt1 with a cell-permeable peptide, TAT-TCL1, blocks the amino-Nogo inhibition. Finally, we provide evidence that these signaling pathways operate in neurons in addition to fibroblasts. Our results suggest that activation of protein kinase D and Akt1 are approaches to promote axonal regeneration after injury.Injured neurons in the adult mammalian central nervous system fail to regenerate, in part due to the existence of myelin-associated inhibitors (MAIs), 2 such as NogoA, myelinassociated glycoprotein, and oligodendrocyte-myelin glycoprotein (1). Two inhibitory regions of NogoA, a C-terminal 66-amino acid region (Nogo-66) and a lengthy N-terminal region (amino-Nogo), have been characterized based on their inhibitory function on cell spreading and neurite outgrowth (2). Nogo-66 and two other major MAIs, myelin-associated glycoprotein and oligodendrocyte-myelin glycoprotein, exert their inhibitory effects by interacting with a receptor complex that contains the Nogo receptor 1 (NgR1), Lingo-1, p75 NTR , and higher order gangliosides (1) or a newly identified receptor, PirB (paired immunoglobulin-like receptor-B) (3). However, amino-Nogo restricts non-neuronal cell spreading and axonal outgrowth (2, 4) through an unknown mechanism independent of the above NgR complex (5, 6). Despite evidence supporting the existence of a specific receptor on neurons and some nonneuronal cells (5), the identity of the amino-Nogo receptor(s) remains obscure. A recent study has provided evidence that amino-Nogo can bind to and selectively block signal transduction initiated by certain integrins (7).The intracellular signaling mechanisms responsible for amino-Nogo-induced inhibition of cell spreading and regeneration are not well understood. So far, Rho GTPases, mainly RhoA and Rac1, remain the most established mediators. Amino-Nogo activates RhoA and suppresses Rac1 in primary neurons (6,8). Activation of RhoA by MAIs has been extensively studied and is generally viewed as perhaps the key convergence point at which various MAIs exert similar functional outcomes (6, 8 -12). Blockade of RhoA signaling improves axonal regeneration in both in vitro and in vivo models (13). Rac1 activity, on the other hand, is required for neurite formation and neurite outgrowth on permissive substrates (14 -17). Although one of the con...