Thus, a nonspecific manipulation of UPS-dependent protein degradation could markedly influence both spontaneous and polarizing factor-induced axon formation. As described below, more specific manipulation of UPS via changes in the E3 ligase activity provides more specific dissection of the proteins involved in axon

formation. We then examined the effects of BDNF or dibutyryl(db)-cAMP, a membrane permeant analog of cAMP, on the UPS-dependent degradation of five proteins that are known to be involved in axon differentiation and growth: partitioning-defective 6 (Par6), atypical protein kinase C (aPKC), Akt/PKB, Bortezomib chemical structure liver kinase B1 (LKB1), and small GTPase RhoA (Arimura and Kaibuchi, 2007, Barnes and Polleux, 2009, Shelly et al., 2007 and Yuan et al., 2003). We found that 10 hr incubation of hippocampal neurons with BDNF (50 ng/ml) or db-cAMP (20 μM) selectively increased the level of Par6 and LKB1 as well as decreased the level of RhoA, without affecting that of aPKC and Akt (Figure 1A). On the other hand, general inhibition of UPS with MG132 (1 μM for 10 hr) markedly increased the level of all five proteins (Figure 1A). These changes induced by db-cAMP/BDNF were due to modulation of protein degradation rather than synthesis, because they were not affected

by the presence of the protein synthesis inhibitor cycloheximide (10 μg/ml, data not shown). The protein stabilization effects of

BDNF were also prevented Digestive enzyme by the specific PKA inhibitor KT5720 R428 (200 nM) (Figure 1A), consistent with the involvement of PKA in BDNF-induced growth cone guidance (Gallo et al., 2002 and Yuan et al., 2003) and axon initiation (Mai et al., 2009 and Shelly et al., 2007). Furthermore, we performed ubiquitination assay on Par6, LKB1, Akt, and RhoA, by transfecting myc-tagged ubiquitin in cultured Neuro2a cells, which exhibited the high transfection efficiency required for this assay. We found that 10 hr treatment of these Neuro2a cells with db-cAMP (20 μM) in the presence of MG132 (1 μM, to block ongoing UPS activity) led to a reduced ubiquitination of Par6 and LKB1 but enhanced RhoA ubiquitination, without affecting Akt ubiquitination (Figure 1B). Pretreatment of KT-5720 also diminished the changes of endogenous Par6 and RhoA protein level induced by BDNF or db-cAMP (see Figure S1C), Together, these findings show that BDNF and db-cAMP could induce a PKA-dependent selective stabilization and degradation of proteins relevant to axon formation, through its effects on the UPS activity. During axon/dendrite differentiation in cultured hippocampal neurons, Par6 accumulates at the axon tip and forms a complex with Par3 and aPKC (Shi et al., 2003) that participates in axon differentiation by interacting with Cdc42 and GSK3β (Garvalov et al., 2007, Joberty et al., 2000, Shi et al.