No abstract

Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates.Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis. Here we demonstrate that reducing expression of endogenous Akt1 and Akt2 by RNA interference (RNAi) reduces neuron generation in P19 cells transfected with a neural bHLH expression vector. The reduction in neuron generation from decreased Akt expression is not solely due to decreased cell survival, since addition of the caspase inhibitor z-VAD-FMK rescues cell death associated with loss of Akt function but does not restore neuron formation. This result indicates that Akt1 and Akt2 have additional functions during neuronal differentiation that are separable from neuronal survival. We show that activated Akt1 enhances complex formation between bHLH proteins and the transcriptional coactivator p300. Activated Akt1 also significantly augments the transcriptional activity of the bHLH protein neurogenin 3 in complex with the coactivators p300 or CBP. In addition, inhibition of endogenous Akt activity by the PI3K/Akt inhibitor LY294002 abolishes transcriptional cooperativity between the bHLH proteins and p300. We propose that Akt regulates the assembly and activity of bHLH-coactivator complexes to promote neuronal differentiation.

Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.Ras proteins (Ha-Ras, K-Ras, N-Ras, TC21, and Ras1/2) regulate cell growth in eukaryotic cells, and perturbation of signaling pathways by mutation and constitutive activation of Ras proteins is a common occurrence in a wide spectrum of human tumors (1). In addition to regulating cell proliferation, Ras proteins also regulate differentiation, cell death, and cell survival (1). The Ras proteins are members of a large superfamily of low molecular weight GTP-binding proteins, which include members that regulate the actin cytoskeleton (Rho and Rac), vesicle trafficking (Rabs), and nuclear transport (Ran).The biological activity of Ras proteins is controlled by a regulated GTP/GDP cycle (2). The GTP-bound, or active, Ras relays signals to downstream effector proteins. In the case of Ha-Ras, numerous effector proteins have been described, including serine/threonine kinases (c-Raf, A-Raf, and B-Raf), lipid kinases (type I phosphatidylinositol 3-kinase), and guanine nucleotide dissociation stimulators for Ral (Ral GDS 1 and RGL) (1). Activation of Ras effector pathways leads to proliferation, differentiation, cell death, and cell survival. Which biological outcome predominates is somewhat of a mystery but seems to depend on cell type and the coordinate integration of signaling pathways activated and/or inhibited within a cell. Members of the Ras superfamily are subject to post-translational modifications. The spectrum of modifications depends on the composition of the carboxyl terminus. In the case of HaRas, the protein is subject to prenylation, proteolysis, carboxylmethylation, and S-acylation/palmitoylation (3). Prenylation is the covalent attachment of farnesyl or geranylgeranyl isoprenoids at or near the carboxyl terminus of the GTPase. For Ras family members, prenylation occurs at a conserv...

How scaffold proteins integrate signaling pathways with cytoskeletal components to drive axon outgrowth is not well understood. We report here that the multidomain scaffold protein Plenty of SH3s (POSH) regulates axon outgrowth. Reduction of POSH function by RNA interference (RNAi) enhances axon outgrowth in differentiating mouse primary cortical neurons and in neurons derived from mouse P19 cells, suggesting POSH negatively regulates axon outgrowth. Complementation analysis reveals a requirement for the third Src homology (SH) 3 domain of POSH, and we find that the actomyosin regulatory protein Shroom3 interacts with this domain of POSH. Inhibition of Shroom3 expression by RNAi leads to increased process lengths, as observed for POSH RNAi, suggesting that POSH and Shroom function together to inhibit process outgrowth. Complementation analysis and interference of protein function by dominant-negative approaches suggest that Shroom3 recruits Rho kinase to inhibit process outgrowth. Furthermore, inhibition of myosin II function reverses the POSH or Shroom3 RNAi phenotype, indicating a role for myosin II regulation as a target of the POSH-Shroom complex. Collectively, these results suggest that the molecular scaffold protein POSH assembles an inhibitory complex that links to the actin-myosin network to regulate neuronal process outgrowth.

We demonstrate that POSH, a scaffold for the JNK signaling pathway, binds to Akt2. A POSH mutant that is unable to bind Akt2 (POSH W489A) exhibits enhancedbinding to MLK3, and this increase in binding is accompanied by increased activation of the JNK signaling pathway. In addition, we show that the association of MLK3 with POSH is increased upon inhibition of the endogenous phosphatidylinositol 3-kinase/Akt signaling pathway. Thus, the assembly of an active JNK signaling complex by POSH is negatively regulated by Akt2.