Increasing complexity of the Ras signaling pathway

Ras is a key regulator of cell growth in all eukaryotic cells. Genetic, biochemical, and molecular studies in Caenorhabditis elegans, Drosophila, and mammalian cells have positioned Ras centrally in signal transduction pathways that respond to diverse extracellular stimuli, including peptide growth factors, cytokines, and hormones. The biological activity of Ras is controlled by a regulated GDP/GTP cycle. Guanine nucleotide exchange factors (GEFs 1; RasGRF1/2 and Sos1/2) promote the formation of the active, GTP-bound form of Ras (1). GTPaseactivating proteins (GAPs; p120 GAP and NF1) accelerate the intrinsic GTP hydrolytic activity of Ras to promote formation of the inactive, GDP-bound form of Ras (1). Mutations in Ras at amino acids 12, 13, or 61 make Ras insensitive to GAP action and, hence, constitutively active in transforming mammalian cells (2, 3). These activating mutations in Ras are prevalent in a wide spectrum of human cancers. It has been estimated that 30 % of all human tumors contain an activating mutation in Ras. The frequency of Ras mutations varies depending on tumor type, with the highest frequencies seen in lung, colon, thyroid, and pancreatic carcinomas (3). The frequency of Ras mutations is likely to be an underestimation of the contribution of aberrant signaling through the Ras pathway to human malignancies because chronic up-regulation of the Ras pathway can occur in the absence of mutations in Ras itself (4–6). Ras Directly Binds Raf and Activates a Kinas

Akt Regulates Basic Helix-Loop-Helix Transcription Factor-Coactivator Complex Formation and Activity during Neuronal Differentiation

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