Phosphatidylinositol (PI) 3-kinase and its downstream effector Akt are thought to be signaling intermediates that link cell surface receptors to p70 S6 kinase. We examined the effect of a G q -coupled receptor on PI 3-kinase/Akt signaling and p70 S6 kinase activation using Rat-1 fibroblasts stably expressing the human ␣ 1A -adrenergic receptor. Treatment of the cells with phenylephrine, a specific ␣ 1 -adrenergic receptor agonist, activated p70 S6 kinase but did not activate PI 3-kinase or any of the three known isoforms of Akt. Furthermore, phenylephrine blocked the insulin-like growth factor-I (IGF-I)-induced activation of PI 3-kinase and the phosphorylation and activation of Akt-1. The effect of phenylephrine was not confined to signaling pathways that include insulin receptor substrate-1, as the ␣ 1 -adrenergic receptor agonist also inhibited the platelet-derived growth factor-induced activation of PI 3-kinase and Akt-1. Although increasing the intracellular Ca 2؉ concentration with the ionophore A23187 inhibited the activation of Akt-1 by IGF-I, Ca 2؉ does not appear to play a role in the phenylephrine-mediated inhibition of the PI 3-kinase/Akt pathway. The differential ability of phenylephrine and IGF-I to activate Akt-1 resulted in a differential ability to protect cells from UV-induced apoptosis. These results demonstrate that activation of p70 S6 kinase by the ␣ 1A -adrenergic receptor in Rat-1 fibroblasts occurs in the absence of PI 3-kinase/Akt signaling. Furthermore, this receptor negatively regulates the PI 3-kinase/Akt pathway, resulting in enhanced cell death following apoptotic insult.Cellular growth requires the generation of new translational machinery to accommodate the increased demand for additional proteins. It has been shown that treatment of cells with growth-promoting agents induces the translational up-regulation of ribosomal proteins and protein synthesis elongation factors (1, 2). This process is controlled in part by phosphorylation of the S6 protein of 40 S ribosomal subunits by the M r ϭ 70,000 S6 kinase (p70 S6 kinase 1 ; Refs. 3 and 4). p70 S6 kinase is activated upon treatment of cells with a variety of growth factors, hormones, mitogens, and phosphatase inhibitors, etc. This increase in activity is due to phosphorylation of p70 S6 kinase at multiple sites presumably by multiple kinases (5, 6). Due to its importance in the growth response, the signal transduction pathways leading to activation of p70 S6 kinase have received considerable attention. A variety of experimental approaches have led to the identification of phosphatidylinositol (PI) 3-kinase and its downstream effector, the protein kinase Akt, as signaling intermediates that link cell surface receptors to p70 S6 kinase. First, treatment of cells with wortmannin or LY294002, two inhibitors of PI 3-kinase, prevents the activation of Akt (7-9) and p70 S6 kinase (10, 11) in response to growth factors or hormones. Second, platelet-derived growth factor (PDGF) receptor mutants that cannot bind PI 3-kinase were unable to indu...

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Protein-mediated endocytosis of membrane is a key event in biological system. The mechanism, however, is still not clear. Using a de novo designed bola-type peptide KKKLLLLLLLLKKK (KLK) as a protein mimic, we studied how it induced giant unilamellar vesicle (GUV) to form inward buds or endocytosis at varying conditions. Results show that the inward budding is initiated as the charged lipids are neutralized by KLK, which results in a negative spontaneous curvature. If the charged lipids have unsaturated tails, the buddings are slim fibrils, which can further wrap into a spherical structure. In the case of saturated charged lipids, the buddings are rigid tubules, stable in the studied time period. The unsaturated lipid to saturated lipid ratio in the mother membrane is another key parameter governing the shape and dynamics of the buds. A complete endocytosis is observed when KLK is attached with a hydrophobic moiety, suggesting that hydrophobic interaction helps the buds to detach from the mother membrane. The molecules in the surrounding medium, such as negatively charged oligonucleotides, are engulfed into the GUV via endocytosis pathway induced by KLK. Our study provides a novel strategy for illustrating the endocytosis mechanism by using peptides of simple sequence.

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