A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70(S6K)). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70(S6K) mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.

Nerve growth factor (NGF) induces both differentiation and survival of neurons by binding to the Trk receptor protein tyrosine kinase. Although Ras is required for differentiation, it was not required for NGF-mediated survival of rat pheochromocytoma PC-12 cells in serum-free medium. However, the ability of NGF to prevent apoptosis (programmed cell death) was inhibited by wortmannin or LY294002, two specific inhibitors of phosphatidylinositol (Pl)-3 kinase. Moreover, platelet-derived growth factor (PDGF) prevented apoptosis of PC-12 cells expressing the wild-type PDGF receptor, but not of cells expressing a mutant receptor that failed to activate Pl-3 kinase. Cell survival thus appears to be mediated by a Pl-3 kinase signaling pathway distinct from the pathway that mediates differentiation.

Growth factor-dependent survival of a variety of mammalian cells is dependent on the activation of phosphatidylinositol (PI) 3-kinase and its downstream effector, the protein kinase Akt. Glycogen synthase kinase-3 (GSK-3) has been previously identified as a physiological target of Akt, which is inhibited by phosphorylation, so we have investigated the role of GSK-3 in cell survival. Overexpression of catalytically active GSK-3 induced apoptosis of both Rat-1 and PC12 cells, whereas dominant-negative GSK-3 prevented apoptosis following inhibition of PI 3-kinase. GSK-3 thus plays a critical role in regulation of apoptosis and represents a key downstream target of the PI 3-kinase/Akt survival signaling pathway.Although many types of mammalian cells are dependent upon growth factors for survival (1), the intracellular signaling pathways that control cell survival by preventing apoptosis have only begun to be elucidated. A role for PI 1 3-kinase in the regulation of cell survival was first indicated by experiments showing that PI 3-kinase was required to prevent apoptosis of PC12 rat pheochromocytoma cells maintained in nerve growth factor (NGF) (2). These findings have been extended by observations demonstrating that PI 3-kinase is required for survival of several other growth factor-dependent cell types, including fibroblasts, epithelial cells, hematopoietic cells, and primary neurons (3-12). In addition, the protein kinase Akt has been identified as a key effector of PI 3-kinase in signaling cell survival (5,13,14).The principal characterized physiological substrate of Akt is glycogen synthase kinase-3 (GSK-3) (15), which was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (16). GSK-3 is a ubiquitously expressed protein-serine/threonine kinase whose activity is inhibited by Akt phosphorylation in response to growth factor stimulation. In addition to glycogen synthase, GSK-3 phosphorylates a broad range of substrates, including several transcription factors and translation initiation factor eIF2B (16). GSK-3 has also been implicated in the regulation of cell fate in Dictyostelium (17) and is a component of the Wnt signaling pathway required for Drosophila and Xenopus development (18 -21). These studies suggest that GSK-3 is involved in multiple cellular processes, including metabolism, proliferation, and differentiation. Here we show that GSK-3 is also involved in the regulation of apoptosis, identifying it as a critical downstream element of the PI 3-kinase/Akt cell survival pathway. EXPERIMENTAL PROCEDURESCell Culture-PC12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 5% horse serum. Rat-1 cells were grown in DMEM supplemented with 10% calf serum.Assay of GSK-3-PC12 cells were plated in 100-mm culture dishes (3 ϫ 10 5 cells/plate) in DMEM containing 10% fetal bovine serum and 5% horse serum. On the next day the medium was changed to DMEM containing 0.5% horse serum. One day later, cells were stimulated by addi...

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20-to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.The mammalian rais gene family contains three members, ras , ras and rasN, which encode similar 21,000-dalton proteins referred to as p21's (for a review, see reference 1). These proteins reside on the inner face of the plasma membrane, bind GTP and GDP with an equally high affinity. and display a weak GTP hydrolysis activity (1). These properties are similar to those of G proteins that transduce signals from a wide variety of cell surface receptors to enzymes which affect the metabolism of second messengers, including adenylate cyclase, cyclic GMP phosphodiesterase, and phospholipases C and A2 and may also directly regulate ion channels (for a review, see reference 21). Based on this analogy, it has been proposed that r-as proteins also act as signal-transducing molecules. The fact that r-as genes activated by point mutations confer upon cultured cells some aspects of an oncogenic phenotype implies that their encoded proteins function in the transduction of signals that regulate cell proliferation. Other pathways are also probably affected, since p21 is expressed in nondividing cells (6. 19) and activated rcas genes can induce neuronal differentiation of PC12 cells (2, 22), concomitant with a cessation of cell division.G proteins characterized to date have a common structural design. They exist as oligomers made up of ox, ,B, and y subunits (21). In the inactive form. the a subunit is bound to GDP as well as to the a and -y subunits (21). An excited membrane receptor activates the G protein by enhancing the rate of exchange of bound GDP for GTP. Go then dissociates from 13 and y and alters the activity of its target enzyme or ion channel. The ot chain then hydrolyzes its bound GTP to GDP, thereby terminating activation (21). Although no 1 a...

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Proteolysis by the ubiquitin/proteasome pathway controls the intracellular levels of a number of proteins that regulate cell proliferation and cell cycle progression. To determine whether this pathway of protein turnover was also linked to apoptosis, we treated Rat-1 and PC12 cells with specific proteasome inhibitors. The peptide aldehydes PSI and MG115, which specifically inhibit the chymotrypsin-like activity of the proteasome, induced apoptosis of both cell types. In contrast, apoptosis was not induced by inhibitors of lysosomal proteases or by an alcohol analog of PSI. The tumor suppressor p53 rapidly accumulated in cells treated with proteasome inhibitors, as did the p53-inducible gene products p21 and Mdm-2. In addition, apoptosis induced by proteasome inhibitors was inhibited by expression of dominant-negative p53, whereas overexpression of wild-type p53 was sufficient to induce apoptosis of Rat-1 cells in transient transfection assays. Although other molecules may also be involved, these results suggest that stabilization and accumulation of p53 plays a key role in apoptosis induced by proteasome inhibitors.

Activation of growth factor receptors by ligand binding initiates a cascade of events leading to cell growth and division. Progression through the cell cycle is controlled by cyclin-dependent protein kinases (Cdks), but the mechanisms that link growth factor signaling to the cell cycle machinery have not been established. We report here that Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G 1 . Ras is required for cell cycle progression and activation of both Cdk2 and Cdk4 until ϳ2 h before the G 1 /S transition, corresponding to the restriction point. Analysis of Cdk-cyclin complexes indicates that Ras signaling is required both for induction of cyclin D1 and for downregulation of the Cdk inhibitor p27 KIP1. Constitutive expression of cyclin D1 circumvents the requirement for Ras signaling in cell proliferation, indicating that regulation of cyclin D1 is a critical target of the Ras signaling cascade.