AKT/PKB signaling: navigating downstream.

The programmed cell death that occurs as part of normal mammalian development was first observed nearly a century ago (Collin 1906). It has since been established that approximately half of all neurons in the neuroaxis and >99.9% of the total number of cells generated during the course of a human lifetime go on to die through a process of apoptosis (for review, see Datta and Greenberg 1998; Vaux and Korsmeyer 1999). The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. The purification in the 1950s of the nerve growth factor (NGF), which promotes the survival of sympathetic neurons, set the stage for the discovery that peptide trophic factors promote the survival of a wide variety of cell types in vitro and in vivo (Levi-Montalcini 1987). The profound biological consequences of growth factor (GF) suppression of apoptosis are exemplified by the critical role of target-derived neurotrophins in the survival of neurons and the maintenance of functional neuronal circuits. (Pettmann and Henderson 1998). Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 38-OH kinase (PI3K)/c-Akt kinase cascade. Several targets of the PI3K/c-Akt signaling pathway have been recently identified that may underlie the ability of this regulatory cascade to promote survival. These substrates include two components of the intrinsic cell death machinery, BAD and caspase 9, transcription factors of the forkhead family, and a kinase, IKK, that regulates the NF-kB transcription factor. This article reviews the mechanisms by which survival factors regulate the PI3K/c-Akt cascade, the evidence that activation of the PI3K/c-Akt pathway promotes cell survival, and the current spectrum of c-Akt targets and their roles in mediating c-Akt-dependent cell survival.

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Cellular survival: a play in three Akts

Phosphatidylinositol 3-kinases (PI3Ks) evolved from a single enzyme that regulates vesicle trafficking in unicellular eukaryotes into a family of enzymes that regulate cellular metabolism and growth in multicellular organisms. In this review, we examine how the PI3K pathway has evolved to control these fundamental processes, and how this pathway is in turn regulated by intricate feedback and crosstalk mechanisms. In light of the recent advances in our understanding of the function of PI3Ks in the pathogenesis of diabetes and cancer, we discuss the exciting therapeutic opportunities for targeting this pathway to treat these diseases.

The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).

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Mechanism of activation of protein kinase B by insulin and IGF-1.

BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) was inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, was activated by IL-3 in a PI 3-kinase-dependent manner. Active, but not inactive, forms of Akt were found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway.

https://science.sciencemag.org/content/sci/278/5338/687.full.pdf

Interleukin-3-Induced Phosphorylation of BAD Through the Protein Kinase Akt

A previous report described the isolation of a directly transforming retrovirus, AKT8, from a spontaneous thymoma of an AKR mouse. The AKT8 provirus has now been molecularly cloned from a transformed, nonproducer cell line. The virus genome contains both viral and nonviral, cell-related sequences; the nonviral sequence has been designated v-akt, the presumed viral oncogene of the AKT8 virus. This gene lacks homology to the 16 other oncogenes tested. The cloned provirus has undergone a partial deletion, during cell passage in vitro, that prevents direct demonstration of the transforming ability of this molecular clone. Two human homologues of the v-akt oncogene, AKT1 and AKT2, were cloned. A survey of 225 human tumors for changes involving AKT1 led to the discovery of a 20-fold amplification of this gene in one of the five gastric adenocarcinomas tested. The results demonstrate that AKT8 has the characteristic structure of a directly transforming retrovirus and that it contains a gene derived from highly conserved cellular sequences that may be involved in the pathogenesis of some human malignancies.

https://www.pnas.org/content/pnas/84/14/5034.full.pdf

Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma.

Murine leukemia viruses capable of malignant transformation of mink tissue culture cells have been isolated from an AKR thymoma cell line and from a spontaneous reticulum cell sarcoma in an NIH Swiss mouse partially congenic for the AKR ecotropic virus-inducing locus Akv-2. In contrast to the recently described mink cell focus-inducing strains of murine leukemia virus, at least one of the two transforming strains is replication defective. Nonproducer mink cells carrying the genome of the transforming virus of AKR origin have been isolated, and pseudotype transforming viruses generated.

https://www.pnas.org/content/pnas/74/7/3065.full.pdf

Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma

A specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 500-base-pair DNA segment, corresponding to a portion of the env region of the AKR ecotropic virus, in a pBR322/Escherichia coli K-12 host/vector system. This probe was used to screen the cellular DNAs of six inbred strains of mice for the presence of ecotropic retroviral DNA sequences by the Southern blot hybridization procedure. Three copies of ecotropic viral DNA were detected in AKR/N (a high-ecotropic virus strain) and two were found in BALB/c (a low-ecotropic virus strain) DNAs. As expected, no sequences reactive with this probe were found in NFS mouse DNA (a virus-negative strain). However, cellular DNA sequences that reacted strongly with the ecotropic-specific DNA probe were detected in certain NZB, C57L, and 129 mice (all virus-negative strains). In contrast to the reactive sequences in AKR and BALB/c, the reactions were chiefly associated with EcoRI segments that were subgenomic in size.

Identification of ecotropic proviral sequences in inbred mouse strains with a cloned subgenomic DNA fragment.

Human T-cell leukemia-lymphoma virus (HTLV) is the first unequivocal human retrovirus. Seroepidemiological and virus isolation studies indicate that HTLV is etiologically associated with a subtype of adult T-cell malignancy. We have molecularly cloned approximately 1 kilobase of sequences derived from the 5' and 3' termini of the HTLV genome. Use of these clones as probes allowed isolation of a 9.8-kilobase EcoRI fragment from a genomic DNA library of an HTLV-infected neoplastic T-cell line (CR). Analysis of this clone revealed the presence of cellular sequences flanking approximately 5 kilobases of viral sequences including one long terminal repeat sequence. The 5' and 3' clones, as well as subclones derived from different regions of the genomic clone, were used as probes to compare integrated proviruses and viral RNA expression in different HLTV-infected neoplastic T cell lines. The results indicate that the infected cells are of clonal origin with respect to the virus integration sites and they express multiple viral mRNA species including a 35S RNA.

Stephen P. Staal

Papers

Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma.

Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma

Identification of ecotropic proviral sequences in inbred mouse strains with a cloned subgenomic DNA fragment.

Human T-cell leukemia-lymphoma virus (HTLV): cloning of an integrated defective provirus and flanking cellular sequences.

Effect of interferon on murine leukemia virus infection. II. Synthesis of viral components in exogenous infection.