Cell signaling by receptor-tyrosine kinases

Akt Phosphorylation of BAD Couples Survival Signals to the Cell-Intrinsic Death Machinery

One signal that is overactivated in a wide range of tumour types is the production of a phospholipid, phosphatidylinositol (3,4,5) trisphosphate, by phosphatidylinositol 3-kinase (PI3K) This lipid and the protein kinase that is activated by it — AKT — trigger a cascade of responses, from cell growth and proliferation to survival and motility, that drive tumour progression Small-molecule therapeutics that block PI3K signalling might deal a severe blow to cancer cells by blocking many aspects of the tumour-cell phenotype

The phosphatidylinositol 3-Kinase AKT pathway in human cancer.

Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation

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

Ras (p21ras) interacts directly with the catalytic subunit of phosphatidylinositol-3-OH kinase in a GTP-dependent manner through the Ras effector site. In vivo, dominant negative Ras mutant N17 inhibits growth factor induced production of 3' phosphorylated phosphoinositides in PC12 cells, and transfection of Ras, but not Raf, into COS cells results in a large elevation in the level of these lipids. Therefore Ras can probably regulate phosphatidylinositol-3-OH kinase, providing a point of divergence in signalling pathways downstream of Ras.

Phosphatidylinositol-3-oh kinase as a direct target of ras

Role of Phosphoinositide 3-OH Kinase in Cell Transformation and Control of the Actin Cytoskeleton by Ras

Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3-OH kinase (PI 3-kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3-kinase or PKB/Akt block anoikis, while inhibition of PI 3-kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3-kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3-kinase lipid products and PKB/Akt activity, both of which remain high in Ras-transformed cells even in suspension. PI 3-kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras-transformed epithelial cells in the absence of attachment, and mediates matrix-induced survival of normal epithelial cells.

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Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway

External signals that control the activity of proteins encoded by the ras proto-oncogenes have not previously been characterized. It is now shown that stimulation of the antigen receptor of T lymphocytes causes a rapid activation of p21ras. The mechanism seems to involve a decrease in the activity of GAP, the GTPase-activating protein, on stimulation of protein kinase C. In lymphocytes, p21ras may therefore be an important mediator of the action of protein kinase C.

Stimulation of p21ras upon T-cell activation

The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.

Patricia H. Warne

Papers

Phosphatidylinositol-3-oh kinase as a direct target of ras

Role of Phosphoinositide 3-OH Kinase in Cell Transformation and Control of the Actin Cytoskeleton by Ras

Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway

Stimulation of p21ras upon T-cell activation

Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro