Stefan Wennström

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

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.

PDGF stimulates an increase in GTP–Rac via activation of phosphoinositide 3-kinase

BACKGROUND Phosphoinositide 3-kinases (PI 3-kinases) are thought to play an important role in coordinating the responses elicited by a variety of growth factors, oncogene products and inflammatory stimuli. These responses include activation of membrane ruffling, chemotaxis, glucose transport, superoxide production, neurite outgrowth and pp70 S6 kinase. Some of these responses are also known to be regulated by Rac, a small GTP-binding protein related to Ras. Neither the transducing elements upstream of Rac, nor those downstream of PI 3-kinase, have been defined. RESULTS We show here that platelet-derived growth factor (PDGF) can stimulate an increase in the level of GTP-Rac by at least two distinct mechanisms: firstly, by increased guanine nucleotide exchange; and secondly, by inhibition of a Rac GTPase activity. The first of these mechanisms is essential for the activation of Rac, and we show that it is dependent upon PDGR-stimulated synthesis of phosphatidylinositol (3,4,5)-trisphosphate. CONCLUSIONS These results suggest that Rac activation lies downstream of PI 3-kinase activation on a PDGF-stimulated signalling pathway. Furthermore, as Rac has been implicated in at least two diverse cellular responses that are also though to require activation of PI 3-kinase--a reorganization of the actin cytoskeleton known as membrane ruffling and the neutrophil oxidative burst--these results suggest that Rac may be a major effector protein for the PI 3-kinase signalling pathway in many cell types.

Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling

BACKGROUND There is substantial evidence that phosphoinositide 3-kinase (PI 3-kinase) is a critical component of signalling pathways used by the cell-surface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a highly polar membrane lipid called phosphatidylinositol (3,4,5)-trisphosphate This lipid has been postulated to act as a second-messenger in cells but its putative targets are still unknown. RESULTS A particular rearrangement of actin filaments, which results in membrane ruffling, is elicited by the activation of PDGF beta-receptors expressed in cultured porcine aortic endothelial cells. We have found that this consequence of PDGF beta-receptor activation is inhibited by three independent manipulations of PI 3-kinase activity: firstly, by the deletion of tyrosine residues in the PDGF beta-receptor to which PI 3-kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-kinase regulatory subunit to which the catalytic kinase subunit cannot bind; and thirdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-kinase. CONCLUSIONS These results argue strongly that phosphatidylinositol (3,4,5)-trisphosphate synthesis is required for growth-factor-stimulated membrane ruffling in porcine aortic endothelial cells, and suggest that synthesis of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.

Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor

ABSTRACT The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110α is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase

p85/p110 phosphoinositide 3‐kinase (PI3K) is a heterodimer composed of a p85‐regulatory and a p110‐catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65‐PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85α‐protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

R-Ras can activate the phosphoinositide 3-kinase but not the MAP kinase arm of the Ras effector pathways

BACKGROUND The small GTPase R-Ras displays a less potent transforming activity than the closely related Ras oncogene products. Although R-Ras has been reported to interact with c-Raf1 and Ral-GDS in vitro, the pathways by which it exerts its effects on cellular proliferation are not known. RESULTS Both Ras and R-Ras interact with phosphoinositide (PI) 3-kinase in vitro, and induce elevation of the levels of PI 3-kinase lipid products in intact cells. Unlike Ras, R-Ras does not activate Raf or mitogen-activated protein (MAP) kinase in cells. In co-transfection assays, the serine/threonine protein kinase PKB (or Akt) is effectively stimulated by R-Ras, Ras, mutants of Ras that activate PI 3-kinase but not other effectors, and activated forms of PI 3-kinase. Ras and R-Ras stimulate PKB/Akt through a non-autocrine mechanism that involves PI 3-kinase. The constitutive activation of PI 3-kinase alone is sufficient to activate PKB/Akt, but not the MAP kinase ERK or the stress-activated protein kinase, Jun N-terminal kinase. Transformation assays in fibroblasts suggest that PKB/Akt and Raf are part of distinct oncogenic signalling pathways. CONCLUSIONS Both the Raf-MAP kinase and PI 3-kinase-PKB/Akt pathways are activated by Ras, but only the PI 3-kinase-PKB/Akt pathway is activated by R-Ras. PI 3-kinase, and downstream targets such as PKB/Akt, are likely to be essential mediators of transformation induced by R-Ras. PI 3-kinase, as well as Raf, is thus implicated also in Ras transformation.

Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.

Identification of the Ras GTPase-activating protein GAP1m as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.

cDNA Cloning and Expression of a Human FGF Receptor which Binds Acidic and Basic FGF

We have isolated and characterized a cDNA clone, phFGFR, encoding a human fibroblast growth factor (FGF) receptor. phFGFR contains an open reading frame which encodes an 820 amino acid polypeptide with three immunoglobulin-like domains in the extracellular part and an intracellular split tyrosine kinase domain. Transient expression in COS-1 cells and immunoprecipitation using an antiserum raised against a C-terminal peptide, gave rise to two components, representing mature (130 kDa) and precursor (115 kDa) forms of the phFGFR encoded polypeptide, which was denoted hFGFR-1. Crosslinking of iodinated acidic FGF (aFGF) and basic FGF (bFGF) to transiently expressing COS-1 cells revealed a major band of 95 kDa, which was competed for by both aFGF and bFGF. From Scatchard analyses, the Kd:s for binding of aFGF and bFGF to hFGFR-1 were estimated to 25 pM and 41 pM, respectively. Thus, phFGFR encodes a human FGF receptor with high affinity for both aFGF and bFGF.