Bernard Payrastre

Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: A comparison with tyrosine kinase and protein kinase C inhibition

Depending on their structure, flavonoids display more or less potent inhibitory effects on the growth and proliferation of certain malignant cells in vitro, and these effects are thought to be due to inhibition of various enzymes. We investigated the inhibitory action of fourteen flavonoids of different chemical classes on phosphatidylinositol 3-kinase alpha (PI 3-kinase alpha) activity, an enzyme recently shown to play an important role in signal transduction and cell transformation. Of the fourteen flavonoids tested, myricetin was the most potent PI 3-kinase inhibitor (IC50 = 1.8 microM), while luteolin and apigenin were also effective inhibitors, with IC50 values of 8 and 12 microM, respectively. Fisetin and quercetin, as previously reported, were also found to significantly inhibit PI 3-kinase activity. The same flavonoids were also analyzed for inhibition of epidermal growth factor receptor (EGF-R), intrinsic tyrosine kinase and bovine brain protein kinase C (PKC). At elevated doses, some of these flavonoids were found to also cause significant inhibition of PKC and tyrosine kinase activity of EGF-R. A structure-activity study indicated that the position, number and substitution of the hydroxyl group of the B ring, and saturation of the C2-C3 bond are important factors affecting flavonoid inhibition of PI 3-kinase. They may also play a significant role in specificity of inhibition and could help to provide a basis for the further design of specific inhibitors of this lipid kinase. Finally, possible relationships between the antitumoral properties of these flavonoids and their biological activities are discussed.

The PHD Finger of the Chromatin-Associated Protein ING2 Functions as a Nuclear Phosphoinositide Receptor

Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.

A Role for Phosphoinositide 3-Kinase in Bacterial Invasion

Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. Inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 3-kinase p85α-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3,4)P2 and PI(3,4,5)P3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein InlB, host cell tyrosine phosphorylation, and association of p85α with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.

Conversion of PtdIns(4,5)P2 into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology

Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization. Here we show that, upon infection of epithelial cells with the Gram‐negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4‐phosphatase that specifically dephosphorylates phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] into phosphatidylinositol 5‐monophosphate [PtdIns(5)P] that then accumulates. Transfection experiments indicate that the transformation of PtdIns(4,5)P2 into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling. These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site.

Flavonoids and the inhibition of PKC and PI 3-kinase

Flavonoids provide a large number of interesting natural compounds that are consumed daily and exhibit more or less potent and selective effects on some signaling enzymes as well as on the growth and proliferation of certain malignant cells in vitro. Among the identified signal transducers, phosphoinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC) are now considered key players in many cellular responses including cell multiplication, apoptosis, and transformation. Despite their lack of strict specificity, some flavonoids provide valuable bases for the design of analogues that could be used to specifically block particular isoforms of PI 3-kinase or PKC and their downstream-dependent cellular responses.

Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.

Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

The yeast Kes1p/Osh4p protein functions as a sterol/PI(4)P exchanger between lipid membranes, which suggests the possibility of creating a sterol gradient via phosphoinositide metabolism.

Phosphoinositide signaling disorders in human diseases

Phosphoinositides (PIs) play an essential role in diverse cellular functions. Their intracellular level is strictly regulated by specific PI kinases, phosphatases and phospholipases. Recent discoveries indicate that dysfunctions in the control of their level often lead to pathologies. This review will focus on some human diseases whose etiologies involve PI‐metabolizing enzymes. The role of PTEN (phosphatase and tensin homolog deleted on chromosome ten) in cancer, the impact of the Src homology 2‐containing inositol‐5‐phosphatase phosphatases in acute myeloid leukemia or diabetes, the involvement of myotubularin family members in genetic diseases and the implication of OCRL1 in Lowe syndrome will be emphasized. We will also review how some bacterial pathogens have evolved strategies to specifically manipulate the host cell PI metabolism to efficiently infect them.

PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4‐phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns(5)P). We show that PtdIns(5)P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri‐induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short‐chain penetrating PtdIns(5)P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns(5)P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD‐expressing cells. Accordingly, IpgD and PtdIns(5)P production specifically activates a class IA PI 3‐kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3‐kinase/Akt activation via PtdIns(5)P production that plays an important role in host cell responses such as survival.

Phosphatidylinositol 3,4,5-Trisphosphate-dependent Stimulation of Phospholipase C-γ2 Is an Early Key Event in FcγRIIA-mediated Activation of Human Platelets

Platelets express a single class of Fcγ receptor (FcγRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcγRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-γ2 (PLC-γ2), and calcium signaling. Here, we show that in response to FcγRIIA cross-linking, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCγ2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin αIIb/β3 engagement. In contrast, the stable association of PLCγ2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCγ2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCγ2 induced by FcγRIIA cross-linking.