Tania Maffucci

Insulin induces phosphatidylinositol‐3‐phosphate formation through TC10 activation

Phosphatidylinositol‐3‐phosphate (PtdIns‐3‐P) is considered as a lipid constitutively present on endosomes; it does not seem to have a dynamic role in signalling. In contrast, phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns‐3,4,5‐P3) plays a crucial role in different signalling pathways including translocation of the glucose transporter protein GLUT4 to the plasma membrane upon insulin receptor activation. GLUT4 translocation requires activation of two distinct pathways involving phosphatidylinositol 3‐kinase (PI 3‐K) and the small GTP‐binding protein TC10, respectively. The contribution of each pathway remains to be elucidated. Here we show that insulin specifically induces the formation of PtdIns‐3‐P in insulin‐ responsive cells. The insulin‐mediated formation of PtdIns‐3‐P occurs through the activation of TC10 at the lipid rafts subdomain of the plasma membrane. Exogenous PtdIns‐3‐P induces the plasma membrane translocation of both overexpressed and endogenous GLUT4. These data indicate that PtdIns‐3‐P is specifically produced downstream from insulin‐mediated activation of TC10 to promote the plasma membrane translocation of GLUT4. These results give a new insight into the intracellular role of PtdIns‐3‐P and shed light on some aspects of insulin signalling so far not completely understood.

Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2β). Both PtdIns-3-P and PI3K-C2β are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2β as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2β–PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.

The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment

Integrating the pleomorphic membranes of the intermediate compartment (IC) into the array of Golgi cisternae is a crucial step in membrane transport, but it is poorly understood. To gain insight into this step, we investigated the dynamics by which cis-Golgi matrix proteins such as GM130 and GRASP65 associate with, and incorporate, incoming IC elements. We found that GM130 and GRASP65 cycle via membranous tubules between the Golgi complex and a constellation of mobile structures that we call late IC stations. These stations are intermediate between the IC and the cis-Golgi in terms of composition, and they receive cargo from earlier IC elements and deliver it to the Golgi complex. Late IC elements are transient in nature and sensitive to fixatives; they are seen in only a fraction of fixed cells, whereas they are always visible in living cells. Finally, late IC stations undergo homotypic fusion and establish tubular connections between themselves and the Golgi. Overall, these features indicate that late IC stations mediate the transition between IC elements and the cis-Golgi face.

The putative cannabinoid receptor GPR55 defines a novel autocrine loop in cancer cell proliferation

Recently, the orphan receptor G protein-coupled receptor 55 (GPR55) has been proposed as a potential cannabinoid receptor, although controversy remains on its physiological roles. Current evidence suggests a role for GPR55 as a receptor for the lysophospholipid lysophosphatidylinositol (LPI). In this study, we show that GPR55 is expressed in several prostate and ovarian cancer cell lines, both at the mRNA and at the protein level, and that it has a critical role in regulating proliferation and anchorage-independent growth. We further show that GPR55 mediates the effects of LPI in prostate and ovarian cancer cells. Indeed we demonstrate that LPI is able to induce calcium mobilization and activation of Akt and extracellular signal-regulated kinase (ERK)1/2 in these cells and that both pharmacological blockade of GPR55 and its downregulation using specific small interfering RNA strongly inhibits these processes. We further identify an autocrine loop by which LPI is synthesized by cytosolic phospholipase A2, pumped out of the cell by the ATP-binding cassette transporter ABCC1/MRP1, and is then able to initialize cascades downstream of GPR55. All together, these data demonstrate a role of LPI and its receptor GPR55 in cancer cells in activating an autocrine loop that regulates cell proliferation. These findings may have important implications for LPI as a novel cancer biomarker and for its receptor GPR55 as a potential therapeutic target.

The Role of Phosphoinositide 3-Kinase C2α in Insulin Signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2α, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2α involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2α and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2α contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.

Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway by Inositol Pentakisphosphate Results in Antiangiogenic and Antitumor Effects

The purpose of this study was to investigate the antiangiogenic and in vivo properties of the recently identified phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor Inositol(1,3,4,5,6) pentakisphosphate [Ins(1,3,4,5,6)P5]. Because activation of the PI3K/Akt pathway is a crucial step in some of the events leading to angiogenesis, the effect of Ins(1,3,4,5,6)P5 on basic fibroblast growth factor (FGF-2)-induced Akt phosphorylation, cell survival, motility, and tubulogenesis in vitro was tested in human umbilical vein endothelial cells (HUVEC). The effect of Ins(1,3,4,5,6)P5 on FGF-2-induced angiogenesis in vivo was evaluated using s.c. implanted Matrigel in mice. In addition, the effect of Ins(1,3,4,5,6)P5 on growth of ovarian carcinoma SKOV-3 xenograft was tested. Here, we show that FGF-2 induces Akt phosphorylation in HUVEC resulting in antiapoptotic effect in serum-deprived cells and increase in cellular motility. Ins(1,3,4,5,6)P5 blocks FGF-2-mediated Akt phosphorylation and inhibits both survival and migration in HUVEC. Moreover, Ins(1,3,4,5,6)P5 inhibits the FGF-2-mediated capillary tube formation of HUVEC plated on Matrigel and the FGF-2-induced angiogenic reaction in BALB/c mice. Finally, Ins(1,3,4,5,6)P5 blocks the s.c. growth of SKOV-3 xenografted in nude mice to the same extent than cisplatin and it completely inhibits Akt phosphorylation in vivo. These data definitively identify the Akt inhibitor Ins(1,3,4,5,6)P5 as a specific antiangiogenic and antitumor factor. Inappropriate activation of the PI3K/Akt pathway has been linked to the development of several diseases, including cancer, making this pathway an attractive target for therapeutic strategies. In this respect, Ins(1,3,4,5,6)P5, a water-soluble, natural compound with specific proapoptotic and antiangiogenic properties, might result in successful anticancer therapeutic strategies.

Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide-protein co-operative mechanism

Pleckstrin homology (PH) domains are protein modules found in proteins involved in many cellular processes. The majority of PH domain‐containing proteins require membrane association for their function. It has been shown that most PH domains interact directly with the cell membrane by binding to phosphoinositides with a broad range of specificity and affinity. While a highly specific binding of the PH domain to a phosphoinositide can be necessary and sufficient for the correct recruitment of the host protein to the membrane, a weaker and less specific interaction may be necessary but not sufficient, thus probably requiring alternative, co‐operative mechanisms.

The Role of Phosphoinositide 3-Kinase C2 in Insulin Signaling *□

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2α, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2α involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2α and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2α contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.

The mechanism involved in the regulation of phospholipase Cγ1 activity in cell migration

Activation of the enzyme phospholipase C (PLC) leads to the formation of second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Tyrosine kinase receptors activate this reaction through PLCγ isoenzymes. PLCγ activity involves its activation with, and phosphorylation by, receptor tyrosine kinases. Recently, it has been shown that phosphoinositide 3-kinase (PI 3-K) may regulate PLCγ activity through the interaction of the PI 3-K product phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) and the PLCγ pleckstrin homology (PH) domain. In an effort to understand the signalling pathway that involves PI 3-K regulation of PLCγ, we found that EGF induces a PI 3-K-dependent translocation of PLCγ1 at the leading edge of migrating cells in a wound healing assay. Similarly, the isolated PH, but not the Src-homology (SH) domains, N-SH2 or SH3, of PLCγ1, translocates at the leading edge. Our experiments also showed that stable PH PLCγ1 expression blocks epidermal growth factor (EGF)- and serum-induced cell motility and increases cell adhesion in MDA-MB-231 cells. This may suggest that influence of PI 3-K on PLCγ1 could be relevant in cell migration, where PLCγ1 seems to play a key role by modulating a series of events involved in actin polymerization.

Role of class II phosphoinositide 3-kinase in cell signalling

Although it is now well established that PI3K (phosphoinositide 3-kinase) is a key enzyme in several intracellular processes, there are still relatively few reports that precisely identify the specific isoforms of PI3K actually involved in such events. The lack of specific inhibitors has made it particularly difficult to address the physiological roles of some isoforms, such as the members of class II. As a consequence, there is still relatively little understanding of the role of these enzymes and the question about the intracellular role of these isoforms still waits for more answers.