Essential Role of Class II Phosphatidylinositol-3-kinase-C2α in Sphingosine 1-Phosphate Receptor-1-mediated Signaling and Migration in Endothelial Cells

The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3′-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110β, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2β, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P_1–3. We studied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P₁ in ECs. Knockdown of either PI3K-C2α or class I p110β markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110β was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110β were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110β markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110β suppressed S1P-induced S1P₁ internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P₁ internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P₁ internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.     

The role of phosphoinositide 3-kinase C2alpha 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-C2alpha, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2alpha 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-C2alpha and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2alpha 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.     

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P₁ and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.     

Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2alpha and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2alpha knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2alpha knockdown and expression of PI3K-C2alpha catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca(2+). We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2alpha is indeed regulated by Ca(2+). We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2alpha occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca(2+) in vitro and in living neurosecretory cells.     

Class II phosphoinositide 3-kinases are downstream targets of activated polypeptide growth factor receptors

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.     

Activation of phosphoinositide 3-kinase C2β in the nuclear matrix during compensatory liver growth

In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable PI3K-C2β activity is observed, which is sensitive to wortmannin (10 Mm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On western blots PI3K-C2β revealed a single immunoreactive band of 180 kD, whereas 20 h after partial hepatectomy gel shift of 18 kDa was noticed in the nuclear matrix, suggesting that observed activation of enzyme is achieved by proteolysis. As it is know that PI3K-C2α is associated with nuclear speckles [Didichenko SA, Thelen M. Phosphatidylinositol 3-kinase C2α contains a nuclear localization sequence and associates with nuclear speckles. J Biol Chem 2001;276:48135-42.], the data presented in this report show that in the nuclear matrix PI3K-C2β is activated during the compensatory liver growth, which clearly demonstrates that different class II PI3K enzymes have different subnuclear localization and therefore might have different intranuclear functions.     

The class II phosphoinositide 3-kinase PI3K-C2alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles

In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozymes has been characterized and cloned. Several of these PI3K enzymes have overlapping tissue distributions and it remains unclear if and how their 3-phosphoinositide products elicit differential, intracellular effects. One possibility is that the PI3K enzymes display a restricted distribution within the cell to produce their 3-phospholipid products in specific, subcellular compartments. In the present study we characterize the subcellular distribution of the novel class II PI3K isozyme PI3K-C2alpha in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2alpha is constitutively associated with phospholipid membranes. Centrifugation of rat brain homogenates and Western blotting revealed that in contrast to the class IA PI3K enzymes, PI3K-C2alpha could be co-purified with a population of clathrin-coated vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin treatment was detected in CCV preparations consistent with the presence of the PI3K-C2alpha isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2alpha to have a punctate distribution and an enrichment of immunoreactivity within a perinuclear site consistent with its presence in the endoplasmic reticulum or Golgi apparatus. Dual label immunofluorescence demonstrated that in this region, the distribution of PI3K-C2alpha closely paralleled that of gamma-adaptin, a component of the AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi network (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2alpha was dependent upon either its COOH-terminal PX or C2 domains. Mutants lacking these domains demonstrated a similar distribution to the wild type enzyme when expressed as recombinant proteins. Treatment of cells with brefeldin A disrupted the perinuclear staining pattern of both PI3K-C2alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.     

Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.     

Presence and activation of nuclear phosphoinositide 3-kinase C2beta during compensatory liver growth

Highly purified liver nuclei incorporated radiolabeled phosphate into phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). When nuclei were depleted of their membrane, no radiolabeling of PtdIns(3,4,5)P(3) could be detected showing that within the intranuclear region there are no class I phosphoinositide 3-kinases (PI3K)s. In membrane-depleted nuclei harvested 20 h after partial hepatectomy, the incorporation of radiolabel into PtdIns(3)P was observed together with an increase in immunoprecipitable PI3K-C2beta activity, which is sensitive to wortmannin (10 nm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On Western blots PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas 20 h after partial hepatectomy gel shift of 18 kDa was noticed, suggesting that observed activation of enzyme is achieved by proteolysis. When intact membrane-depleted nuclei were subjected to short term (20 min) exposure to micro-calpain, similar gel shift together with an increase in PI3K-C2beta activity was observed, when compared with the nuclei harvested 20 h after partial hepatectomy. Moreover, the above-mentioned gel shift and increase in PI3K-C2beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that, in the membrane-depleted nuclei during the compensatory liver growth, there is an increase in PtdIns(3)P formation as a result of PI3K-C2beta activation, which may be a calpain-mediated event.     

The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+ Channel KCa3.1 and CD4 T-Cells

The Ca²⁺-activated K⁺ channel KCa3.1 is required for Ca²⁺ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca²⁺ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.     

Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases

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-C2beta). Both PtdIns-3-P and PI3K-C2beta are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2beta as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2beta-PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.     

Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2alpha (PI3K-C2alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2alpha is required for acquisition of fusion competence in neurosecretion.     

The Endosomal Sorting Complex Required for Transport Pathway Mediates Chemokine Receptor CXCR4-promoted Lysosomal Degradation of the Mammalian Target of Rapamycin Antagonist DEPTOR

G protein-coupled receptor (GPCR) signaling mediates many cellular functions, including cell survival, proliferation, and cell motility. Many of these processes are mediated by GPCR-promoted activation of Akt signaling by mammalian target of rapamycin complex 2 (mTORC2) and the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase 1 (PDK1) pathway. However, the molecular mechanisms by which GPCRs govern Akt activation by these kinases remain poorly understood. Here, we show that the endosomal sorting complex required for transport (ESCRT) pathway mediates Akt signaling promoted by the chemokine receptor CXCR4. Pharmacological inhibition of heterotrimeric G protein Gα_i or PI3K signaling and siRNA targeting ESCRTs blocks CXCR4-promoted degradation of DEPTOR, an endogenous antagonist of mTORC2 activity. Depletion of ESCRTs by siRNA leads to increased levels of DEPTOR and attenuated CXCR4-promoted Akt activation and signaling, consistent with decreased mTORC2 activity. In addition, ESCRTs likely have a broad role in Akt signaling because ESCRT depletion also attenuates receptor tyrosine kinase-promoted Akt activation and signaling. Our data reveal a novel role for the ESCRT pathway in promoting intracellular signaling, which may begin to identify the signal transduction pathways that are important in the physiological roles of ESCRTs and Akt.     

A class II phosphoinositide 3-kinase plays an indispensable role in hepatitis C virus replication

Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the β isoform of class II PI3K (PI3K-C2β) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2β abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2β substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2β plays an indispensable role in the HCV replication cycle through the binding to HCV core protein.     

IKBKE protein activates Akt independent of phosphatidylinositol 3-kinase/PDK1/mTORC2 and the pleckstrin homology domain to sustain malignant transformation

Serine/threonine kinase Akt regulates key cellular processes such as cell growth, proliferation, and survival. Activation of Akt by mitogenic factor depends on phosphatidylinositol 3-kinase (PI3K). Here, we report that IKBKE (also known as IKKε and IKKi) activates Akt through a PI3K-independent pathway. IKBKE directly phosphorylates Akt-Thr308 and Ser473 independent of the pleckstrin homology (PH) domain. IKBKE activation of Akt was not affected by inhibition of PI3K, knockdown of PDK1 or mTORC2 complex. Further, this activation could be inhibited by Akt inhibitors MK-2206 and GSK690693 but not the compounds (perifosine and triciribine) targeting the PH domain of Akt. Expression of IKBKE largely correlates with activation of Akt in breast cancer. Moreover, inhibition of Akt suppresses IKBKE oncogenic transformation. These findings indicate that IKBKE is an Akt-Thr308 and -Ser473 kinase and directly activates Akt independent of PI3K, PDK1, and mTORC2 as well as PH domain. Our data also suggest that Akt inhibitors targeting the PH domain have no effect on the tumors in which hyperactive Akt resulted from elevated IKBKE.     

Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G₁/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G₁/S block, which correlates with G₂/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G₁/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃. On Western blots, PI3K-C2β revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with μ-calpain in vitro, the similar gel shift and increase in PI3K-C2β activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2β but did not prevent the gel shift. When HL-60 cells were released from G₁/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2β was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2β during G₂/M phase of the cell cycle in HL-60 cells.     

Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L

Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.     

Insulin activates the alpha isoform of class II phosphoinositide 3-kinase.

The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2-3-fold increase in the activity of PI 3-kinase C2alpha (PI3K-C2alpha) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2alpha was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2alpha by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2alpha to associate with IRS-1 or insulin receptor. PI3K-C2alpha existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2alpha. The finding that insulin activates PI3K-C2alpha in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2alpha is a novel component of insulin-stimulated signaling cascades.     

The class II phosphoinositide 3-kinase PI3K-C2beta regulates cell migration by a PtdIns3P dependent mechanism

The biological and pathophysiological significance of class II phosphoinositide 3-kinase enzyme expression currently remains unclear. Using an in vitro scrape wound assay and time-lapse video microscopy, we demonstrate that cell motility is increased in cultures expressing recombinant PI3K-C2beta enzyme. In addition, overexpression of PI3K-C2beta transiently decreased cell adhesion, stimulated the formation of cytoplasmic processes, and decreased the rate of cell proliferation. Consistent with these observations, expression of PI3K-C2beta also decreased expression of alpha4 beta1 integrin subunits. Using asynchronous cultures, we show that endogenous PI3K-C2beta is present in lamellipodia of motile cells. When cells expressing recombinant PI3K-C2beta were plated onto fibronectin, cortical actin staining increased markedly and actin rich lamellipodia and filopodia became evident. Overexpression of a 2xFYVE(Hrs) domain fusion protein abolished this response demonstrating that the effect of PI3K-C2beta on the reorganization of actin filaments is dependent upon PtdIns3P. Finally, overexpression of PI3K-C2beta increased GTP loading of Cdc42. Our data demonstrates for the first time, that PI3K-C2beta plays a regulatory role in cell motility and that the mechanism by which it reorganizes the actin cytoskeleton is dependent upon PtdIns3P production.