Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.     

PIKE/nuclear PI 3-kinase signaling mediates the antiapoptotic actions of NGF in the nucleus

PI 3-kinase (PI3K) occurs in the nuclei of a broad range of cell types, and various stimuli elicit PI3K nuclear translocation. However, little is known about the biological function of nuclear PI3K. Here we show that nuclear PI3K and its upstream regulator PIKE mediate the antiapoptotic activity of nerve growth factor (NGF) in the isolated nuclei. The nuclei from NGF-treated PC12 cells, EGF-treated HEK293 cells and HeLa cells are resistant to DNA fragmentation initiated by activated cell-free apoptosome. Nuclei from constitutively active PI3K adenovirus-infected cells display the same resistance as those treated by NGF, whereas PI3K inhibitors, dominant-negative PI3K or PIKE abolishes it. Knockdown of either PI3K or PIKE diminishes the antiapoptotic activity of NGF. PI (3,4,5)P3 alone mimics the antiapoptotic activity of NGF, for which nuclear Akt is required. These results demonstrate that PIKE/nuclear PI3K signaling through nuclear PI (3,4,5)P3 and Akt plays an essential role in promoting cell survival.     

Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth

Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only approximately 20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.     

PIKE/nuclear PI 3-kinase signaling in preventing programmed cell death

PI 3-kinase enhancer (PIKE) is a nuclear GTPase that enhances PI 3-kinase (PI3K) activity. Nerve growth factor (NGF) treatment leads to PIKE activation by triggering the nuclear translocation of PLC-gamma1, which acts as a physiological guanine nucleotide exchange factor (GEF) for PIKE. PI3K occurs in the nuclei of a broad range of cell types, and various stimuli elicit PI3K nuclear translocation. While cytoplasmic PI3K has been well characterized, little is known about the biological function of nuclear PI3K. Surprisingly, nuclei from 30 min NGF-treated PC12 cells are resistant to DNA fragmentation initiated by the activated cell-free apoptosome, and both PIKE and nuclear PI3K are sufficient and necessary for this effect. Moreover, pretreatment of the control nucleus with PI(3,4,5)P3 alone mimics the anti-apoptotic activity of NGF by selectively preventing apoptosis, for which nuclear Akt is required but not sufficient. Recently, a nuclear PI(3,4,5)P3 receptor, nucleophosmin/B23, has been identified from NGF-treated PC12 nuclear extract. PI(3,4,5)P3/B23 complex mediates the anti-apoptotic effects of NGF by inhibiting DNA fragmentation activity of caspase-activated DNase (CAD). Thus, PI(3,4,5)P3/B23 complex and nuclear Akt effectors might coordinately mediate PIKE/nuclear PI3K signaling in promoting cell survival by NGF.     

Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation.

We investigated the role of Ras in vascular endothelial growth factor (VEGF)-mediated signal transduction and the promotion of angiogenic changes primary endothelial cells. We find that VEGF potently induces Ras activation and that this step is essential for the stimulation by VEGF of several cellular changes associated with angiogenesis, including proliferation, migration, and branching morphogenesis in three-dimensional culture. Inhibition of Ras signaling induced subtle changes in the actin architecture but had no effect on the phosphatidylinositol 3-kinase (PI3K) or p38 signaling pathways. In contrast, activation of ERK was largely dependent on Ras. Although inhibiting ERK activity completely suppressed cell proliferation and partially blocked in vitro differentiation, neither ERK nor PI3K activity was required for VEGF-induced migration. These data provide the first direct demonstration that inhibition of Ras signal transduction is anti-angiogenic. Interestingly, VEGF signal transduction bifurcates both upstream and downstream of Ras, with different Ras-dependent signals controlling endothelial cell proliferation and migration, essential components of the angiogenic response.     

Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells

The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-iota was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-iota with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-iota by NGF. At the level of Raf-1, neither PKC-iota nor PI3 kinase was required for activation; however, PKC-iota could weakly activate MEK. Inhibitors of PKC-iota activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-iota were likewise required for NGF-induced NF-kappaB activation and cell survival, whereas Ras was not required for either survival or NF-kappaB activation but was required for differentiation. IKK existed as a complex with PKC-iota, Src and IkappaB. Consistent with a role for Src in regulating NF-kappaB activation, an absence of Src activity impaired recruitment of PKC-iota into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-kappaB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-kappaB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-iota and the NF-kappaB pathway.     

Nerve growth factor signals through TrkA,phosphatidylinositol 3-kinase,and Rac1 to inactivate RhoA during the initiation of neuronal differentiation of PC12 cells

In PC12 rat pheochromocytoma cells, nerve growth factor (NGF)-induced neuronal differentiation is blocked by constitutively active dominant mutants of RhoA but augmented by negative ones, suggesting a not yet elucidated inhibitory signaling link between NGF receptors and RhoA. Here we show that NGF treatment rapidly translocates RhoA from the plasma membrane to the cytosol and simultaneously decreases RhoA affinity to its target Rho-associated kinase (ROK), a key mediator of neurite outgrowth. This effect was transient, because after 2 days of NGF treatment, RhoA relocated from the cytosol to the plasma membrane, and its GTP loading returned to a level found in undifferentiated cells. Inhibition of RhoA is mediated by activation of the TrkA receptor, because NGF failed to induce RhoA translocation and inhibition of ROK binding in nnr5 cells that lack TrkA, whereas the inhibition was reconstituted in receptor add-back B5 cells. In MM17-26 cells, which due to expression of dominant negative Ras do not differentiate, NGF-stimulated transient RhoA inhibition was unaffected. The inhibitory pathway from TrkA to RhoA involves phosphatidylinositol-3-kinase (PI3K), because the inhibitors LY294002 or wortmannin prevented NGF-induced RhoA translocation and increased RhoA association with ROK. Furthermore, inhibition of PI3K significantly reduced NGF- mediated Rac1 activation, whereas dominant negative Rac1 abolished the inhibitory signaling to RhoA. Taken together, these data indicate that NGF-mediated activation of TrkA receptor stimulates PI3K, which in turn increases Rac1 activity to induce transient RhoA inactivation during the initial phase of neurite outgrowth.     

G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility

Phosphoinositide 3-kinase (PI3K)gamma and Dictyostelium PI3K are activated via G protein-coupled receptors through binding to the Gbetagamma subunit and Ras. However, the mechanistic role(s) of Gbetagamma and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gbeta null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P(3). Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras-guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.     

Functional links between diacylglycerol and phosphatidylinositol signaling systems in human leukocyte-derived cell lines

In leukocytes, diacylglycerol (DAG) regulates the small GTPase Ras through the agency of Ras guanyl releasing proteins (RasGRPs). Ras is thought to regulate phosphatidylinositol 3-kinase (PI3K). Therefore, DAG signaling is hypothesized to impact PI3K activity. The DAG analogue “pico” was used to activate RasGRPs in leukocyte-derived cell lines. PI3K signaling was monitored using antibodies that recognize the activated form of the PI3K-regulated protein kinase Akt. Diverse responses were documented. Some cell lines exhibit a DAG analogue-stimulated increase in phospho-Akt but this response proceeded even when Ras activation was blocked. In some Epstein-Barr virus-associated malignant cell lines and transformed B cells, high basal phospho-Akt was decreased by DAG analogue treatment. The pan-PKC inhibitor Bisindolymaleimide I blocked this effect. Basal phospho-Akt was also decreased by treatment with Go6976, an inhibitor of conventional protein kinase C and protein kinase D. While the proposed RasGRP-Ras-PI3K-Akt signaling axis may operate in some situations, our results indicate that alternative links between DAG targets such as protein kinases and the PI3K signaling system are more prominent.     

Activation of PI3K and R‐ras signaling promotes the extension of sensory axons on inhibitory chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are extracellular inhibitors of axon extension and plasticity, and cause growth cones to exhibit dystrophic behaviors. Phosphoinositide 3‐kinase (PI3K) is a lipid kinase activated by axon growth promoting signals. In this study, we used embryonic chicken dorsal root ganglion neurons to determine if CSPGs impair signaling through PI3K. We report that CSPGs inhibit PI3K signaling in axons and growth cones, as evidenced by decreased levels of phosphorylated downstream kinases (Akt and S6). Direct activation of PI3K signaling, using a cell permeable phosphopeptide (PI3Kpep), countered the effects of CSPGs on growth cones and axon extension. Both overnight and acute treatment with PI3Kpep promoted axon extension on CSPG‐coated substrates. The R‐Ras GTPase is an upstream positive regulator of PI3K signaling. Expression of constitutively active R‐Ras promoted axon extension and growth cone elaboration on CSPGs and permissive substrata. In contrast, an N‐terminus‐deleted constitutively active R‐Ras, deficient in PI3K activation, promoted axon extension but not growth cone elaboration on CSPGs and permissive substrata. These data indicate that activation of R‐Ras‐PI3K signaling may be a viable approach for manipulating axon extension on CSPGs. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 918–933, 2014     

Pathway crosstalk between Ras/Raf and PI3K in promotion of M-CSF-induced MEK/ERK-mediated osteoclast survival

While M-CSF-mediated MEK/ERK activation promotes osteoclast survival, the signaling pathway by which M-CSF activates MEK/ERK is unresolved. Functions for PI3K, Ras, and Raf have been implicated in support of osteoclast survival, although interaction between these signaling components has not been examined. Therefore, the interplay between PI3K, Ras and Raf in M-CSF-promoted MEK/ERK activation and osteoclast survival was investigated. M-CSF activates Ras to coordinate activation of PI3K and Raf/MEK/ERK, since Ras inhibition decreased PI3K activation and PI3K inhibition did not block M-CSF-mediated Ras activation. As further support for Ras-mediated signaling, constitutively active (ca) Ras promoted MEK/ERK activation and osteoclast survival, which was blocked by inhibition of PI3K or Raf. Moreover, PI3K-selective or Raf-selective caRas were only partially able to promote osteoclast survival when compared to parental caRas. We then examined whether PI3K and Raf function linearly or in parallel downstream of Ras. Expression of caPI3K increased MEK/ERK activation and promoted osteoclast survival downstream of M-CSF, supporting this hypothesis. Blocking Raf did not decrease osteoclast survival and MEK/ERK activation promoted by caPI3K. In addition, PI3K-selective Ras-mediated survival was not blocked by Raf inhibition. Taken together, our data support that Raf signaling is separate from Ras/PI3K signaling and PI3K signaling is separate from Ras/Raf signaling. These data therefore support a role for Ras in coordinate activation of PI3K and Raf acting in parallel to mediate MEK/ERK-promoted osteoclast survival induced by M-CSF.     

Role of Per1-interacting protein of the suprachiasmatic nucleus in NGF mediated neuronal survival

We previously identified Per1-interacting protein of the suprachiasmatic nucleus (PIPS) in rats. To reveal its role, its tissue distribution was examined by immunoblotting. PIPS-like immunoreactive substance (PIPSLS) was observed in the brain, adrenal gland, and PC12 cells. Since PIPS, which has no nuclear localization signal (NLS), is translocated into nuclei of COS-7 cells in the presence of mPer1, the effect of NGF on nuclear localization of PIPS was examined using PC12 cells. NGF caused nuclear translocation of either PIPSLS or GFP-PIPS. NGF mediated nuclear translocation of PIPSLS was blocked by K252a, a TrkA-inhibitor, or wortmannin, a PI3K-inhibitor. Gab1, which is implicated in TrkA signaling and has NLS, co-immunoprecipitated with PIPSLS from PC12 cells using an anti-PIPS antibody. Inhibition of PIPS expression by RNAi increased levels of apoptosis in PC12 cells. These findings suggest that nuclear translocation of PIPS is involved in NGF mediated neuronal survival via TrkA, PI3K, and Gab1 signaling pathway.     

LET-60 RAS modulates effects of insulin/IGF-1 signaling on development and aging in Caenorhabditis elegans

The DAF-2 insulin/insulin-like growth factor 1 (IGF-1) receptor signals via a phosphatidylinositol 3-kinase (PI3K) pathway to control dauer larva formation and adult longevity in Caenorhabditis elegans. Yet epistasis analysis suggests signal bifurcation downstream of DAF-2. We have used epistasis analysis to test whether the Ras pathway (which plays a role in signaling from mammalian insulin receptors) acts downstream of DAF-2. We find that an activated Ras mutation, let-60(n1046gf), weakly suppresses constitutive dauer diapause in daf-2 and age-1 (PI3K) mutants. Moreover, increased Ras pathway signaling partially suppresses the daf-2 mutant feeding defect, while reduced Ras pathway signaling enhances it. By contrast, activated Ras extends the longevity induced by mutation of daf-2, while reduced Ras pathway signaling partially suppresses it. Thus, Ras pathway signaling appears to act with insulin/IGF-1 signaling during larval development, but against it during aging.     

Signal transduction at point-blank range: analysis of a spatial coupling mechanism for pathway crosstalk

The plasma membrane provides a physical platform for the orchestration of molecular interactions and biochemical conversions involved in the early stages of receptor-mediated signal transduction in living cells. In that context, we introduce here the concept of spatial coupling, wherein simultaneous recruitment of different enzymes to the same receptor scaffold facilitates crosstalk between different signaling pathways through the local release and capture of activated signaling molecules. To study the spatiotemporal dynamics of this mechanism, we have developed a Brownian dynamics modeling approach and applied it to the receptor-mediated activation of Ras and the cooperative recruitment of phosphoinositide 3-kinase (PI3K) by activated receptors and Ras. Various analyses of the model simulations show that cooperative assembly of multimolecular complexes nucleated by activated receptors is facilitated by the local release and capture of membrane-anchored signaling molecules (such as active Ras) from/by receptor-bound signaling proteins. In the case of Ras/PI3K crosstalk, the model predicts that PI3K is more likely to be recruited by activated receptors bound or recently visited by the enzyme that activates Ras. By this mechanism, receptor-bound PI3K is stabilized through short-range, diffusion-controlled capture of active Ras and Ras/PI3K complexes released from the receptor complex. We contend that this mechanism is a means by which signaling pathways are propagated and spatially coordinated for efficient crosstalk between them.     

NGF Attenuates High Glucose-Induced ER Stress,Preventing Schwann Cell Apoptosis by Activating the PI3K/Akt/GSK3β and ERK1/2 Pathways

Diabetic peripheral neuropathy (DPN) is one of the most common and troublesome complications of diabetes mellitus. It has been demonstrated that nerve growth factor (NGF) exerts a pivotal role in the regulation of neuronal growth and the promotion of DPN recovery. However, the exact molecular mechanisms are not well understood. Recent studies have indicated that as a novel therapeutic target, endoplasmic reticulum (ER) stress participates in the onset and progression of DPN. In the present study, it has been demonstrated that NGF prevents the sciatic nerve from degeneration and demyelination in DPN rats. Thus, RSC 96 cells, which retain the characteristic features of Schwann cells (SCs), were cultured in medium containing 30 mM glucose (high glucose, HG) to mimic SCs in DPN mice. The 50-ng/ml dose of NGF was identified to be the optimal concentration for treating an excessive ER stress level under HG conditions for 24 h. We found that NGF treatment significantly inhibits HG-induced ER stress and subsequently suppresses ER-related apoptosis. Further, NGF administration also activates the upstream signaling pathway of ER stress, PI3K/Akt/GSK3β signaling and ERK1/2 signaling. Co-treatment with the PI3K inhibitor LY294002 or ERK1/2 inhibitor U0126 significantly reverses the protective role of NGF on HG-induced excessive ER stress and subsequent apoptosis. These observations suggest that the neuroprotective role of NGF in DPN is mediated by the inhibition of excessive ER stress via the activation of the PI3K/Akt/GSK3β and ERK1/2 signaling pathways.     

NGF/PI3K signaling-mediated epigenetic regulation of delta opioid receptor gene expression

The G protein-coupled delta opioid receptor gene (dor) has been associated with neuronal survival, differentiation, and neuroprotection. Our previous study identified PI3K/Akt/NF-κB signaling is a main downstream signaling pathway in nerve growth factor (NGF)-induced temporal expression of the dor gene in the PC12 cell model. It is still unknown how NGF/PI3K signaling regulates the expression of the dor gene in the nucleus. In the current study, we investigated how PI3K signaling affected epigenetic modifications of histone H3 Lys⁹ (H3K9) in the 5′-UTR region of the rat dor gene locus. NGF treatment resulted in the global reversal of H3K9 trimethylation in cells. Moreover, the locus-specific reversal of H3K9 trimethylation and acetylation of H3K9 were dependent upon NGF/PI3K signaling and temporally well correlated with NGF-induced gene expression. These results indicate the importance of epigenetic modifications of H3K9, particularly the reversal of trimethylated H3K9, in the regulation of NGF/PI3K-dependent genes during neuronal differentiation.     

NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC

Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. Here we demonstrate that a tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of glycogen synthase kinase 3beta (GSK-3beta). These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). Our results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway.     

Molecularly targeting the PI3K-Akt-mTOR pathway can sensitize cancer cells to radiotherapy and chemotherapy

Radiotherapy and chemotherapeutic agents that damage DNA are the current major non-surgical means of treating cancer. However, many patients develop resistances to chemotherapy drugs in their later lives. The PI3K and Ras signaling pathways are deregulated in most cancers, so molecularly targeting PI3K-Akt or Ras-MAPK signaling sensitizes many cancer types to radiotherapy and chemotherapy, but the underlying molecular mechanisms have yet to be determined. During the multi-step processes of tumorigenesis, cancer cells gain the capability to disrupt the cell cycle checkpoint and increase the activity of CDK4/6 by disrupting the PI3K, Ras, p53, and Rb signaling circuits. Recent advances have demonstrated that PI3K-Akt-mTOR signaling controls FANCD2 and ribonucleotide reductase (RNR). FANCD2 plays an important role in the resistance of cells to DNA damage agents and the activation of DNA damage checkpoints, while RNR is critical for the completion of DNA replication and repair in response to DNA damage and replication stress. Regulation of FANCD2 and RNR suggests that cancer cells depend on PI3K-Akt-mTOR signaling for survival in response to DNA damage, indicating that the PI3K-AktmTOR pathway promotes resistance to chemotherapy and radiotherapy by enhancing DNA damage repair.     

Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells.