Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer.

The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2 and PI(3,4,5)P3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.

PtdIns(4,5)P2 signaling regulates ATG14 and autophagy.

Autophagy is a regulated self-digestion pathway with fundamental roles in cell homeostasis and diseases. Autophagy is regulated by coordinated actions of a series of autophagy-related (ATG) proteins. The Barkor/ATG14(L)-VPS34 (a class III phosphatidylinositol 3-kinase) complex and its product phosphatidylinositol 3-phosphate [PtdIns(3)P] play key roles in autophagy initiation. ATG14 contains a C-terminal Barkor/ATG14(L) autophagosome-targeting sequence (BATS) domain that senses the curvature of PtdIns(3)P-containing membrane. The BATS domain also strongly binds PtdIns(4,5)P2, but the functional significance has been unclear. Here we show that ATG14 specifically interacts with type Iγ PIP kinase isoform 5 (PIPKIγi5), an enzyme that generates PtdIns(4,5)P2 in mammalian cells. Autophagosomes have associated PIPKIγi5 and PtdIns(4,5)P2 that are colocalized with late endosomes and the endoplasmic reticulum. PtdIns(4,5)P2 generation at these sites requires PIPKIγi5. Loss of PIPKIγi5 results in a loss of ATG14, UV irradiation resistance-associated gene, and Beclin 1 and a block of autophagy. PtdIns(4,5)P2 binding to the ATG14-BATS domain regulates ATG14 interaction with VPS34 and Beclin 1, and thus plays a key role in ATG14 complex assembly and autophagy initiation. This study identifies an unexpected role for PtdIns(4,5)P2 signaling in the regulation of ATG14 complex and autophagy.

IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration.

Phosphatidylinositol 4,5 bisphosphate (PIP2) is a key lipid messenger for regulation of cell migration. PIP2 modulates many effectors, but the specificity of PIP2 signalling can be defined by interactions of PIP2-generating enzymes with PIP2 effectors. Here, we show that type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP2 effector that directly binds PIP2 through a polybasic motif and PIP2 binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP2 binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.

Emerging roles of PtdIns(4,5)P2--beyond the plasma membrane.

Phosphoinositides are a collection of lipid messengers that regulate most subcellular processes. Amongst the seven phosphoinositide species, the roles for phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at the plasma membrane, such as in endocytosis, exocytosis, actin polymerization and focal adhesion assembly, have been extensively studied. Recent studies have argued for the existence of PtdIns(4,5)P2 at multiple intracellular compartments, including the nucleus, endosomes, lysosomes, autolysosomes, autophagic precursor membranes, ER, mitochondria and the Golgi complex. Although the generation, regulation and functions of PtdIns(4,5)P2 are less well-defined in most other intracellular compartments, accumulating evidence demonstrates crucial roles for PtdIns(4,5)P2 in endolysosomal trafficking, endosomal recycling, as well as autophagosomal pathways, which are the focus of this Commentary. We summarize and discuss how phosphatidylinositol phosphate kinases, PtdIns(4,5)P2 and PtdIns(4,5)P2-effectors regulate these intracellular protein and membrane trafficking events.

Phosphatidylinositol Phosphate 5-Kinase Iγ and Phosphoinositide 3-Kinase/Akt Signaling Couple to Promote Oncogenic Growth.

The assembly of signaling complexes at the plasma membrane is required for the initiation and propagation of cellular signaling upon cell activation. The class I PI3K and the serine/threonine-specific protein kinase Akt signaling pathways (PI3K/Akt) are often activated in tumors. These pathways are initiated by the generation of phosphatidylinositol 3,4,5-triphosphate (PIP3) by PI3K-mediated phosphorylation of phosphatidylinositol 4,5-biphosphate (PIP2), synthesized by phosphatidylinositol 4-phosphate 5-kinase (PIPKI) enzymes. The mechanism of how tumor cells recruit and organize the PIP2-synthesizing enzymes with PI3K in the plasma membrane for activation of PI3K/Akt signaling is not defined. Here, we demonstrated a role for the phosphatidylinositol 4-phosphate 5-kinase Iγ (PIPKIγ) in PI3K/Akt signaling. PIPKIγ is overexpressed in triple-negative breast cancers. Loss of PIPKIγ or its focal adhesion-targeting variant, PIPKIγi2, impaired PI3K/Akt activation upon stimulation with growth factors or extracellular matrix proteins in different tumor cells. PIPKIγi2 assembles into a complex containing Src and PI3K; Src was required for the recruitment of PI3K enzyme into the complex. PIPKIγi2 interaction with Src and its lipid kinase activity were required for promoting PI3K/Akt signaling. These results define a mechanism by which PIPKIγi2 and PI3K are integrated into a complex regulated by Src, resulting in the spatial generation of PIP2, which is the substrate PI3K required for PIP3 generation and subsequent Akt activation. This study elucidates the mechanism by which PIP2-generating enzyme controls Akt activation upstream of a PI3K enzyme. This pathway may represent a signaling nexus required for the survival and growth of metastasizing and circulating tumor cells in vivo.

PIP kinases define PI4,5P2signaling specificity by association with effectors.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P2) is an essential lipid messenger with roles in all eukaryotes and most aspects of human physiology. By controlling the targeting and activity of its effectors, PI4,5P2modulates processes, such as cell migration, vesicular trafficking, cellular morphogenesis, signaling and gene expression. In cells, PI4,5P2has a much higher concentration than other phosphoinositide species and its total content is largely unchanged in response to extracellular stimuli. The discovery of a vast array of PI4,5P2 binding proteins is consistent with data showing that the majority of cellular PI4,5P2is sequestered. This supports a mechanism where PI4,5P2functions as a localized and highly specific messenger. Further support of this mechanism comes from the de novo synthesis of PI4,5P2which is often linked with PIP kinase interaction with PI4,5P2effectors and is a mechanism to define specificity of PI4,5P2signaling. The association of PI4,5P2-generating enzymes with PI4,5P2effectors regulate effector function both temporally and spatially in cells. In this review, the PI4,5P2effectors whose functions are tightly regulated by associations with PI4,5P2-generating enzymes will be discussed. This article is part of a Special Issue entitled Phosphoinositides.

Isoform 5 of PIPKIγ regulates the endosomal trafficking and degradation of E-cadherin.

Phosphatidylinositol phosphate kinases (PIPKs) have distinct cellular targeting, allowing for site-specific synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to activate specific signaling cascades required for cellular processes. Several C-terminal splice variants of PIPKIγ (also known as PIP5K1C) exist, and have been implicated in a multitude of cellular roles. PI(4,5)P2 serves as a fundamental regulator of E-cadherin transport, and PI(4,5)P2-generating enzymes are important signaling relays in these pathways. We present evidence that the isoform 5 splice variant of PIPKIγ (PIPKIγi5) associates with E-cadherin and promotes its lysosomal degradation. Additionally, we show that the endosomal trafficking proteins SNX5 and SNX6 associate with PIPKIγi5 and inhibit PIPKIγi5-mediated E-cadherin degradation. Following HGF stimulation, activated Src directly phosphorylates PIPKIγi5. Phosphorylation of the PIPKIγi5 C-terminus regulates its association with SNX5 and, consequently, E-cadherin degradation. Additionally, this PIPKIγi5-mediated pathway requires Rab7 to promote degradation of internalized E-cadherin. Taken together, the data indicate that PIPKIγi5 and SNX5 are crucial regulators of E-cadherin sorting and degradation. PIPKIγi5, SNX and phosphoinositide regulation of lysosomal sorting represent a novel area of PI(4,5)P2 signaling and research. PIPKIγi5 regulation of E-cadherin sorting for degradation might have broad implications in development and tissue maintenance, and enhanced PIPKIγi5 function might have pathogenic consequences due to downregulation of E-cadherin.

Phosphatidylinositol phosphate 5-kinase Iγi2 in association with Src controls anchorage-independent growth of tumor cells.

A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.

Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells.

Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesion-dependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesion-dependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer.

Cross-talk between TGFß1 and EGFR signalling pathways induces TM4SF5 expression and epithelial-mesenchymal transition.

The EMT (epithelial-mesenchymal transition) is involved in fibrosis and cancer, and is regulated by different signalling pathways mediated through soluble factors, actin reorganization and transcription factor actions. Because the tetraspan (also called tetraspanin) TM4SF5 (transmembrane 4 L6 family member 5) is highly expressed in hepatocellular carcinoma and induces EMT, understanding how TM4SF5 expression in hepatocytes is regulated is important. We explored the mechanisms that induce TM4SF5 expression and whether impaired signalling pathways for TM4SF5 expression inhibit the acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. We found that TGFß1 (transforming growth factor ß1)-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the EGFR [EGF (epidermal growth factor) receptor] pathway. Inhibition of EGFR activity following TGFß1 treatment abolished acquisition of EMT, suggesting a link from Smads to EGFR for TM4SF5 expression. Further, TGFß1-mediated EGFR activation and TM4SF5 expression were abolished by EGFR suppression or extracellular EGF depletion. Smad overexpression mediated EGFR activation and TM4SF5 expression in the absence of serum, and EGFR kinase inactivation or EGF depletion abolished Smad-overexpression-induced TM4SF5 and mesenchymal cell marker expression. Inhibition of Smad, EGFR or TM4SF5 using Smad7 or small compounds also blocked TM4SF5 expression and/or EMT. These results indicate that TGFß1- and growth factor-mediated signalling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features, suggesting that TM4SF5 induction may be involved in the development of liver pathologies.

Electromagnetic drag forces between HTS magnet and tube infrastructure for hyperloop.

Maglevs are typically accelerated using electromagnetic propulsion and levitation. High-temperature superconducting (HTS) magnets along with electrodynamic suspension (EDS) and linear synchronous motors are one of the best options for Hyperloop. However, the strong magnetic fields generated by HTS magnets on the pods inevitably interact with the magnetic and conductive structures in the vacuum tubes, along with the tube itself, while the pods move through the tubes. This interaction is observed as a drag force on the pods, significantly reducing the propulsion efficiency. This study comprehensively analyzes the electromagnetic drag force (EDF) generated by HTS magnets on pods, which accounts for most of the drag forces faced by Hyperloop. Theoretical analysis and 3D FEA simulations are performed to analyze the propulsion forces with HTS magnets and all the drag forces on the pods. The EDF generated by AISI 1010 steel rebars in concrete guideways is even greater than the designed propulsion forces of 40 kN. Consequently, high-manganese (Hi-Mn) steel and insulated steel rebars are adopted and analyzed using 3D FEA simulations. The EDFs generated by the AISI 1010 steel and Hi-Mn steel vacuum tubes are determined by varying the distance between the HTS magnets and tubes at 50 and 1200 km/h, respectively; a minimum distance of 0.75 m is determined by a drag force below 8 kN within their operating velocities. Lastly, the total EDFs of the AISI 1010 steel and Hi-Mn steel tubes with EDS rails are obtained through the optimal design of rebars and tubes. The simulation results show that the total EDFs can be significantly reduced to below 10 kN (approximately 25% of the designed propulsion force after the levitation of pods).

A p53-phosphoinositide signalosome regulates nuclear AKT activation.

The tumour suppressor p53 and PI3K-AKT pathways have fundamental roles in the regulation of cell growth and apoptosis, and are frequently mutated in cancer. Here, we show that genotoxic stress induces nuclear AKT activation through a p53-dependent mechanism that is distinct from the canonical membrane-localized PI3K-AKT pathway. Following genotoxic stress, a nuclear PI3K binds p53 in the non-membranous nucleoplasm to generate a complex of p53 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which recruits AKT, PDK1 and mTORC2 to activate AKT and phosphorylate FOXO proteins, thereby inhibiting DNA damage-induced apoptosis. Wild-type p53 activates nuclear AKT in an on/off fashion following stress, whereas mutant p53 dose-dependently stimulates high basal AKT activity. The p53-PtdIns(3,4,5)P3 complex is dephosphorylated to p53-phosphatidylinositol 4,5-bisphosphate by PTEN to inhibit AKT activation. The nuclear p53-phosphoinositide signalosome is distinct from the canonical membrane-localized pathway and insensitive to PI3K inhibitors currently in the clinic, which underscores its therapeutic relevance.

Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma.

The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27Kip1, as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT.

Cooperation between integrin alpha5 and tetraspan TM4SF5 regulates VEGF-mediated angiogenic activity.

Tetraspan TM4SF5 is highly expressed in a diverse number of tumor types. Here we explore the mechanistic roles of TM4SF5 in angiogenesis. We found that TM4SF5 overexpression correlates with vascular endothelial growth factor (VEGF) expression in SNU449 hepatocytes and with vessel formation in clinical hepatocarcinoma samples. Conditioned media from TM4SF5-expressing cells enhanced viability and tube formation of primary human umbilical vein endothelial cells, and outgrowth of endothelial cells from aorta ring segments, which was abolished by treatment with an anti-VEGF antibody. TM4SF5 retained integrin alpha(5) on the cell surface for VEGF induction, and preincubation with anti-integrin alpha(5) antibody abolished TM4SF5-mediated VEGF expression and secretion. TM4SF5-mediated effects required integrin alpha(5), c-Src, and signal transducer and activator of transcription 3 (STAT3). In addition, tumors from nude mice injected with TM4SF5-expressing cells and from clinical human hepatocarcinoma tissues showed enhanced integrin alpha(5) expression, vessel formation, and signaling activity, which were inhibited by administration of anti-integrin alpha(5) or -VEGF antibody. This study suggests that TM4SF5 facilitates angiogenesis of neighboring endothelial cells through VEGF induction, mediated by cooperation between TM4SF5 and integrin alpha(5) of epithelial cells.

Equivalent inductance model for the design analysis of electrodynamic suspension coils for hyperloop.

Hyperloop is a new concept of ground transportation. In Hyperloop, travelling occurs in near-vacuum tubes under 0.001 atm at a subsonic speed of up to 1200 km/h. During acceleration to and driving at a subsonic speed, magnetic levitation is employed. Thus far, various levitation technologies in existing high-speed maglev trains have been considered. Among those technologies, superconducting (SC) electrodynamic suspension (EDS) is a highly effective levitation system for Hyperloop owing to its advantages of a large levitation gap, levitation stability, and control being unnecessary. However, analyzing an EDS system requires the electromagnetic transient analysis of complex three-dimensional (3D) features, and its computational load generally limits the use of numerical methods, such as the 3D finite element method (FEM) or dynamic circuit theory. In this study, a novel model that can rapidly and accurately calculate the frequency-dependent equivalent inductance was developed. The developed model was then applied to design an EDS system using the decoupled resistance-inductance equations of levitation coils. Next, levitation coils of SC-EDS were designed and analyzed for use in Hyperloop. The obtained results were compared with the FEM results to validate the developed model. In addition, the model was experimentally validated by measuring currents induced by moving pods.

Role of IQGAP1 in Papillomavirus-Associated Head and Neck Tumorigenesis.

Approximately 25% of head and neck squamous cell carcinomas (HNSCC) are associated with human papillomavirus (HPV) infection. In these cancers as well as in HPV-associated anogenital cancers, PI3K signaling is highly activated. We previously showed that IQ motif-containing GTPase activating protein 1 (IQGAP1), a PI3K pathway scaffolding protein, is overexpressed in and contributes to HNSCC and that blocking IQGAP1-mediated PI3K signaling reduces HPV-positive HNSCC cell survival and migration. In this study, we tested whether IQGAP1 promotes papillomavirus (PV)-associated HNSCCs. IQGAP1 was necessary for optimal PI3K signaling induced by HPV16 oncoproteins in transgenic mice and MmuPV1 infection, a mouse papillomavirus that causes HNSCC in mice. Furthermore, we found that, at 6 months post-infection, MmuPV1-infected Iqgap1-/- mice developed significantly less severe tumor phenotypes than MmuPV1-infected Iqgap1+/+ mice, indicating a role of IQGAP1 in MmuPV1-associated HNSCC. The tumors resulting from MmuPV1 infection showed features consistent with HPV infection and HPV-associated cancer. However, such IQGAP1-dependent effects on disease severity were not observed in an HPV16 transgenic mouse model for HNC. This may reflect that IQGAP1 plays a role in earlier stages of viral pathogenesis, or other activities of HPV16 oncogenes are more dominant in driving carcinogenesis than their influence on PI3K signaling.

Specific tyrosine phosphorylation of focal adhesion kinase mediated by Fer tyrosine kinase in suspended hepatocytes.

Cell adhesion to the extracellular matrix (ECM) can activate signaling via focal adhesion kinase (FAK) leading to dynamic regulation of cellular morphology. Mechanistic basis for the lack of effective intracellular signaling by non-attached epithelial cells is poorly understood. To examine whether signaling in suspended cells is regulated by Fer cytoplasmic tyrosine kinase, we investigated the effect of ectopic Fer expression on signaling in suspended or adherent hepatocytes. We found that ectopic Fer expression in Huh7 hepatocytes in suspension or on non-permissive poly-lysine caused significant phosphorylation of FAK Tyr577, Tyr861, or Tyr925, but not Tyr397 or Tyr576. Fer-mediated FAK phosphorylation in suspended cells was independent of c-Src activity or growth factor stimulation, but dependent of cortactin expression. Consistent with these results, complex formation between FAK, Fer, and cortactin was observed in suspended cells. The Fer-mediated effect correlated with multiple membrane protrusions, even on poly-lysine. Together, these observations suggest that Fer may allow a bypass of anchorage-dependency for intracellular signal transduction in hepatocytes.

Blockade of four-transmembrane L6 family member 5 (TM4SF5)-mediated tumorigenicity in hepatocytes by a synthetic chalcone derivative.

UNLABELLED: We previously reported that the four-transmembrane L6 family member 5 (TM4SF5) was highly expressed in hepatocarcinoma, induced morphological elongation and epithelial-mesenchymal transition, and caused abnormal cell growth in multilayers in vitro and tumor formation in vivo. In this study, we identified a synthetic compound, 4'-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC) that antagonized both the TM4SF5-mediated multilayer growth and TM4SF5-enhanced migration/invasion. TSAHC treatment induced multilayer-growing cells to grow in monolayers, recovering contact inhibition without accompanying apoptosis, and inhibited chemotactic migration and invasion. Tumor formation in nude mice injected with TM4SF5-expressing cells and the growth of cells expressing endogenous TM4SF5, but not of TM4SF5-null cells, was suppressed by treatment with TSAHC, but not by treatment with its analogs. The structure-activity relationship indicated the significance of 4'-p-toluenesulfonylamido and 4-hydroxy groups for the anti-TM4SF5 effects of TSAHC. Point mutations of the putative N-glycosylation sites abolished the TM4SF5-specific TSAHC responsiveness. CONCLUSION: These observations suggest that TM4SF5-enhanced tumorigenic proliferation and metastatic potential can be blocked by TSAHC, likely through targeting the extracellular region of TM4SF5, which is important for protein-protein interactions.

Phosphatidylinositol-3-OH kinase signalling is spatially organized at endosomal compartments by microtubule-associated protein 4.

The canonical model of agonist-stimulated phosphatidylinositol-3-OH kinase (PI3K)-Akt signalling proposes that PI3K is activated at the plasma membrane, where receptors are activated and phosphatidylinositol-4,5-bisphosphate is concentrated. Here we show that phosphatidylinositol-3,4,5-trisphosphate generation and activated Akt are instead largely confined to intracellular membranes upon receptor tyrosine kinase activation. Microtubule-associated protein 4 (MAP4) interacts with and controls localization of membrane vesicle-associated PI3Kα to microtubules. The microtubule-binding domain of MAP4 binds directly to the C2 domain of the p110α catalytic subunit. MAP4 controls the interaction of PI3Kα with activated receptors at endosomal compartments along microtubules. Loss of MAP4 results in the loss of PI3Kα targeting and loss of PI3K-Akt signalling downstream of multiple agonists. The MAP4-PI3Kα assembly defines a mechanism for spatial control of agonist-stimulated PI3K-Akt signalling at internal membrane compartments linked to the microtubule network.