A p85 isoform switch enhances PI3K activation on endosomes by a MAP4- and PI3P-dependent mechanism.

Phosphatidylinositol 3-kinase α (PI3Kα) is a heterodimer of p110α catalytic and p85 adaptor subunits that is activated by agonist-stimulated receptor tyrosine kinases. Although p85α recruits p110α to activated receptors on membranes, p85α loss, which occurs commonly in cancer, paradoxically promotes agonist-stimulated PI3K/Akt signaling. p110α localizes to microtubules via microtubule-associated protein 4 (MAP4), facilitating its interaction with activated receptor kinases on endosomes to initiate PI3K/Akt signaling. Here, we demonstrate that in response to agonist stimulation and p85α knockdown, the residual p110α, coupled predominantly to p85ß, exhibits enhanced recruitment with receptor tyrosine kinases to endosomes. Moreover, the p110α C2 domain binds PI3-phosphate, and this interaction is also required to recruit p110α to endosomes and for PI3K/Akt signaling. Stable knockdown of p85α, which mimics the reduced p85α levels observed in cancer, enhances cell growth and tumorsphere formation, and these effects are abrogated by MAP4 or p85ß knockdown, underscoring their role in the tumor-promoting activity of p85α loss.

Regulation of Cell Adhesion and Migration via Microtubule Cytoskeleton Organization, Cell Polarity, and Phosphoinositide Signaling.

The capacity for cancer cells to metastasize to distant organs depends on their ability to execute the carefully choreographed processes of cell adhesion and migration. As most human cancers are of epithelial origin (carcinoma), the transcriptional downregulation of adherent/tight junction proteins (e.g., E-cadherin, Claudin and Occludin) with the concomitant gain of adhesive and migratory phenotypes has been extensively studied. Most research and reviews on cell adhesion and migration focus on the actin cytoskeleton and its reorganization. However, metastasizing cancer cells undergo the extensive reorganization of their cytoskeletal system, specifically in originating/nucleation sites of microtubules and their orientation (e.g., from non-centrosomal to centrosomal microtubule organizing centers). The precise mechanisms by which the spatial and temporal reorganization of microtubules are linked functionally with the acquisition of an adhesive and migratory phenotype as epithelial cells reversibly transition into mesenchymal cells during metastasis remains poorly understood. In this Special Issue of "Molecular Mechanisms Underlying Cell Adhesion and Migration", we highlight cell adhesion and migration from the perspectives of microtubule cytoskeletal reorganization, cell polarity and phosphoinositide signaling.

Regulation of Phosphoinositide Signaling by Scaffolds at Cytoplasmic Membranes.

Cytoplasmic phosphoinositides (PI) are critical regulators of the membrane-cytosol interface that control a myriad of cellular functions despite their low abundance among phospholipids. The metabolic cycle that generates different PI species is crucial to their regulatory role, controlling membrane dynamics, vesicular trafficking, signal transduction, and other key cellular events. The synthesis of phosphatidylinositol (3,4,5)-triphosphate (PI3,4,5P3) in the cytoplamic PI3K/Akt pathway is central to the life and death of a cell. This review will focus on the emerging evidence that scaffold proteins regulate the PI3K/Akt pathway in distinct membrane structures in response to diverse stimuli, challenging the belief that the plasma membrane is the predominant site for PI3k/Akt signaling. In addition, we will discuss how PIs regulate the recruitment of specific scaffolding complexes to membrane structures to coordinate vesicle formation, fusion, and reformation during autophagy as well as a novel lysosome repair pathway.

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.

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.

The nuclear phosphoinositide response to stress.

Accumulating evidence reveals that nuclear phosphoinositides (PIs) serve as central signaling hubs that control a multitude of nuclear processes by regulating the activity of nuclear proteins. In response to cellular stressors, PIs accumulate in the nucleus and multiple PI isomers are synthesized by the actions of PI-metabolizing enzymes, kinases, phosphatases and phospholipases. By directly interacting with effector proteins, phosphoinositide signals transduce changes in cellular functions. Here we describe nuclear phosphoinositide signaling in multiple sub-nuclear compartments and summarize the literature that demonstrates roles for specific kinases, phosphatases, and phospholipases in the orchestration of nuclear phosphoinositide signaling in response to cellular stress. Additionally, we discuss the specific PI-protein complexes through which these lipids execute their functions by regulating the configuration, stability, and transcription activity of their effector proteins. Overall, our review provides a detailed landscape of the current understanding of the nuclear PI-protein interactome and its role in shaping the coordinated response to cellular stress.

The Specificity of EGF-Stimulated IQGAP1 Scaffold Towards the PI3K-Akt Pathway is Defined by the IQ3 motif.

Epidermal growth factor receptor (EGFR) and its downstream phosphoinositide 3-kinase (PI3K) pathway are commonly deregulated in cancer. Recently, we have shown that the IQ motif-containing GTPase-activating protein 1 (IQGAP1) provides a molecular platform to scaffold all the components of the PI3K-Akt pathway and results in the sequential generation of phosphatidylinositol-3,4,5-trisphosphate (PI3,4,5P3). In addition to the PI3K-Akt pathway, IQGAP1 also scaffolds the Ras-ERK pathway. To define the specificity of IQGAP1 for the control of PI3K signaling, we have focused on the IQ3 motif in IQGAP1 as PIPKIα and PI3K enzymes bind this region. An IQ3 deletion mutant loses interactions with the PI3K-Akt components but retains binding to ERK and EGFR. Consistently, blocking the IQ3 motif of IQGAP1 using an IQ3 motif-derived peptide mirrors the effect of IQ3 deletion mutant by reducing Akt activation but has no impact on ERK activation. Also, the peptide disrupts the binding of IQGAP1 with PI3K-Akt pathway components, while IQGAP1 interactions with ERK and EGFR are not affected. Functionally, deleting or blocking the IQ3 motif inhibits cell proliferation, invasion, and migration in a non-additive manner to a PIPKIα inhibitor, establishing the functional specificity of IQ3 motif towards the PI3K-Akt pathway. Taken together, the IQ3 motif is a specific target for suppressing activation of the PI3K-Akt but not the Ras-ERK pathway. Although EGFR stimulates the IQGAP1-PI3K and -ERK pathways, here we show that IQGAP1-PI3K controls migration, invasion, and proliferation independent of ERK. These data illustrate that the IQ3 region of IQGAP1 is a promising therapeutic target for PI3K-driven cancer.

Phosphoinositide spatially free AKT/PKB activation to all membrane compartments.

Ser and Thr kinase AKT also known as protein kinase B (PKB) was discovered more than two and half decades ago and is one of the key downstream molecules in the phosphoinositide 3-kinase signaling pathways. The pleiotropic effects of this kinase have attracted intense interest and limelight in cancer biology, cancer therapy, diabetes, and cardiovascular diseases. Authors may refer to other more comprehensive and recent reviews on AKT/PKB (Manning and Cantley, 2007; Manning and Toker, 2017). AKT/PKB is one of the most enigmatic and most studied signaling molecule in cancers and is a significant therapeutic target (Brown and Banerji, 2017). Yet, how AKT/PKB activation couples with its downstream target/substrate molecules that function in diverse subcellular compartments remains obscure. Recent studies indicate the continuous interaction of AKT/PKB with PI3,4,5P3 or PI3,4P2 in a lipid membrane is required for its activation throughout the cells (Ebner et al., 2017). Here, we summarize the recent progress on the mechanism for phosphoinositide (PI3,4,5P3 and PI3,4P2) spatial control of AKT/PKB activation on the plasma membrane and endomembrane compartments.

PLD and PA Take MT1-MMP for a Metastatic Ride.

Polarized targeting and deposition of MT1-MMP is pivotal for metastasis. In this issue of Developmental Cell, Wang et al. (2017) reveal that a signaling molecule generated by phospholipase D2 drives deposition of MT1-MMP at the site of invadopodia formation and is critical for metastasis in a transgenic breast cancer model.

The Hidden Conundrum of Phosphoinositide Signaling in Cancer.

Phosphoinositide 3-kinase (PI3K) generation of PI(3,4,5)P3 from PI(4,5)P2 and the subsequent activation of Akt and its downstream signaling cascades (e.g. mTORC1) dominates the landscape of phosphoinositide signaling axis in cancer research. However, PI(4,5)P2 is breaking its boundary as merely a substrate for PI3K and phospholipase C (PLC), and is now an established lipid messenger pivotal for different cellular events in cancer. Here, we review the phosphoinositide signaling axis in cancer, giving due weight to PI(4,5)P2 and its generating enzymes, the phosphatidylinositol phosphate (PIP) kinases (PIPKs). We highlighted how PI(4,5)P2 and PIP kinases serve as a proximal node in phosphoinositide signaling axis and how its interaction with cytoskeletal proteins regulates migratory and invasive nexus of metastasizing tumor cells.

Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases.

Generation of the lipid messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) is crucial for development, cell growth and survival, and motility, and it becomes dysfunctional in many diseases including cancers. Here we reveal a mechanism for PtdIns(3,4,5)P3 generation by scaffolded phosphoinositide kinases. In this pathway, class I phosphatidylinositol-3-OH kinase (PI(3)K) is assembled by IQGAP1 with PI(4)KIIIα and PIPKIα, which sequentially generate PtdIns(3,4,5)P3 from phosphatidylinositol. By scaffolding these kinases into functional proximity, the PtdIns(4,5)P2 generated is selectively used by PI(3)K for PtdIns(3,4,5)P3 generation, which then signals to PDK1 and Akt that are also in the complex. Moreover, multiple receptor types stimulate the assembly of this IQGAP1-PI(3)K signalling complex. Blockade of IQGAP1 interaction with PIPKIα or PI(3)K inhibited PtdIns(3,4,5)P3 generation and signalling, and selectively diminished cancer cell survival, revealing a target for cancer chemotherapy.

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.

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.

LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling, lysosomal sorting, and degradation.

Lysosomal degradation is essential for the termination of EGF-stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi-vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF-induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT-0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P2 and recruiting SNX5. PtdIns(4,5)P2 and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P2 signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression.

A kinase-independent role for EGF receptor in autophagy initiation.

The epidermal growth factor receptor (EGFR) is upregulated in numerous human cancers. Inhibition of EGFR signaling induces autophagy in tumor cells. Here, we report an unanticipated role for the inactive EGFR in autophagy initiation. Inactive EGFR interacts with the oncoprotein LAPTM4B that is required for the endosomal accumulation of EGFR upon serum starvation. Inactive EGFR and LAPTM4B stabilize each other at endosomes and recruit the exocyst subcomplex containing Sec5. We show that inactive EGFR, LAPTM4B, and the Sec5 subcomplex are required for basal and starvation-induced autophagy. LAPTM4B and Sec5 promote EGFR association with the autophagy inhibitor Rubicon, which in turn disassociates Beclin 1 from Rubicon to initiate autophagy. Thus, the oncoprotein LAPTM4B facilitates the role of inactive EGFR in autophagy initiation. This pathway is positioned to control tumor metabolism and promote tumor cell survival upon serum deprivation or metabolic stress.

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.

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.

Phosphatidylinositol 4,5-bisphosphate: targeted production and signaling.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) is a key lipid signaling molecule that regulates a vast array of biological activities. PI4,5P(2) can act directly as a messenger or can be utilized as a precursor to generate other messengers: inositol trisphosphate, diacylglycerol, or phosphatidylinositol 3,4,5-trisphosphate. PI4,5P(2) interacts with hundreds of different effector proteins. The enormous diversity of PI4,5P(2) effector proteins and the spatio-temporal control of PI4,5P(2) generation allow PI4,5P(2) signaling to control a broad spectrum of cellular functions. PI4,5P(2) is synthesized by phosphatidylinositol phosphate kinases (PIPKs). The array of PIPKs in cells enables their targeting to specific subcellular compartments through interactions with targeting factors that are often PI4,5P(2) effectors. These interactions are a mechanism to define spatial and temporal PI4,5P(2) synthesis and the specificity of PI4,5P(2) signaling. In turn, the regulation of PI4,5P(2) effectors at specific cellular compartments has implications for understanding how PI4,5P(2) controls cellular processes and its role in diseases.