Shunsuke Takasuga

Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) regulate cell migration, but the role of PtdIns(3,4,5)P3-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P3 phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P3 phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P3 metabolism in living primary cells, we generated a novel transgenic mouse (AktPH–GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P3. Time-lapse footage showed rapid, localized binding of AktPH–GFP to the leading edge membrane of chemotaxing ship1+/+AktPH–GFP Tg neutrophils, but only diffuse localization in ship1−/−AktPH–GFP Tg neutrophils. By directing where PtdIns(3,4,5)P3 accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.

Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis.

During chemotaxis, activation of the small guanosine triphosphatase Rac is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate–dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D–mediated synthesis of phosphatidic acid, which stabilized DOCK2 there by means of interaction with a polybasic amino acid cluster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.

Mammalian phosphoinositide kinases and phosphatases.

Phosphoinositides are lipids that are present in the cytoplasmic leaflet of a cells plasma and internal membranes and play pivotal roles in the regulation of a wide variety of cellular processes. Phosphoinositides are molecularly diverse due to variable phosphorylation of the hydroxyl groups of their inositol rings. The rapid and reversible configuration of the seven known phosphoinositide species is controlled by a battery of phosphoinositide kinases and phosphoinositide phosphatases, which are thus critical for phosphoinositide isomer-specific localization and functions. Significantly, a given phosphoinositide generated by different isozymes of these phosphoinositide kinases and phosphatases can have different biological effects. In mammals, close to 50 genes encode the phosphoinositide kinases and phosphoinositide phosphatases that regulate phosphoinositide metabolism and thus allow cells to respond rapidly and effectively to ever-changing environmental cues. Understanding the distinct and overlapping functions of these phosphoinositide-metabolizing enzymes is important for our knowledge of both normal human physiology and the growing list of human diseases whose etiologies involve these proteins. This review summarizes the structural and biological properties of all the known mammalian phosphoinositide kinases and phosphoinositide phosphatases, as well as their associations with human disorders.

The PtdIns(3,4)P2 phosphatase INPP4A is a suppressor of excitotoxic neuronal death

Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P2 phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a-/- mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P2, PtdIns(3,4,5)P3 and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P2 metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P2 metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders.

Regulation of anaphylactic responses by phosphatidylinositol phosphate kinase type I α

The membrane phospholipid phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] is a critical signal transducer in eukaryotic cells. However, the physiological roles of the type I phosphatidylinositol phosphate kinases (PIPKIs) that synthesize PI(4,5)P2 are largely unknown. Here, we show that the α isozyme of PIPKI (PIPKIα) negatively regulates mast cell functions and anaphylactic responses. In vitro, PIPKIα-deficient mast cells exhibited increased degranulation and cytokine production after Fcɛ receptor-I cross-linking. In vivo, PIPKIα−/− mice displayed enhanced passive cutaneous and systemic anaphylaxis. Filamentous actin was diminished in PIPKIα−/− mast cells, and enhanced degranulation observed in the absence of PIPKIα was also seen in wild-type mast cells treated with latrunculin, a pharmacological inhibitor of actin polymerization. Moreover, the association of FcɛRI with lipid rafts and FcɛRI-mediated activation of signaling proteins was augmented in PIPKIα−/− mast cells. Thus, PIPKIα is a negative regulator of FcɛRI-mediated cellular responses and anaphylaxis, which functions by controlling the actin cytoskeleton and dynamics of FcɛRI signaling. Our results indicate that the different PIPKI isoforms might be functionally specialized.

Delivery of endosomes to lysosomes via microautophagy in the visceral endoderm of mouse embryos

The differentiation and patterning of murine early embryos are sustained by the visceral endoderm, an epithelial layer of polarised cells that has critical roles in multiple signalling pathways and nutrient uptake. Both nutritional and signalling functions rely upon the endocytosis of various molecules from the cell surface via the endocytic pathway. However, endocytic membrane dynamics in this embryonic tissue remain poorly understood. Here we show that the functions of rab7, a small GTP-binding protein regulating the late endocytic pathway, are essential for embryonic patterning during gastrulation. The endosomes of visceral endoderm cells are delivered via a unique microautophagy-like process to the apical vacuole, a large compartment exhibiting lysosomal characteristics. Loss of rab7 function results in severe inhibition of this endocytic pathway. Our results indicate that the microautophagic process and flow of the endocytic membrane have essential roles in early embryonic development.

Activation of PI 3-kinase by G protein βγ subunits

Abstract We have reported that fMLP-induced activation of pertussis toxin-sensitive GTP-binding proteins in THP-1 cells potentiates the insulin-induced accumulation of PtdIns(3,4,5)P 3 , a product of phosphoinositide 3-kinase (T. Okada et al., Biochem. J. 317 , 475–480, 1996). The synergism in PtdIns(3,4,5)P 3 accumulation was observed in Chinese hamster ovary cells expressing both insulin and fMLP receptors. In rat adipocytes, which represent the physiological target cells of insulin, receptor-mediated activation of GTP-binding protein by adenosine and prostaglandin E 2 potentiated the insulin-induced PtdIns(3,4,5)P 3 accumulation. In cell-free systems, the activity of the p85/p110β subtype of phosphoinositide 3-kinase was, while that of p85/p110α was not, stimulated by the βγ subunits of the GTP-binding proteins. We propose here a hypothesis that the p85/p110β subtype is under the control of both the insulin receptors and the GTP-binding protein-coupled receptors in intact cell systems.

3′ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP)

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5′ position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] upon voltage depolarization. However, it is unclear whether VSPs also have 3′ phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P3. TLC assay showed that the 3′ phosphate of PI(3,4,5)P3 was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] was removed by VSPs. Monitoring of PI(3,4)P2 levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PHTAPP1-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5′ phosphatase activity of VSP toward PI(3,4,5)P3. However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P3 is dephosphorylated at the 5′ position, PI(3,4)P2 is then dephosphorylated at the 3′ position. These results suggest that substrate specificity of the VSP changes with membrane potential.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P2 in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P2, is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell’s apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P2 metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

Phosphatidylinositol-3,5-bisphosphate : metabolism and physiological functions

Phosphatidylinositol (PtdIns) is a membrane phospholipid composed of diacylglycerol and a D-myo-inositol head group. In mammals, the hydroxyl groups at the D3, D4 and D5 positions of the inositol ring can be phosphorylated to yield seven phosphoinositide derivatives. PtdIns-3,5-bisphosphate [PtdIns(3,5)P2] is the most recently discovered species of phosphoinositide that is generated by the phosphorylation of PtdIns(3)P at the D5 position by PtdIns phosphate kinase and catabolized through the dephosphorylation by myotubularin family of phosphatases. Genetic and biochemical analyses of the enzymes metabolizing PtdIns(3,5)P2 have revealed that this phospholipid is involved in the control of endolysosomal systems and plays crucial roles in various mammalian tissues. In this article, we review the current state of knowledge of the metabolic/physiological functions of PtdIns(3,5)P2, and describe how disruption of these functions may contribute to human diseases.