Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin

Muscle and fat cells develop insulin resistance when cultured under hyperinsulinemic conditions for sustained periods. Recent data indicate that early insulin signaling defects do not fully account for the loss of insulin action. Given that cortical filamentous actin (F-actin) represents an essential aspect of insulin regulated glucose transport, we tested to see whether cortical F-actin structure was compromised during chronic insulin treatment. The acute effect of insulin on GLUT4 translocation and glucose uptake was diminished in 3T3-L1 adipocytes exposed to a physiological level of insulin (5 nm) for 12 h. This insulin-induced loss of insulin responsiveness was apparent under both low (5.5 mm) and high (25 mm) glucose concentrations. Microscopic and biochemical analyses revealed that the hyperinsulinemic state caused a marked loss of cortical F-actin. Since recent data link phosphatidylinositol 4,5-bisphosphate (PIP(2)) to actin cytoskeletal mechanics, we tested to see whether the insulin-resistant condition affected PIP(2) and found a noticeable loss of this lipid from the plasma membrane. Using a PIP(2) delivery system, we replenished plasma membrane PIP(2) in cells following the sustained insulin treatment and observed a restoration in cortical F-actin and insulin responsiveness. These data reveal a novel molecular aspect of insulin-induced insulin resistance involving defects in PIP(2)/actin regulation.     

Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP(2(4,5)), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM WT but not by 10 nM WT, whereas PI3K activity and PIP(3(3,4,5)) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP(2(4,5)) and F-actin formation. The increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP(2(4,5)), PIP(3(3,4,5)), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP(3(3,4,5)) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.     

Phosphoinositides regulate clathrin-dependent endocytosis at the tip of pollen tubes in Arabidopsis and tobacco

Using the tip-growing pollen tube of Arabidopsis thaliana and Nicotiana tabacum as a model to investigate endocytosis mechanisms, we show that phosphatidylinositol-4-phosphate 5-kinase 6 (PIP5K6) regulates clathrin-dependent endocytosis in pollen tubes. Green fluorescent protein-tagged PIP5K6 was preferentially localized to the subapical plasma membrane (PM) in pollen tubes where it apparently converts phosphatidylinositol 4-phosphate (PI4P) to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. RNA interference-induced suppression of PIP5K6 expression impaired tip growth and inhibited clathrin-dependent endocytosis in pollen tubes. By contrast, PIP5K6 overexpression induced massive aggregation of the PM in pollen tube tips. This PM abnormality was apparently due to excessive clathrin-dependent membrane invagination because this defect was suppressed by the expression of a dominant-negative mutant of clathrin heavy chain. These results support a role for PI(4,5)P(2) in promoting early stages of clathrin-dependent endocytosis (i.e., membrane invagination). Interestingly, the PIP5K6 overexpression-induced PM abnormality was partially suppressed not only by the overexpression of PLC2, which breaks down PI(4,5)P(2), but also by that of PI4Kβ1, which increases the pool of PI4P. Based on these observations, we propose that a proper balance between PI4P and PI(4,5)P(2) is required for clathrin-dependent endocytosis in the tip of pollen tubes.     

Activation of protein kinase C in rat basophilic leukemia cells stimulates increased production of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: correlation with actin polymerization.

Cross-linking of the immunoglobulin E receptor on rat basophilic leukemia (RBL)1 cells by multivalent antigen activates phosphatidylinositol (PI) kinase and phosphatidylinositol 4-phosphate (PIP) kinase leading to the increased production of PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). Activators of protein kinase C (PKC), such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol, 1,2-dioctanoyl-sn-glycerol (diC8), were found to have the same effect even though PMA and diC8 do not cause the activation of phospholipase C. Although the kinetics are different depending on the stimulant, activation of PKC using multivalent antigen, PMA or diC8 also causes the polymerization of actin and an increase in the F-actin content of the cells. In all cases, a good correlation was observed between F-actin levels, activation of PI and PIP kinases, and the increased production of PIP and PIP2. However, in the case of antigen, there is no correlation between actin polymerization and the total amount of PIP and PIP2. Staurosporine, an inhibitor of protein kinases, blocks the F-actin response and the increased synthesis of PIP and PIP2 with similar dose dependencies. Furthermore, depletion of PKC activity through long-term exposure to PMA, inhibited both the F-actin response and the increased synthesis of PIP and PIP2 induced by either DNP-BSA or diC8. These results suggest that activation of PKC precedes the activation of PI and PIP kinases and that under certain circumstances activation of the kinases and the increased synthesis of PIP and PIP2 may be involved in the polymerization of actin in RBL cells, possibly through the interaction of the polyphosphoinositides with actin-binding proteins such as gelsolin and profilin.     

Inositol phospholipid-induced suppression of F-actin-gelating activity of smooth muscle filamin.

Filamin, a high molecular weight actin-binding protein, cross-links actin filaments and produces a gel composed of F-actin. The effects of polyphosphoinositides on the gelating activity of smooth muscle filamin were examined by measuring the low shear viscosity of the F-actin solutions containing filamin incubated with phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), or phosphatidylinositol 4,5-bisphosphate (PIP2). Micelles of these inositol phospholipids bound to filamin inhibited the ability to form a gel of F-actin. The inhibiting activity of each phospholipid was in the following order, PIP2 greater than PIP greater than PI. The F-actin binding assay of filamin revealed that the inhibition of F-actin-gelation resulted in the loss of the F-actin-binding activity of filamin. Thus, polyphosphoinositides may play important roles in regulating the gelating activity of filamin.     

Overexpression of murine phosphatidylinositol 4-phosphate 5-kinase type Ibeta disrupts a phosphatidylinositol 4,5 bisphosphate regulated endosomal pathway

The type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K) phosphorylate phosphatidylinositol 4-phosphate [PI(4)P] to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Ibeta (mPIP5K-Ibeta) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand-dependent downregulation of the colony-stimulating factor-1 receptor. The present study shows that enforced expression of mPIP5K-Ibeta in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2-binding pleckstrin homology (PH) domain of phospholipase-Cdelta (PLC-delta). Analysis of the conserved domains of mPIP5K-Ibeta led to the identification of dimerization domains in the N- and C-terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K-Ibeta and the PLC-delta-PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K-Ibeta acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion.     

Inhibition of the interactions of cofilin, destrin, and deoxyribonuclease I with actin by phosphoinositides

Cofilin is a widely distributed actin-modulating protein that has the ability to bind along the side of F-actin and to depolymerize F-actin in a pH-dependent manner. We found that phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) inhibited both actions of cofilin in a dose-dependent manner, while inositol 1,4,5-triphosphate (IP3), 1-oleoyl-2-acetylglycerol (OAG), phosphatidylserine (PS), or phosphatidylcholine (PC) had little or no effect on them. Gel filtration analyses showed that PIP2 bound to cofilin and thereby inhibited the binding of cofilin to G-actin. Destrin is a mammalian, pH-independent actin-depolymerizing protein. The actin-depolymerizing activity of destrin was also inhibited by PI, PIP, and PIP2, but not by IP3, OAG, PS, or PC. In addition, we found further that an actin-depolymerizing activity of bovine pancreas deoxyribonuclease I, a G-actin-sequestering protein, was inhibited by PIP and PIP2, but not by PI, IP3, OAG, PS, or PC. These results together with previous findings (Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474; Janmey, P. A., and Stossel, T. P. (1987) Nature 325, 362-364) suggest that the sensitivity to polyphosphoinositides may be a common feature in vitro among actin-binding proteins that can bind to G-actin and regulate the state of actin polymerization.     

Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism

Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction.     

Regulation of PIP5K activity by Arf6 and its physiological significance

The phospholipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the phosphorylation of the membrane phospholipid phosphatidylinositol 4-phosphate to generate the pleiotropic phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2) ]. To date, three mammalian PIP5K isozymes, α, β, and γ, and several splicing variants of the γ isozyme have been identified. These PIP5K isozymes and PIP5Kγ variants play critical roles in various cellular functions through their product PI(4,5)P(2) . The small GTPase Arf6 is one of the key activators of PIP5K. Increasing evidence suggests that PIP5K functions as a downstream effector of Arf6 to regulate a wide variety of cellular functions, such as exocytosis, endocytosis, endosomal recycling, membrane ruffle formation, immune response, and bacterial invasion. In this review, we place our focus on the recent advances in Arf6/PIP5K signaling and its linkage to cellular functions.     

Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis

Synaptic vesicles (SVs) are retrieved by clathrin-mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP-2 complex directly interacts with phosphatidylinositol 4-phosphate 5-kinase gamma661 (PIP5Kgamma661), the major PI(4,5)P2-producing enzyme in the brain. The beta2 subunit of AP-2 was found to bind to the C-terminal tail of PIP5Kgamma661 and cause PIP5Kgamma661 activation. The interaction is regulated by PIP5Kgamma661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kgamma661 C-terminal region in hippocampal neurons suppresses depolarization-dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kgamma661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.     

C5a reduces formyl peptide-induced actin polymerization and phosphatidylinositol(3,4,5)trisphosphate formation, but not phosphatidylinositol (4,5) bisphosphate hydrolysis and superoxide production, in human neutrophils.

We investigated phospholipid signal transduction, calcium flux, O2- anion production and actin polymerization after stimulation with the C fragment and chemoattractant, C5a, and then determined how C5a pretreatment affected subsequent responses to formyl peptide in human neutrophils. We have previously demonstrated that the novel lipids, phosphatidylinositol trisphosphate (PIP3) and phosphatidylinositol(3,4)P2 (PI(3,4)P2), rise transiently in neutrophils after activation with formyl peptide. Furthermore, the rise in PIP3 parallels actin polymerization. In this study, neutrophils activated with C5a exhibited two distinct G protein-dependent signal pathways involving different phosphoinositides: 1) [32P]PI(4,5)P2 hydrolysis and [32P]PA production, and 2) the transient formation of D-3-phosphorylated phosphoinositides, [32P]PIP3 and [32P]PI(3,4)P2. When neutrophils were preincubated with C5a for 5 min before stimulation with formyl peptide, [32P]PI(4,5)P2 hydrolysis was unchanged, and [32P]PA production and O2- formation were slightly enhanced compared with controls stimulated with formyl peptide in the absence of C5a. In contrast, [32P]PIP3 production, right angle light scatter, and actin polymerization were all reduced 35 to 40%. Therefore, these data support the hypothesis that PIP3 plays a role in chemotaxis but not superoxide formation.     

A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling.

Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-DeltaWA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-DeltaWA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.     

Phosphatidic acid regulates the affinity of the murine phosphatidylinositol 4-phosphate 5-kinase-Ibeta for phosphatidylinositol-4-phosphate

Type I phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) catalyzes the phosphorylation of phosphatidylinositol 4 phosphate [PI(4)P] at carbon 5, producing phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Phosphatidic acid (PA) activates PI4P5K in vitro and plays a central role in the activation of PIP5K pathways in vivo. This report demonstrates that actin fiber formation in murine fibroblasts involves PA activation of PIP5Ks and defines biochemical interactions between PA and the PIP5Ks. Inhibition of phospholipase D production of PA results in the loss of actin fibers. Overexpression of the beta isoform of the type I murine phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta) maintains actin fiber structure in the face of phospholipase D inhibition. PA activates mPIP5K-Ibeta by direct binding to mPIP5K-Ibeta through both electrostatic and hydrophobic interactions, with the fatty acid acyl chain length and degree of saturation acting as critical determinants of binding and activation. Furthermore, kinetic analysis suggests that phosphorylation of the PI(4)P substrate does not follow classical Michaelis-Menten kinetics. Instead, the kinetic data are consistent with a model in which mPIP5K-Ibeta initially binds to the lipid micelle and subsequently binds the PI(4)P substrate. In addition, the kinetics indicate substrate inhibition, suggesting that mPIP5K-Ibeta contains an inhibitory PI(4)P-binding site. These results suggest a model in which mPIP5K-Ibeta is surrounded by PI(4)P, but is unable to catalyze its conversion to PI(4,5)P2 unless PA is bound.     

A short sequence responsible for both phosphoinositide binding and actin binding activities of cofilin.

Cofilin is a widely distributed actin-modulating protein that has abilities to bind along the side of F-actin and to depolymerize F-actin. Both abilities of cofilin can be inhibited by phosphoinositides such as phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate (PIP2). We have previously shown that the synthetic dodecapeptide corresponding to Trp104-Met115 of cofilin is a potent inhibitor of actin polymerization (Yonezawa, N., Nishida, E., Iida, K., Kumagai, H., Yahara, I., and Sakai, H. (1991) J. Biol. Chem. 266, 10485-10489). In this study, we have found that the inhibitory effect of the synthetic dodecapeptide on actin polymerization is canceled specifically by phosphatidylinositol, phosphatidylinositol 4-monophosphate and PIP2. We further show that the dodecapeptide as well as cofilin binds to PIP2 molecules and inhibits PIP2 hydrolysis by phospholipase C. Thus, the actin-binding dodecapeptide sequence of cofilin may constitute a multifunctional domain in cofilin.     

Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils?

Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated [32P]PIP3 levels decayed toward initial values. However, recovery of [32P]PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of [32P] PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.     

Polyamines, PI(4,5)P2, and actin polymerization

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.     

Phosphatidylinositol-4,5 bisphosphate produced by PIP5KIgamma regulates gelsolin, actin assembly, and adhesion strength of N-cadherin junctions

Phosphoinositides regulate several actin-binding proteins but their role at intercellular adhesions has not been defined. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was generated at sites of N-cadherin-mediated intercellular adhesion and was a critical regulator of intercellular adhesion strength. Immunostaining for PI(4,5)P2 or transfection with GFP-PH-PLCdelta showed that PI(4,5)P2 was enriched at sites of N-cadherin adhesions and this enrichment required activated Rac1. Isoform-specific immunostaining for type I phosphatidylinositol 4-phosphate 5 kinase (PIP5KI) showed that PIP5KIgamma was spatially associated with N-cadherin-Fc beads. Association of PIP5KIgamma with N-cadherin adhesions was in part dependent on the activation of RhoA. Transfection with catalytically inactive PIP5KIgamma blocked the enrichment of PI(4,5)P2 around beads. Catalytically inactive PIP5KIgamma or a cell-permeant peptide that mimics and competes for the PI(4,5)P2-binding region of the actin-binding protein gelsolin inhibited incorporation of actin monomers in response to N-cadherin ligation and reduced intercellular adhesion strength by more than twofold. Gelsolin null fibroblasts transfected with a gelsolin severing mutant containing an intact PI(4,5)P2 binding region, demonstrated intercellular adhesion strength similar to wild-type transfected controls. We conclude that PIP5KIgamma-mediated generation of PI(4,5)P2 at sites of N-cadherin contacts regulates intercellular adhesion strength, an effect due in part to PI(4,5)P2-mediated regulation of gelsolin.     

An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis

Plasmalemmal phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P₂) synthesized by PI 4-phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-γ prevented the disappearance of PI4,5P₂ and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.     

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P₂ is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P₂-containing liposomes of the PtdIns(4,5)P₂ binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P₂ on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P₂ mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P₂ may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.     

Gα11 Signaling through ARF6 Regulates F-Actin Mobilization and GLUT4 Glucose Transporter Translocation to the Plasma Membrane

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.