Identification of a functional phosphatidylinositol 3,4,5-trisphosphate binding site in the epithelial Na+ channel

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na(+) channel (ENaC). Whereas PI(3,4,5)P(3) directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of gamma-mENaC just following the second trans-membrane domain (residues 569-583) important to PI(3,4,5)P(3) binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P(3), increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P(3) binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P(3). Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within gamma-mENaC as being part of a functional PI(3,4,5)P(3) binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.     

Rapid translocation and insertion of the epithelial Na+ channel in response to RhoA signaling

Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ absorption across many epithelia. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. Two members of the Ras superfamily of small GTPases, K-Ras and RhoA, activate ENaC. K-Ras activates ENaC via a signaling pathway involving phosphatidylinositol 3-kinase and production of phosphatidylinositol 3,4,5-trisphosphate with the phospholipid directly interacting with the channel to increase open probability. How RhoA increases ENaC activity is less clear. Here we report that RhoA and K-Ras activate ENaC through independent signaling pathways and final mechanisms of action. Activation of RhoA signaling rapidly increases the membrane levels of ENaC likely by promoting channel insertion. This process dramatically increases functional ENaC current, resulting in tight spatial-temporal control of these channels. RhoA signals to ENaC via a transduction pathway, including the downstream effectors Rho kinase and phosphatidylinositol-4-phosphate 5-kinase. Phosphatidylinositol 4,5-biphosphate produced by activated phosphatidylinositol 4-phosphate 5-kinase may play a role in targeting vesicles containing ENaC to the plasma membrane.     

Stimulation of phosphatidylinositol-4-phosphate 5-kinase by Rho-kinase

The serine/threonine kinase Rho-kinase was recently identified as a downstream effector of the small GTPase Rho, mediating effects of Rho on the actin cytoskeleton. Also phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) has been implicated in the regulation of actin polymerization. As the synthesis of PI(4,5)P(2) has been suggested to be affected by Rho proteins, we investigated whether Rho-kinase is involved in the control of PI(4,5)P(2) levels. Overexpression of RhoA in HEK-293 cells increased phosphatidylinositol 4-phosphate (PI4P) 5-kinase activity and concomitantly enhanced cellular PI(4,5)P(2) levels, whereas overexpression of the Rho-inactivating C3 transferase decreased both PI4P 5-kinase activity and PI(4,5)P(2) levels. These effects of RhoA could be mimicked by overexpression of wild-type Rho-kinase and of the constitutively active catalytic domain of Rho-kinase, Rho-kinase-CAT. In contrast, a kinase-deficient mutant of Rho-kinase had no effect on PI4P 5-kinase activity. Importantly, the increase in PI4P 5-kinase activity and PI(4,5)P(2) levels by wild-type Rho-kinase, but not by Rho-kinase-CAT, was completely prevented by coexpression of C3 transferase, indicating that the effect of Rho-kinase was under the control of endogenous Rho. In cell lysates, addition of recombinant RhoA and Rho-kinase-CAT stimulated PI4P 5-kinase activity. Finally, the increase in PI(4,5)P(2) levels induced by both Rho-kinase-CAT and RhoA was reversed by the Rho-kinase inhibitor HA-1077. Our data suggest that Rho-kinase is involved in the Rho-controlled synthesis of PI(4,5)P(2) by PI4P 5-kinase.     

Direct activation of the epithelial Na(+) channel by phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by phosphoinositide 3-OH kinase

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P(2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), in contrast, are not. We report here activation of the epithelial Na(+) channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na(+) reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns(3,4)P(2)/PtdIns(3,4,5)P(3), as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.     

Ras couples phosphoinositide 3-OH kinase to the epithelial Na channel

Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells ∼3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPβS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110α) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.     

Ras couples phosphoinositide 3-OH kinase to the epithelial Na+ channel

Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells approximately 3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPbetaS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110alpha) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.     

Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.     

Regulation of podosomes by integrin alphavbeta3 and Rho GTPase-facilitated phosphoinositide signaling

In osteoclasts, polyphosphoinositides such as phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) are produced in response to integrin alphavbeta3 signaling and they have a critical role in actin cytoskeleton remodeling. The levels of PI(4,5)P2 and PI(3,4,5)P3 are regulated by Rho GTPase through the activation of phosphatidylinositol 4-phosphate 5-kinase (PI4P-5 kinase) and phospatidylinositol 3-kinase (PI3 kinase), respectively. Interaction of PI(4,5)P2 with gelsolin and Wiscott-Aldrich syndrome protein (WASP) is critical for podosome assembly/disassembly and actin ring formation in osteoclasts. Interaction of PI(3,4,5)P3 with gelsolin functions in orchestrating the podosome signaling complex consisting of several key signaling molecules. Gelsolin deficiency has been shown to block podosome assembly and motility in mouse osteoclasts. However, these osteoclasts are able to form a WASP-containing actin ring and retain their resorptive function. The TAT-mediated delivery of gelsolin phosphoinositide-binding domains into osteoclasts resulted in production of podosome clusters and disruption of actin ring formation. Hence, these osteoclasts were hypomotile and less resorptive. Our observations suggest that both PI(4,5)P2 and PI(3,4,5)P3 are involved in regulating osteoclast functions through modulation of severing, capping, and nucleating functions of actin-binding proteins.     

Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases.

When activated, ERM (ezrin, radixin, moesin) proteins are recruited to the plasma membrane, with concomitant carboxy-terminal threonine phosphorylation, where they crosslink actin filaments to the plasma membrane to form microvilli (reviewed in [1] [2] [3] [4] [5]). Here, we report that, when NIH3T3 or HeLa cells were transfected with a constitutively active mutant of the small GTPase RhoA (V14RhoA), microvilli were induced and the level of carboxy-terminal threonine-phosphorylated ERM proteins (CPERM) [6] [7] increased approximately 30-fold. This increase was not observed following transfection of constitutively active forms of two other Rho-family GTPases, Rac1 and Cdc42, or of a direct effector of Rho, Rho-kinase (also known as ROKalpha or ROCK-II) [8] [9] [10]. The V14RhoA-induced phosphorylation of ERM proteins was not suppressed by Y-27632, a specific inhibitor of ROCK kinases including Rho-kinase [11]. Overexpression of another direct effector of Rho, phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) type Ialpha [12] [13] [14], but not a kinase-inactive mutant [15], increased approximately sixfold the level of CPERM, and induced microvilli. Together with the previous finding that the PI4P5K product phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates ERM proteins in vitro [16], our data suggest that PIP(2), and not ROCK kinases, is involved in the RhoA-dependent activation of ERM proteins in vivo. The active state of ERM proteins is maintained through threonine phosphorylation by as yet undetermined kinases, leading to microvillus formation.     

Active Rho kinase (ROK-alpha ) associates with insulin receptor substrate-1 and inhibits insulin signaling in vascular smooth muscle cells.

Recent studies from our laboratory have shown that insulin stimulates myosin-bound phosphatase (MBP) in vascular smooth muscle cells (VSMCs) by decreasing site-specific phosphorylation of the myosin-bound subunit (MBS) of MBP via nitric oxide/cGMP-mediated Rho/Rho kinase inactivation. Here we tested potential interactions between Rho kinase and insulin signaling pathways. In control VSMCs, insulin inactivates ROK-alpha, the major Rho kinase isoform in VSMCs, and inhibits thrombin-induced increase in ROK-alpha association with the insulin receptor substrate-1 (IRS-1). Hypertension (in spontaneous hypertensive rats) or expression of an active RhoA(V14) up-regulates Rho kinase activity and increases ROK-alpha/IRS-1 association resulting in IRS-1 serine phosphorylation that leads to inhibition of both insulin-induced IRS-1 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activation. In contrast, expression of dominant negative RhoA or cGMP-dependent protein kinase type I alpha inactivates Rho kinase, abolishes ROK-alpha/IRS-1 association, and potentiates insulin-induced tyrosine phosphorylation and PI3-kinase activation leading to decreased MBS(T695) phosphorylation and decreased MBP inhibition. Collectively, these results suggest a novel function for ROK-alpha in insulin signal transduction at the level of IRS-1 and potential cross-talk between cGMP-dependent protein kinase type I alpha, Rho/Rho kinase signaling, and insulin signaling at the level of IRS-1/PI3-kinase.     

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.     

Tumor Necrosis Factor-α Induces Stress Fiber Formation through Ceramide Production: Role of Sphingosine Kinase

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.     

Interruption of inositol sphingolipid synthesis triggers Stt4p-dependent protein kinase C signaling

The protein kinase C (PKC)-MAPK signaling cascade is activated and is essential for viability when cells are starved for the phospholipid precursor inositol. In this study, we report that inhibiting inositol-containing sphingolipid metabolism, either by inositol starvation or treatment with agents that block sphingolipid synthesis, triggers PKC signaling independent of sphingoid base accumulation. Under these same growth conditions, a fluorescent biosensor that detects the necessary PKC signaling intermediate, phosphatidylinositol (PI)-4-phosphate (PI4P), is enriched on the plasma membrane. The appearance of the PI4P biosensor on the plasma membrane correlates with PKC activation and requires the PI 4-kinase Stt4p. Like other mutations in the PKC-MAPK pathway, mutants defective in Stt4p and the PI4P 5-kinase Mss4p, which generates phosphatidylinositol 4,5-bisphosphate, exhibit inositol auxotrophy, yet fully derepress INO1, encoding inositol-3-phosphate synthase. These observations suggest that inositol-containing sphingolipid metabolism controls PKC signaling by regulating access of the signaling lipids PI4P and phosphatidylinositol 4,5-bisphosphate to effector proteins on the plasma membrane.     

Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade

Production of the essential phospholipid PI4P at the Golgi by the Pik1 kinase is required for protein secretion, while a distinct pool of PI4P generated by the Stt4 kinase is critical for normal actin cytoskeleton organization. We identify a transmembrane protein that stabilizes Stt4 at the plasma membrane where it directs synthesis of PI4P, which is required for activation of the Rho1/Pkc1-mediated MAP kinase cascade. Inactivation of Stt4 or the PI4P 5-kinase Mss4 results in mislocalization of the Rho-GTPase GEF Rom2. Rom2 binds PI4,5P(2) through its PH domain and represents the first identified effector in the Stt4-Mss4 pathway. Based on these results, we propose that Stt4-Mss4 generates PI4,5P(2) at the plasma membrane, required to recruit/activate effector proteins such as Rom2.     

Ras activates the epithelial Na(+) channel through phosphoinositide 3-OH kinase signaling

Aldosterone induces expression and activation of the GTP-dependent signaling switch K-Ras. This small monomeric G protein is both necessary and sufficient for activation of the epithelial Na(+) channel (ENaC). The mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras activates human ENaC reconstituted in Chinese hamster ovary cells in a GTP-dependent manner. K-Ras influences ENaC activity most likely by affecting open probability. Inhibition of phosphoinositide 3-OH kinase (PI3K) abolished K-Ras actions on ENaC. In contrast, inhibition of other K-Ras effector cascades, including the MAPK and Ral/Rac/Rho cascades, did not affect K-Ras actions on ENaC. Activation of ENaC by K-Ras, moreover, was sensitive to co-expression of dominant negative p85(PI3K). The G12:C40 effector-specific double mutant of Ras, which preferentially activates PI3K, enhanced ENaC activity in a manner sensitive to inhibition of PI3K. Other effector-specific mutants preferentially activating MAPK and RalGDS signaling had no effect. Constitutively active PI3K activated ENaC independent of K-Ras with the effects of PI3K and K-Ras on ENaC not being additive. We conclude that K-Ras activates ENaC via the PI3K cascade.     

Molecular determinants of PI(4,5)P2 and PI(3,4,5)P3 regulation of the epithelial Na+ channel

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na(+) channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P(3) and PI(4,5)P(2) to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in beta- and gamma- but not alpha-ENaC as necessary for PI(3,4,5)P(2) but not PI(4,5)P(2) modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in beta- and gamma-ENaC are critical to PI(3,4,5)P(3) augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of beta- and gamma-ENaC were identified as being critical to down-regulation of ENaC activity and P(o) in response to depletion of membrane PI(4,5)P(2). These regions of the channel played no identifiable role in a PI(3,4,5)P(3) response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P(2) to increase open probability. We conclude that beta and gamma subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P(3) and PI(4,5)P(2). This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.     

Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.     

Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.     

Local change in phospholipid composition at the cleavage furrow is essential for completion of cytokinesis

Cell division ends up with the membrane separation of two daughter cells, presumably by a membrane fusion that requires dynamic changes of the distribution and the composition of membrane lipids. We have previously shown that a membrane lipid phosphatidylethanolamine (PE) is exposed on the cell surface of the cleavage furrow during late cytokinesis and that this PE movement is involved in regulation of the contractile ring disassembly. Here we show that immobilization of cell surface PE by a PE-binding peptide blocks the RhoA inactivation in the late stage of cytokinesis. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), but not other RhoA effectors, is co-localized with RhoA in the peptide-treated cells. Indeed, PIP5K and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are localized to the cleavage furrow of normally dividing cells. Both overexpression of a kinase-deficient PIP5K mutant and microinjection of anti-PI(4,5)P(2) antibodies compromise cytokinesis by preventing local accumulation of PI(4,5)P(2) in the cleavage furrow. These findings demonstrate that the localized production of PI(4,5)P(2) is required for the proper completion of cytokinesis and that the possible formation of a unique lipid domain in the cleavage furrow membrane may play a crucial role in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.     

Mechanism by which cAMP activates PI3-kinase and increases bile acid secretion in WIF-B9 cells

Previous studies in rat bile canalicular membrane vesicles and WIF-B9 cells revealed that cAMP-induced trafficking of ATP-binding cassette (ABC) transporters to the canalicular membrane and their activation require phosphoinositide 3-kinase (PI3-K) products. In the present studies, canalicular secretion of fluorescein isothiocyanate-glycocholate in WIF-B9 cells was increased by cAMP and a decapeptide that enhances PI3-K activity; these effects were inhibited by wortmannin. To determine the mechanism(s) whereby cAMP activates PI3-K, we examined signal transduction pathways in WIF-B9 and COS-7 cells. cAMP activated PI3-K in both cell lines in a phosphotyrosine-independent manner. PI3-K activity increased in association with p110 beta in both cell lines. The effect of cAMP was KT-5720 sensitive, suggesting involvement of protein kinase A. Expression of a dominant-negative beta-adrenergic receptor kinase COOH terminus (beta-ARKct), which blocks G beta gamma signaling, decreased PI3-K activation in both cell lines. cAMP increased GTP-bound Ras in COS-7 but not WIF-B9 cells. Expression of dominant-negative Ras abolished cAMP-mediated PI3-K, which suggests that the effect is downstream of Ras and G beta gamma. These data indicate that cAMP activates PI3-K in a cell type-specific manner and provide insight regarding mechanisms of PI3-K activation required for bile acid secretion.