Rgf1p is a specific Rho1-GEF that coordinates cell polarization with cell wall biogenesis in fission yeast

Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. We have identified a new GEF, designated Rgf1p, which specifically regulates Rho1p during polarized growth. The phenotype of rgf1 null cells was very similar to that seen after depletion of Rho1p, 30% of cells being lysed. In addition, rgf1(+) deletion caused hypersensitivity to the antifungal drug Caspofungin and defects in the establishment of bipolar growth. rho1(+), but none of the other GTPases of the Rho-family, suppressed the rgf1Delta phenotypes. Moreover, deletion of rgf1(+) suppressed the severe growth defect in rga1(+) null mutants (a Rho1-GAP, negative regulator). Rgf1p and Rho1p coimmunoprecipitated and overexpression of rgf1(+) specifically increased the GTP-bound Rho1p; it caused changes in cell morphology, and a large increase in beta(1,3)-glucan synthase activity. These effects were similar to those elicited when the hyperactive rho1-G15V allele was expressed. A genetic relationship was observed between Rgf1p, Bgs4p (beta[1,3]-glucan synthase), and Pck1p (protein kinase C [PKC] homologue); Bgs4p and Pck1p suppressed the hypersensitivity to Caspofungin in rgf1Delta mutants. Rgf1p localized to the growing ends and the septum, where Rho1, Pck1p, and Bgs4p are known to function. Our results suggest that Rgf1p probably activates the Rho functions necessary for coordinating actin deposition with cell wall biosynthesis during bipolar growth, allowing the cells to remodel their wall without risk of rupture.     

Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in platelets

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the rate-limiting step in the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a signaling phospholipid that contributes to actin dynamics. We have shown in transfected tissue culture cells that PIP5K translocates from the cytosol to the plasma membrane following agonist-induced stimulation of Rho family GTPases. Nonetheless, it is unclear whether Rho GTPases induce PIP5K relocalization in platelets. We used PIP5K isoform-specific immunoblotting and lipid kinase assays to examine the intracellular localization of PIP5K in resting and activated platelets. Using differential centrifugation to separate the membrane skeleton, actin filaments and associated proteins, and cytoplasmic fractions, we found that PIP5K isoforms were translocated from cytosol to actin-rich fractions following stimulation of the thrombin receptor. PIP5K translocation was detectable within 30 s of stimulation and was complete by 2-5 min. This agonist-induced relocalization and activation of PIP5K was inhibited by 8-(4-parachlorophenylthio)-cAMP, a cAMP analogue that inhibits Rho and Rac. In contrast, 8-(4-parachlorophenylthio)-cGMP, a cGMP analogue that inhibits Rac but not Rho, did not affect PIP5K translocation and activation. This suggests that Rho GTPase may be an essential regulator of PIP5K in platelets. Consistent with this hypothesis, we found that C3 exotoxin (a Rho-specific inhibitor) and HA1077 (an inhibitor of the Rho effector, Rho-kinase) also eliminated PIP5K activation and trafficking into the membrane cytoskeleton. Thus, these data indicate that Rho GTPase and its effector Rho-kinase have an intimate relationship with the trafficking and activation of platelet PIP5K. Moreover, these data suggest that relocalization of platelet PIP5K following agonist stimulation may play an important role in regulating the assembly of the platelet cytoskeleton.     

The Rho1p Exchange Factor Rgf1p Signals Upstream from the Pmk1 Mitogen-activated Protein Kinase Pathway in Fission Yeast

The Schizosaccharomyces pombe exchange factor Rgf1p specifically regulates Rho1p during polarized growth. Rgf1p activates the β-glucan synthase (GS) complex containing the catalytic subunit Bgs4p and is involved in the activation of growth at the second end, a transition that requires actin reorganization. In this work, we investigated Rgf1p signaling and observed that Rgf1p acted upstream from the Pck2p-Pmk1p MAPK signaling pathway. We noted that Rgf1p and calcineurin play antagonistic roles in Cl⁻ homeostasis; rgf1Δ cells showed the vic phenotype (viable in the presence of immunosuppressant and chlorine ion) and were unable to grow in the presence of high salt concentrations, both phenotypes being characteristic of knockouts of the MAPK components. In addition, mutations that perturb signaling through the MAPK pathway resulted in defective cell integrity (hypersensitivity to caspofungin and β-glucanase). Rgf1p acts by positively regulating a subset of stimuli toward the Pmk1p-cell integrity pathway. After osmotic shock and cell wall damage HA-tagged Pmk1p was phosphorylated in wild-type cells but not in rgf1Δ cells. Finally, we provide evidence to show that Rgf1p regulates Pmk1p activation in a process that involves the activation of Rho1p and Pck2p, and we demonstrate that Rgf1p is unique in this signaling process, because Pmk1p activation was largely independent of the other two Rho1p-specific GEFs, Rgf2p and Rgf3p.     

G-protein-coupled receptor activation induces the membrane translocation and activation of phosphatidylinositol-4-phosphate 5-kinase I alpha by a Rac- and Rho-dependent pathway.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) mediates cell motility and changes in cell shape in response to extracellular stimuli. In platelets, it is synthesized from PI4P by PIP5K in response to stimulation of a G-protein-coupled receptor by an agonist, such as the thrombin. In the present study, we have addressed the pathway that induces PIP5K I alpha activation following the addition of thrombin. Under resting condition expressed PIP5K I alpha was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of G alpha(q), PIP5K I alpha translocated to the plasma membrane. Movement of PIP5K I alpha to the cell membrane was dependent on both GTP-bound Rac and Rho, but not Arf, because: 1) inactive GDP-bound variants of either Rac or Rho blocked the translocation induced by constitutively active G alpha(q), 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K I alpha translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K I alpha. In addition, a dominant negative variant of Rho blocked the PIP5K I alpha membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K I alpha membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K I alpha by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K I alpha, the related lipid kinase PIP4K failed to undergo translocation after stimulation by small GTP-binding proteins Rac or Rho. We also tested whether membrane localization of PIP5K I alpha correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K I alpha with either constitutively active G alpha(q), Rac, or Rho led to a 5- to 7-fold increase in PIP5K I alpha activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of G alpha(q), Rac, Rho, and PIP5K I alpha. Once activated and translocated to the cell membrane, PIP5K I alpha becomes available to phosphorylate PI4P to generate PI4,5P(2) on the plasma membrane.     

Interaction of protein kinase C isozymes with phosphatidylinositol 4,5-bisphosphate.

Interaction of protein kinase C (PKC) isozymes with phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated by monitoring the changes in the intrinsic fluorescence of the enzyme, the kinase activity, and phorbol ester binding. Incubation of PKC I, II, and III with PIP2 resulted in different rates of quenching of PKC fluorescence and different degrees of inactivation of these enzymes. Other inositol-containing phospholipids such as phosphatidylinositol and phosphatidylinositol 4-phosphate also caused differential rates of quenching of the intrinsic fluorescence of these enzymes. These latter two phospholipids were, however, less potent in the inactivation of PKCs than PIP2. The IC50 of PIP2 were 2, 4, and 11 microM for PKC I, II, and III, respectively. Inactivation of PKCs by PIP2 cannot be reversed by extensive dilution of PIP2 with Nonidet P-40 nor by digestion of PIP2 with phospholipase C. Interaction of PIP2 with the various PKC isozymes was greatly facilitated in the presence of Mg2+ or Ca2+ as evidenced by the accelerated quenching of the PKC fluorescence, however, these divalent metal ions protected PKC from the PIP2-induced inactivation. Binding of PIP2 to PKC in the absence of divalent metal ion also caused a reduction of [3H]phorbol 12,13-dibutyrate binding as a result of reducing the affinity of the enzyme for phorbol ester. Based on gel filtration chromatography, it was estimated that one molecule of PKC interacted with one PIP2 micelle with an aggregation number of 80-90. The PIP2-bound PKC could further interact with phosphatidylserine in the presence of Ca2+ to form a larger complex. Binding of PKC to both PIP2 and phosphatidylserine in the presence of Ca2+ was also evident by changes in the intrinsic fluorescence of PKC. As the interaction of PKC with PIP2, but not with phosphatidylserine, could be enhanced by millimolar concentrations of Mg2+, we propose that PIP2 may be a component of the membrane anchor for PKC under basal physiological conditions when [Ca2+]i is low and Mg2+ is plentiful. Under the in vitro assay conditions, PIP2 could stimulate PKC activity to a level approximately 10-20% of that by diacylglycerol. The stimulatory effect of PIP2 on PKC apparently is not due to binding to the same site recognized by diacylglycerol or phorbol ester, because PIP2 cannot effectively compete with phorbol 12,13-dibutyrate in the binding assay.     

Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.     

Rho1 GTPase and PKC Ortholog Pck1 Are Upstream Activators of the Cell Integrity MAPK Pathway in Fission Yeast

In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.     

NEDD4‐induced degradative ubiquitination of phosphatidylinositol 4‐phosphate 5‐kinase α and its implication in breast cancer cell proliferation

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) family members generate phosphatidylinositol 4,5‐bisphosphate (PIP2), a critical lipid regulator of diverse physiological processes. The PIP5K‐dependent PIP2 generation can also act upstream of the oncogenic phosphatidylinositol 3‐kinase (PI3K)/Akt pathway. Many studies have demonstrated various mechanisms of spatiotemporal regulation of PIP5K catalytic activity. However, there are few studies on regulation of PIP5K protein stability. Here, we examined potential regulation of PIP5Kα, a PIP5K isoform, via ubiquitin‐proteasome system, and its implication for breast cancer. Our results showed that the ubiquitin ligase NEDD4 (neural precursor cell expressed, developmentally down‐regulated gene 4) mediated ubiquitination and proteasomal degradation of PIP5Kα, consequently reducing plasma membrane PIP2 level. NEDD4 interacted with the C‐terminal region and ubiquitinated the N‐terminal lysine 88 in PIP5Kα. In addition, PIP5Kα gene disruption inhibited epidermal growth factor (EGF)‐induced Akt activation and caused significant proliferation defect in breast cancer cells. Notably, PIP5Kα K88R mutant that was resistant to NEDD4‐mediated ubiquitination and degradation showed more potentiating effects on Akt activation by EGF and cell proliferation than wild‐type PIP5Kα. Collectively, these results suggest that PIP5Kα is a novel degradative substrate of NEDD4 and that the PIP5Kα‐dependent PIP2 pool contributing to breast cancer cell proliferation through PI3K/Akt activation is negatively controlled by NEDD4.     

Physical association of the small GTPase Rho with a 68-kDa phosphatidylinositol 4-phosphate 5-kinase in Swiss 3T3 cells.

Our previous work showed that post-translationally modified Rho in its GTP-bound state stimulated phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether Rho physically interacts with PIP5K, we incubated immobilized Rho-GST with Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho-GST, but not Ras-GST or GST alone, bound significant PIP5K activity. The binding of PIP5K was independent of whether Rho was in a GTP- or GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognized a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responded to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitated with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhanced PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown.     

Coupling of M2 muscarinic receptors to membrane ion channels via phosphoinositide 3-kinase gamma and atypical protein kinase C.

We report a novel signaling pathway linking M2 muscarinic receptors to metabotropic ion channels. Stimulation of heterologously expressed M2 receptors, but not other Gi/Go-associated receptors (M4 or alpha2c), activates a calcium- and voltage-independent chloride current in Xenopus oocytes. We show that the stimulatory pathway linking M2 receptors to these chloride channels consists of Gbeta gamma stimulation of phosphoinositide 3-kinase gamma (PI-3Kgamma), formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), and activation of atypical protein kinase C (PKC). The chloride current is activated in the absence of M2 receptor stimulation by the injection of PIP3, and PIP3 current activation is blocked by a pseudosubstrate inhibitory peptide of atypical PKC but not other PKCs. Moreover, the current is activated by injection of recombinant PKCzeta at concentrations as low as 1 nM. M2 receptor-current coupling was disrupted by inhibiton of PI-3K and by injection of beta gamma binding peptides, but it was not affected by expression of dominant negative p85 cRNA. We also show that this pathway mediates M2 receptor coupling to metabotropic nonselective cation channels in mammalian smooth muscle cells, thus demonstrating the broad relevance of this signaling cascade in neurotransmitter signaling.     

skittles,a Drosophila phosphatidylinositol 4-phosphate 5-kinase,is required for cell viability,germline development and bristle morphology,but not for neurotransmitter release.

The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion.     

Rac1 and calmodulin interactions modulate dynamics of ARF6-dependent endocytosis

The main cellular Ca(2+) sensor, calmodulin (CaM), interacts with and regulates several small GTPases, including Rac1. The present study revealed high binding affinity of Rac1 for CaM and uncovered two new essential binding domains in Rac1: the polybasic region, important for phosphatidylinositol-4-phosphate 5-kinase (PIP5K) interaction, and the adjacent prenyl group. CaM inhibition increased Rac1 binding to PIP5K and induced an extensive phosphatidylinositol 4,5-bisphosphate (PI4,5P(2) )-positive tubular membrane network. Immunofluorescence demonstrated that the tubules were plasma membrane invaginations resulting from an ADP-ribosylation factor 6 (ARF6)-dependent and clathrin-independent pathway. The role of Rac1 in this endocytic route was analyzed by expressing constitutively active and inactive mutants. While active Rac1 impaired tubulation, the inactive mutant enhanced it. Intriguingly, inactive mutant expression elicited tubulation by recruiting PIP5K and inhibiting Rac1 at the plasma membrane. Accordingly, CaM inhibition inactivated Rac1 and increased Rac1/PIP5K interaction. Therefore, our findings highlight an important new role for Rac1 and CaM in controlling clathrin-independent endocytosis.     

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.     

Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.     

Dibasic amino acid residues at the carboxy-terminal end of kinase homology domain participate in the plasma membrane localization and function of phosphatidylinositol 5-kinase gamma

Type I phosphatidylinositol 4-phosphate (PI(4)P) 5-kinases (PIP5Ks) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), an essential lipid molecule involved in various cellular processes such as regulation of actin cytoskeleton and membrane traffic. The protein localizes to the plasma membrane where its activity has been shown to be regulated by small GTPase ARFs and/or phosphatidic acid. Deletion analysis of amino- or carboxy-terminal sequences of PIP5Kgamma fused with EGFP demonstrated that the presence of central kinase homology domain (KHD), a 380 amino acid-long region highly conserved among PIP5K family, was necessary and sufficient for the plasma membrane localization of PIP5Kgamma. Particularly, the dibasic Arg-Lys sequence located at the carboxy-terminal end of KHD was shown to be crucial for the plasma membrane targeting of PIP5Kgamma, since the deletion or charge-reversal mutation of this dibasic sequence resulted in the mislocalization of the protein to the cytoplasm. Mislocalized mutants also failed to complement the temperature-sensitive growth of Saccharomyces cerevisiae mss4-1 mutant defective in PIP5K function. The presence of dibasic residues at the C-terminal end of KHD was conserved among mammalian as well as invertebrate PIP5K family members, but not in the type II PIPKs that are not targeted to the plasma membrane, suggesting that the conserved dibasic motif provides a mechanism essential for the proper localization and cellular function of PIP5Ks.     

A unique phosphatidylinositol 4-phosphate 5-kinase is activated by ADP-ribosylation factor in Plasmodium falciparum

In eukaryotes, calcium signalling has been linked to hydrolysis of the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). The final enzyme in the synthesis of this phosphoinositide, a Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), is activated by the small G protein ADP-ribosylation factor 1 (ARF1). In mammals, the ARF-PIP5K pathway is a key regulator of cell motility, secretion and cell signalling. We report the characterisation of a unique, putative bifunctional PIP5K in the human malaria parasite Plasmodium falciparum. The protein comprises a C-terminal, functional PIP5K domain with catalytic specificity for phosphatidylinositol 4-phosphate. The recombinant enzyme is activated by ARF1 but not phosphatidic acid. The protein also incorporates an unusual N-terminal domain with potential helix-loop-helix EF-hand-like motifs that is a member of the neuronal calcium sensor family (NCS). Intriguingly, NCS-1 has been shown to stimulate phosphatidylinositol 4-phosphate synthesis by activating mammalian and yeast phosphatidylinositol 4-kinase β in vitro in a calcium-dependent manner. The unexpected physical attachment of an NCS-like domain to the plasmodial PIP5K might reflect a unique functional link between the calcium and PtdIns(4,5)P₂ pathways allowing modulation of PtdIns(4,5)P₂ production in response to changes in intracellular calcium concentrations within the parasite.     

Phosphatidylinositol 4,5-bisphosphate regulates adipocyte actin dynamics and GLUT4 vesicle recycling

To investigate the potential role of phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) in the regulation of actin polymerization and GLUT4 translocation, the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) were expressed in 3T3L1 adipocytes. In preadipocytes (fibroblasts) PIP5K expression promoted actin polymerization on membrane-bound vesicles to form motile actin comets. In contrast, expression of PIP5K in differentiated 3T3L1 adipocytes resulted in the formation of enlarged vacuole-like structures coated with F-actin, cortactin, dynamin, and N-WASP. Treatment with either latrunculin B (an inhibitor for actin polymerization) or Clostridium difficile toxin B (a general Rho family inhibitor) resulted in a relatively slower disappearance of coated F-actin from these vacuoles, but the vacuoles themselves remained unaffected. Functionally, the increased PI(4,5)P2 levels resulted in an inhibition of transferrin receptor and GLUT4 endocytosis and a slow accumulation of these proteins in the PI(4,5)P2-enriched vacuoles along with the non-clathrin-derived endosome marker (caveolin) and the AP-2 adaptor complex. However, these structures were devoid of early endosome markers (EEA1, clathrin) and the biosynthetic membrane secretory machinery markers p115 (Golgi) and syntaxin 6 (trans-Golgi Network). Taken together, these data demonstrate that PI(4,5)P2 has distinct morphologic and functional properties depending upon specific cell context. In adipocytes, altered PI(4,5)P2 metabolism has marked effects on GLUT4 endocytosis and intracellular vesicle trafficking due to the derangement of actin dynamics.     

NMDA receptor-mediated PIP5K activation to produce PI(4,5)P₂ is essential for AMPA receptor endocytosis during LTD

NMDA receptor activation leads to clathrin-dependent endocytosis of postsynaptic AMPA receptors. Although this process controls long-term depression (LTD) induction in the hippocampus, how it is regulated by neuronal activities is not completely clear. Here, we show that Ca2? influx through the NMDA receptor activates calcineurin and protein phosphatase 1 to dephosphorylate phosphatidylinositol 4-phosphate 5-kinaseγ661 (PIP5Kγ661), the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P?)-producing enzyme in the brain. Bimolecular fluorescence complementation analysis revealed that the dephosphorylated PIP5Kγ661 became associated with the clathrin adaptor protein complex AP-2 at postsynapses in situ. NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced LTD were completely blocked by inhibiting the association between dephosphorylated PIP5Kγ661 and AP-2 and by overexpression of a kinase-dead PIP5Kγ661 mutant in hippocampal neurons. Furthermore, knockdown of PIP5Kγ661 inhibited the NMDA-induced AMPA receptor endocytosis. Therefore, NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating PIP5Kγ661 activity at postsynapses.     

Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis

Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are essential regulators of polarized cell growth, cytokinesis, and many other cellular processes. However, the regulation of Rho-GEFs themselves is not well understood. Rgf3 is an essential GEF for Rho1 GTPase in fission yeast. We show that Rgf3 protein levels and localization are regulated by arrestin-related protein Art1. art1∆ cells lyse during cell separation with a thinner and defective septum. As does Rgf3, Art1 concentrates to the contractile ring starting at early anaphase and spreads to the septum during and after ring constriction. Art1 localization depends on its C-terminus, and Art1 is important for maintaining Rgf3 protein levels. Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1. Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site. As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells. Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.     

Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

Phosphatidylinositol monophosphate 5-kinase (PIP5K) catalyzes the synthesis of PI-4,5-bisphosphate (PtdIns(4,5)P(2)) by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring, and is involved in regulating multiple developmental processes and stress responses. We here report on the functional characterization of Arabidopsis PIP5K2, which is expressed during lateral root initiation and elongation, and whose expression is enhanced by exogenous auxin. The knockout mutant pip5k2 shows reduced lateral root formation, which could be recovered with exogenous auxin, and interestingly, delayed root gravity response that could not be recovered with exogenous auxin. Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2. In addition, analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P(2) reduction, which hence results in suppressed cycling of PIN proteins (PIN2 and 3), and delayed redistribution of PIN2 and auxin under gravistimulation in pip5k2 roots. On the contrary, PtdIns(4,5)P(2) significantly enhanced the vesicle trafficking and cycling of PIN proteins. These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response, and reveal a critical role of PIP5K2/PtdIns(4,5)P(2) in root development through regulation of PIN proteins, providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response, and new insights into the control of polar auxin transport.