G-protein-coupled Receptor Activation Induces the Membrane Translocation and Activation of Phosphatidylinositol-4-phosphate 5-Kinase Iα by a Rac- and Rho-dependent Pathway
2001; Elsevier BV; Volume: 276; Issue: 36 Linguagem: Inglês
10.1074/jbc.m104917200
ISSN1083-351X
AutoresNour.-E.-H. Chatah, Charles S. Abrams,
Tópico(s)Cellular transport and secretion
ResumoPhosphatidylinositol 4,5-bisphosphate (PI4,5P2) 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 Iα activation following the addition of thrombin. Under resting condition expressed PIP5K Iα was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of Gαq, PIP5K Iα translocated to the plasma membrane. Movement of PIP5K Iα 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 Gαq, 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K Iα translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K Iα. In addition, a dominant negative variant of Rho blocked the PIP5K Iα membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K Iα membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K Iα by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K Iα, 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 Iα correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K Iα with either constitutively active Gαq, Rac, or Rho led to a 5- to 7-fold increase in PIP5K Iα activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of Gαq, Rac, Rho, and PIP5K Iα. Once activated and translocated to the cell membrane, PIP5K Iα becomes available to phosphorylate PI4P to generate PI4,5P2 on the plasma membrane. Phosphatidylinositol 4,5-bisphosphate (PI4,5P2) 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 Iα activation following the addition of thrombin. Under resting condition expressed PIP5K Iα was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of Gαq, PIP5K Iα translocated to the plasma membrane. Movement of PIP5K Iα 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 Gαq, 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K Iα translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K Iα. In addition, a dominant negative variant of Rho blocked the PIP5K Iα membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K Iα membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K Iα by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K Iα, 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 Iα correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K Iα with either constitutively active Gαq, Rac, or Rho led to a 5- to 7-fold increase in PIP5K Iα activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of Gαq, Rac, Rho, and PIP5K Iα. Once activated and translocated to the cell membrane, PIP5K Iα becomes available to phosphorylate PI4P to generate PI4,5P2 on the plasma membrane. 5P2, phosphatidylinositol 4,5-bisphosphate phosphatidylinositol-4-phosphate 5-kinase phosphatidylinositol-5-phosphate 4-kinase phosphatidylinositol 4-phosphate phosphatidylinositol 5-phosphate guanosine 5′-3-O-(thio)triphosphate hemagglutinin polymerase chain reaction expressed sequence tag phosphate-buffered saline The synthesis of the membrane-bound phospholipid, phosphatidylinositol 4,5-bisphosphate (PI4,5P2)1 is a critical step in signal transduction. PI4,5P2 serves as a substrate for phospholipase C and phosphatidylinositol 3-kinase to generate lipid second messengers (1Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Crossref PubMed Scopus (4254) Google Scholar, 2Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3313) Google Scholar, 3Auger K.R. Serunian L.A. Soltoff S.P. Libby P. Cantley L.C. 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Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar) demonstrated that the small G-protein ADP-ribosylation factor, Arf, directly activates PIP5K Iα in the presence of phosphatidic acid, whereas Rho and Rac had no effect. In contrast, Tolias et al. (34Tolias K.F. Hartwig J.H. Ishihara H. Shibasaki Y. Cantley L.C. Carpenter C.L. Curr. Biol. 2000; 10: 153-156Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar) showed that the addition of recombinant Rac to permeabilized platelets led to PIP5K Iα activation and PI4,5P2 production. Therefore, it appears that small GTP-binding proteins affect PIP5K I activity and PI4,5P2production. However, it is unclear which GTPase is predominantly responsible. Both PIP5K I and PIP4K appear to localize to distinct subcellular compartments. PIP5K Iα has been reported to specifically localize at the plasma membranes or in the nucleus, whereas PIP4K has been reported to be present diffusely within the cell or to be present in the ER, actin cytoskeleton, cytosol, plasma membrane, or nucleus (35Shibasaki Y. Ishihara H. Kizuki N. Asano T. Oka Y. Yazaki Y. J. Biol. Chem. 1997; 272: 7578-7581Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 36Boronenkov I.V. Loijens J.C. Umeda M. Anderson R.A. Mol. Biol. Cell. 1998; 9: 3547-3560Crossref PubMed Scopus (282) Google Scholar, 37Itoh T. Ijuin T. Takenawa T. J. Biol. Chem. 1998; 273: 20292-20299Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 38Hinchliffe K.A. Irvine R.F. Divecha N. EMBO J. 1996; 15: 6516-6524Crossref PubMed Scopus (63) Google Scholar). We asked whether localization of PIP5K Iα played a role in its regulation, because its only known substrates are membrane-bound phospholipids. In the present study, we demonstrate that stimulation of the PAR1 receptor induces a translocation of PIP5K Iα from the Golgi to the cell membrane. This process involves heterotrimeric G-proteins as well as both Rac and Rho, but not Arf. Coincident with the intracellular translocation of PIP5K Iα, we found that its enzymatic activity increased 5- to 7-fold when co-expressed with either large or small GTP-binding proteins. Therefore, these results delineate a signaling pathway that is initiated by a G-protein-coupled receptor and leads to the production of PI4,5P2. The anti-myc, anti-HA, or the biotinylated monoclonal anti-FLAG (M2) antibodies were purchased from Covance, the anti-GM 130 was purchased from Transduction Laboratories, and the anti-PAR1 monoclonal antibody was a gift from L. F. Brass (University of Pennsylvania, Philadelphia, PA (39Hoxie J.A. Ahuja M. Belmonte E. Pizarro S. Parton R.G. Brass L.F. J. Biol. Chem. 1993; 268: 13756-13763Abstract Full Text PDF PubMed Google Scholar)). The fluorescein isothiocyanate- and rhodamine-conjugated goat secondary antibodies were obtained from BIOSOURCE. Alexa 350, the cascade blue-labeled goat anti-rabbit secondary antibody, and Alexa 546, the red dye-conjugated streptavidin were purchased from Molecular Probes. The PI4P was obtained from Roche Molecular Biochemicals, and all remaining chemicals were obtained from Sigma Chemical Co. The human PIP5K Iα cDNA was generated by PCR using EST AA054379 (Genome Systems) as template and the following oligonucleotide primers: 5′-CG CCA GGA TCC GCC ACC ATG TCT TCT GCT GCT GAA-3′ and 5′-GG CGC TCT AGA TTA CAA GTC TTC TTC AGA AAT CAA CTT TTG TTC TAA ATA GAC GTC AAG CAC-3′. The primers incorporated BamHI and XbaI sites, as well as a carboxyl-terminal myc epitope (EQKLISEEDL). This product was cloned into BglII- and XbaI-digested pCMV5 (a gift of Mark Stinski, University of Iowa). Cloning of PIP4K (also known as PIP5K Type II or Type C) was performed by PCR using EST W00481 from Genome Systems. Full-length sequencing revealed that two nucleotide changes that differed from the published sequence, and altered the predicted translated product from101CGK103 to 101LRE103and V109 to D. These substitutions likely represent polymorphisms, because multiple independent EST clones (N28765, N33825, and THC154497) contained the identical sequence. The cDNA was amplified using PCR primers CG GCA G GTA CCG GCC ATG GCG ACC CCC GGC and GG CGC TCT AGA TTA CAA GTC TTC TTC AGA AAT CAA CTT TTG TTC CGT CAA GAT GTG GCC AAT, which incorporated an myc-epitope onto the carboxyl terminus and KpnI andXbaI restriction sites to facilitate cloning into pCMV5. The amino terminus epitope-tagged small G-proteins HA-Rac1 L61, HA-Rac1 V12N17, and FLAG-Rac1 V12N17 were subcloned into KpnI- andXbaI-digested pCMV5. HA-RhoA L63 and FLAG-RhoA N19 were subcloned into EcoRI- and BamHI-digested pCDNA 3.1+. HA-Arf 1 Q71L was cloned by PCR mutagenesis using EST clone R19462 as the template and ligated into BamHI- andEcoRI-digested pCDNA 3.1+. The sequence of all PCR-generated cDNA was fully confirmed after cloning. The constitutively active HA-Gαq Q209L was a gift from Dr. J. Silvio Gutkind (National Institutes of Health, Bethesda, MD). HA-epitope-tagged dominant negative Arf6 (HA-Arf6 N27) and constitutively active Arf6 (HA-Arf6 L67) were a gift of Dr. Morris Birnbaum (University of Pennsylvania, Philadelphia, PA). The cDNA human thrombin receptor cloned into pRK7 was previously described (40Abrams C.S. Wu H. Zhao W. Belmonte E. White D. Brass L.F. J. Biol. Chem. 1995; 270: 14485-14492Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Human Embryonic Kidney HEK 293T and Cos-7 cells grown in Dulbecco's modified Eagle medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies, Inc.) were transiently transfected by the calcium phosphate technique using a total of 4 μg of plasmid per 30-mm tissue culture dish. Care was always taken to include empty vector to some transfections to allow identical quantities of plasmid to be used in every transfection. Twenty-four hours after transfection, the medium was removed and replaced with a fresh medium, and analyzed after an additional 24 h. All cells were grown at 37 °C in the presence of 5% of CO2. Transiently transfected Cos-7 cells were rinsed in PBS, fixed in 10% Buffered Formalin Acetate (Fisher Scientific) for 30 min, rinsed again twice with PBS, and then permeabilized in 0.2% Triton X-100-containing PBS for 10 min. Cells were double-stained with a polyclonal anti-myc epitope antibody and a monoclonal antibody directed against either PAR1 (WEDE15), γ-adaptin (a Golgi protein), or the HA-epitope (12CA5). Anti-myc was visualized by fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. Anti-PAR1, -adaptin, or -HA was identified by rhodamine-conjugated goat anti-mouse antibody. For the triple staining, cells were first stained with monoclonal anti-HA followed by fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody. The cells were then incubated with polyclonal anti-myc and biotinylated monoclonal anti-FLAG IgG1 (M2) antibodies, followed by staining with the cascade blue-labeled goat anti-rabbit secondary antibody along with Alexa 546-conjugated streptavidin. Localization experiments were performed a minimum of three times, and ∼30–60 cells per experiment were examined by two or more viewers. Confocal micrographs of at least six to eight representative cells of each experimental condition were performed by the University of Pennsylvania Cancer Center Confocal Microscopy Core Facility. Confocal images were acquired from a TCS 4D Upright microscope and processed on an IBM OS9 workstation, using Scanware software. After transient transfection, 10-cm tissue culture plastic dishes of confluent HEK 293T cells were washed three times with cold PBS and lysed with 1 ml buffer A (1% Triton X-100; 0.14 m NaCl; 1 mmMgCl2; 1 mm EGTA; 20 mm HEPES, pH 7.5, containing 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 2 μg/ml aprotinin, 0.2 mmNa3VO4, and 50 mm NaF). The lysates were clarified by centrifugation at 13,000 × g for 30 min at 4 °C. Immunoprecipitation was performed using 9E10, a monoclonal antibody against the myc epitope. The immune complexes are collected on protein G-Sepharose, washed twice with buffer A, three times with the kinase buffer B (40 mm HEPES, 20 mm EGTA, 0.2 mm EDTA, 0.1 mm NaCl, 10 mm MgCl2, pH 7.5, containing 1 mm dithiothreitol) and subjected to immunoblotting and kinase lipid assay. A standard assay for phosphorylation of PI4P was carried out in an incubation medium of 80 μl of buffer B containing 125 μm ATP, 125 μm PI4P, and 2.5 μCi of [γ-32P]ATP. The enzyme reactions were incubated at 37 °C for 20 min. After stopping the reaction with 1 m HCl (2 volumes) and extracting the lipids with an equal volume of chloroform-methanol (1:1, v/v), the32P-labeled PI4,5P2 products were resolved by thin-layer chromatography using water-chlorophorm-methanol-NH4OH (25:70:100:15, v/v) as a solvent system. Immediately before chromatography, the thin-layer plates were precoated with 1% potassium oxalate in water-methanol (1:1) and baked at 110 °C for 35 min. Unlabeled PI4P and PI4,5P2 standards were run in parallel to samples to monitor lipid migration and were visualized by exposure to iodine vapor. The regions of the TLC plates that contained PI4,5P2spots were carefully excised, placed in a screw-capped scintillation vial, and then subjected to scintillation counting. Counts were normalized for immunoprecipitated myc-PIP5K Iα as determined by quantitative 125I-immunoblotting. Previous reports demonstrated that stimulation of the predominant thrombin receptor on human platelet, PAR1, leads to an increase in PI4,5P2 synthesis (41Grondin P. Plantavid M. Sultan C. Breton M. Mauco G. Chap H. J. Biol. Chem. 1991; 266: 15705-15709Abstract Full Text PDF PubMed Google Scholar, 42Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (613) Google Scholar). Using Cos-7 cells transfected with tagged PIP5K, we investigated whether stimulation of this G-protein-coupled receptor was also associated with an intracellular redistribution of PIP5K Iα. Under resting conditions, expressed PIP5K Iα was located adjacent to the cell nucleus (Fig. 1). Because of the eccentric perinuclear distribution, we simultaneously co-stained cells with antibodies against the Golgi marker, adaptin, as well as the epitope tag on PIP5K Iα. We found that resting PIP5K Iα localized in proximity with γ-adaptin (Fig. 1). Confocal microscopy verified this localization and showed that PIP5K Iα colocalized most closely with a marker of the cis-Golgi cisternae, GM-130 (Fig.2 A). Thus, under resting conditions, PIP5K Iα is co-localized within the earlytrans-Golgi network. It should be noted that the Golgi has previously been described to contain PIP5K I activity (43Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (454) Google Scholar, 44Jones D.H. Morris J.B. Morgan C.P. Kondo H. Irvine R.F. Cockcroft S. J. Biol. Chem. 2000; 275: 13962-13966Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and that this intracellular localization is also similar to the related lipid kinase PI4Kβ (45Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 43Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (454) Google Scholar).Figure 2Confocal microscopy PIP5K Iα and Gαq. A, Cos-7 cells were transiently transfected with myc epitope-tagged PIP5K Iα and stained with polyclonal anti-myc (green) and monoclonal anti-GM130 (red). Shown is an overlay confocal micrograph demonstrating that unstimulated PIP5K Iα localizes with GM130. B, Cos-7 cells were transiently transfected with myc epitope-tagged PIP5K Iα along with constitutively active HA-tagged Gαq. The cells were fixed and stained with both polyclonal anti-myc and monoclonal anti-HA. Shown is an overlay confocal image of anti-myc (red) and anti-HA (green) staining. This demonstrates that active Gαq stimulates PIP5K Iα to localize on the cell membrane.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next tested whether stimulation of the PAR1 thrombin receptor would alter the location of PIP5K Iα. As shown in Fig.3, stimulation of PAR1 with the receptor-specific activating peptide SFLLRN resulted in the translocation of PIP5K Iα to the cellular membrane. After 1 h of stimulation with the peptide agonist, 30–40% of cells redistributed PIP5K Iα toward their plasma membrane. Additional studies using transfected HEK-293 cells yielded the same result (not shown). Therefore, these data indicate that stimulation of the thrombin receptor initiates a signaling cascade that can induce the movement of PIP5K Iα toward the cell membrane. PAR1 is typically coupled to phospholipase C by Gαq. To determine whether PAR1 mediated its affect on the intracellular distribution of PIP5K Iα via Gαq, we tested whether the relocalization of PIP5K Iα could be mimicked by co-expression of PIP5K Iα with an active variant of the α subunit. The Q209L mutation in Gαq (HA-Gαq Q209L) is constitutively in the GTP-bound state. In 100% of cells expressing Gαq Q209L and PIP5K Iα, PIP5K Iα was easily identified as being associated with the cell membrane (Fig.4). Confocal microscopy of cells expressing active Gαq and PIP5K Iα, verified PIP5K Iα was present on the cell membrane (Fig. 2 B). Although these confocal images revealed that the majority of PIP5K Iα was now on the cell membrane, in some cells a fraction of PIP5K Iα was still localized with the Golgi. These results indicate that the subcellular distribution of the PIP5K Iα can be regulated by the thrombin receptor and at least one of the G-proteins which normally associate with it. We also found that stimulation of the EGF receptor by 1-h stimulation of 25 ng/ml EGF initiated cell membrane association of PIP5K Iα (data not shown). This demonstrates that the relocalization of PIP5K Iα is not limited to stimulation of a G-protein-coupled receptor. In addition, we have found that expressed PIP5K Iα in Jurkat T-cells has a perinuclear distribution when the cells are plated on poly-l-lysine. However, PIP5K Iα translocates to the cellular membrane when the cells are plated on either fibronectin or C305 (an IgM antibody that binds and activates the T-cell antigen receptor). This implies that the signaling pathway leading to membrane association of PIP5K Iα ultimately involves effectors common to multiple receptor families, including G-protein-coupled, growth factor, immunoglobulin supergene, and integrin receptors. Although there is agreement that low molecular weight GTP-binding proteins contribute to PIP5K Iα activation, the identity of the relevant proteins is controversial. Because the low molecular weight GTP-binding proteins Rac and Rho become activated after stimulation of G-protein-coupled, growth factor, immunoglobulin supergene, and integrin receptors (42Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 46Zhang J. King W.G. Dillon S. Hall A. Feig L. Rittenhouse S.E. J. Biol. Chem. 1993; 268: 22251-22254Abstract Full Text PDF PubMed Google Scholar, 47Dustin M.L. Chan A.C. Cell. 2000; 103: 283-294Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 48Penninger J.M. Crabtree G.R. Cell. 1999; 96: 9-12Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 49Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar), we tested whether the membrane localization of PIP5K Iα could be influenced by these proteins. As shown in Fig. 5, when PIP5K Iα was co-expressed along with constitutively GTP-bound variants of Rac (Rac L61) or Rho (Rho L63), PIP5K Iα was recruited from the Golgi to the cellular membrane in 100% of cells. This effect by Rac or Rho was regulated by the nucleotide-bound state, because overexpression of constitutively GDP-bound variants of either Rac (Rac V12N17) or Rho (Rho N19) failed to induce the PIP5K Iα translocation (Fig. 5). Arf is another low molecular weight GTP-binding protein that has been shown to interact in vitro with PIP5K Iα. In contrast to the effect of Rac and Rho on PIP5K Iα intracellular localization, we found that neither constitutively GTP-bound variant of Arf1 (Fig. 5) or Arf6 (not shown) induced PIP5K Iα translocation. Therefore, this demonstrates that Rac and Rho, but not Arf, are capable of inducing membrane translocation of PIP5K Iα. This also shows that PIP5K translocation is regulated by which guanine nucleotide is bound to the low molecular weight GTP-binding protein. Because PIP4K has also been reported to become stimulated after thrombin stimulation of platelets (38Hinchliffe K.A. Irvine R.F. Divecha N. EMBO J. 1996; 15: 6516-6524Crossref PubMed Scopus (63) Google Scholar), we next tested whether small GTP-binding proteins also affected the intracellular distribution of this lipid kinase. In contrast to our findings with PIP5K Iα, neither the GTP- nor GDP-bound variants of Rac or Rho affected the localization of PIP4K (Fig. 6). Therefore, these observations suggest that, although Rac and Rho can contribute to the membrane localization of PIP5K Iα, they have no effect on the intracellular distribution of PIP4K. Because activation of both high, and low, molecular weight GTP-binding proteins lead to the translocation of PIP5K Iα, we examined whether they are components of the same signaling pathway. To begin to address this question, we tested whether dominant negative variants of Rac or Rho affected the ability of Gαq to induce membrane association of PIP5K Iα. As shown in Fig. 7, GDP-bound variants of either Rac or Rho functioned as competitive inhibitors (dominant negatives) and blocked the ability of Gαq to induce cell mem
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