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Hypertonic Stress Increases Phosphatidylinositol 4,5-Bisphosphate Levels by Activating PIP5KIβ

2006; Elsevier BV; Volume: 281; Issue: 43 Linguagem: Inglês

10.1074/jbc.m605928200

ISSN

1083-351X

Autores

Masaya Yamamoto, Mark Z. Chen, Yingjie Wang, Hui-Qiao Sun, Yongjie Wei, Manuel Martínez, Helen L. Yin,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

Hyperosmotic stress increases phosphoinositide levels, reorganizes the actin cytoskeleton, and induces multiple acute and adaptive physiological responses. Here we showed that phosphatidylinositol 4,5-bisphosphate (PIP2) level increased rapidly in HeLa cells during hypertonic treatment. Depletion of the human type I phosphatidylinositol 4-phosphate 5-kinase β isoform (PIP5KIβ) by RNA interference impaired both the PIP2 and actin cytoskeletal responses. PIP5KIβ was recruited to membranes and was activated by hypertonic stress through Ser/Thr dephosphorylation. Calyculin A, a protein phosphatase 1 inhibitor, blocked the hypertonicity-induced PIP5KIβ dephosphorylation/activation as well as PIP2 increase in cells. Urea, which raises osmolarity without inducing cell shrinkage, did not promote dephosphorylation nor increase PIP2 levels. Disruption or stabilization of the actin cytoskeleton, or inhibition of the Rho kinase, did not block the PIP2 increase nor PIP5KIβ dephosphorylation. Therefore, PIP5KIβ is dephosphorylated in a volume-dependent manner by a calyculin A-sensitive protein phosphatase, which is activated upstream of actin remodeling and independently of Rho kinase activation. Our results establish a cause-and-effect relation between PIP5KIβ dephosphorylation, lipid kinase activation, and PIP2 increase in cells. This PIP2 increase can orchestrate multiple downstream responses, including the reorganization of the actin cytoskeleton. Hyperosmotic stress increases phosphoinositide levels, reorganizes the actin cytoskeleton, and induces multiple acute and adaptive physiological responses. Here we showed that phosphatidylinositol 4,5-bisphosphate (PIP2) level increased rapidly in HeLa cells during hypertonic treatment. Depletion of the human type I phosphatidylinositol 4-phosphate 5-kinase β isoform (PIP5KIβ) by RNA interference impaired both the PIP2 and actin cytoskeletal responses. PIP5KIβ was recruited to membranes and was activated by hypertonic stress through Ser/Thr dephosphorylation. Calyculin A, a protein phosphatase 1 inhibitor, blocked the hypertonicity-induced PIP5KIβ dephosphorylation/activation as well as PIP2 increase in cells. Urea, which raises osmolarity without inducing cell shrinkage, did not promote dephosphorylation nor increase PIP2 levels. Disruption or stabilization of the actin cytoskeleton, or inhibition of the Rho kinase, did not block the PIP2 increase nor PIP5KIβ dephosphorylation. Therefore, PIP5KIβ is dephosphorylated in a volume-dependent manner by a calyculin A-sensitive protein phosphatase, which is activated upstream of actin remodeling and independently of Rho kinase activation. Our results establish a cause-and-effect relation between PIP5KIβ dephosphorylation, lipid kinase activation, and PIP2 increase in cells. This PIP2 increase can orchestrate multiple downstream responses, including the reorganization of the actin cytoskeleton. All cells experience fluctuations in osmolarity. Unicellular organisms and plants continuously confront osmotic challenges in their environment. In higher animals, the kidney and the gastrointestinal system are routinely exposed to severe osmotic fluctuation, while the majority of cells in other organs are protected from large tonicity changes. Nevertheless, these other organs are also confronted with transient osmolarity variations due to changes in the transmembrane transport of solutes or shifts in the balance between low molecular weight pre-cursors and their macromolecular products. Recently, there has been a renewed interest in understanding the mechanism of hypertonic response in the clinical arena (1Rhee P. Koustova E. Alam H.B. J. Trauma. 2003; 54: S52-S62Crossref PubMed Scopus (195) Google Scholar), due to the discovery that treatments using hypertonic resuscitation in experimental models of trauma, hemorrhagic shock, sepsis, and burn injury are more beneficial than conventional isotonic resuscitation (2Horton J.W. Maass D.L. White D.J. Am. J. Physiol. 2006; 290: H1642-H1650Crossref PubMed Scopus (18) Google Scholar, 3Shukla A. Hashguchi N. Chen Y. Coimbra R. Hoyt D.B. Junger W.G. Shock. 2004; 21: 391-400Crossref PubMed Scopus (73) Google Scholar). While the fundamental mechanism for such protection is not completely understood, the actin cytoskeleton, which is reorganized during hypertonic stress, has been implicated (3Shukla A. Hashguchi N. Chen Y. Coimbra R. Hoyt D.B. Junger W.G. Shock. 2004; 21: 391-400Crossref PubMed Scopus (73) Google Scholar, 4Di Ciano C. Nie Z. Szaszi K. Lewis A. Uruno T. Zhan X. Rotstein O.D. Mak A. Kapus A. Am. J. Physiol. 2002; 283: C850-C865Crossref PubMed Scopus (133) Google Scholar). Actin remodeling as well as many of the other hyperosmotic responses are evolutionary conserved. These include large shifts in phosphoinositide metabolism, activation of the mitogen-activated protein and tyrosine kinase pathways, volume regulation and the reprogramming of gene transcription (3Shukla A. Hashguchi N. Chen Y. Coimbra R. Hoyt D.B. Junger W.G. Shock. 2004; 21: 391-400Crossref PubMed Scopus (73) Google Scholar, 5Sheikh-Hamad D. Gustin M.C. Am. J. Physiol. 2004; 287: F1102-F1110Crossref PubMed Scopus (141) Google Scholar). Phosphatidylinositol 4-phosphate (PI4P) 4The abbreviations used are: PI4P, phosphatidylinositol 4-phosphate; PIP5KI, type I phosphatidyoinositol 4-phosphate 5-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; caly A, calyculin A; HA, hemagglutinin; HPLC, high performance liquid chromatography; siRNA, small interfering RNA; PM, plasma membrane; RNAi, RNA interference. and phosphatidylinositol 4,5-bisphosphate (PIP2) levels increase dramatically in mammalian cardiac muscle and tissue culture cells that were exposed to hypertonic sucrose or NaCl (6Nasuhoglu C. Feng S. Mao Y. Shammat I. Yamamato M. Earnest S. Lemmon M. Hilgemann D.W. Am. J. Physiol. 2002; 283: C223-C234Crossref PubMed Scopus (77) Google Scholar). Other phosphoinositides, including phosphatidylinositol 3,5-bisphosphate (7Sbrissa D. Shisheva A. J. Biol. Chem. 2005; 280: 7883-7889Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Ikonomov O.C. Sbrissa D. Yoshimori T. Cover T.L. Shisheva A. J. Biol. Chem. 2002; 277: 46785-46790Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate are increased in some other types of cells as well (9Van der Kaay J. Beck M. Gray A. Downes C.P. J. Biol. Chem. 1999; 274: 35963-35968Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Hyperosmotic stress acutely induces cell shrinkage, which is subsequently corrected by volume regulation. The response cascade can be classified into four mechanistic components: volume sensing, signal generation, signal transduction, and effector activation (10Papakonstanti E.A. Vardaki E.A. Stournaras C. Cell. Physiol. Biochem. 2000; 10: 257-264Crossref PubMed Scopus (83) Google Scholar). Given that PIP2 is a multifunctional regulator and adaptor, the PIP2 increase may coordinate many of the responses to hypertonic stress. Nevertheless, the role of PIP2 in the response hierarchy, and the mechanism for PIP2 increase, have yet to be elucidated. PIP2 levels are maintained by a dynamic balance between PIP2 synthesis and degradation. PIP2 is synthesized primarily by the type I PIP5Ks, which phosphorylate PI4P on the D5 position of the inositol ring. PIP2 is degraded primarily by phosphoinositide phosphatases and by PI-phospholipase Cs. Three major PIP5KI isoforms, called α, β, and γ, have been identified (11Ishihara H. Shibasaki Y. Kizuki N. Wada T. Yazaki Y. Asano T. Oka Y. J. Biol. Chem. 1998; 273: 8741-8748Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Additionally, the γ isoform is differentially spliced to generate several variants (12Ling K. Doughman R.L. Firestone A.J. Bunce M.W. Anderson R.A. Nature. 2002; 420: 89-93Crossref PubMed Scopus (388) Google Scholar, 13Di Paolo G. Pellegrini L. Letinic K. Cestra G. Zoncu R. Voronov S. Chang S. Guo J. Wenk M.R. De Camilli P. Nature. 2002; 420: 85-89Crossref PubMed Scopus (382) Google Scholar). PIP5KIs are predominantly cytosolic proteins that are recruited to plasma and organelle membranes through association with small GTPases such as Rho, Rac (14Yang S.-A. Carpenter C.L. Abrams C.S. J. Biol. Chem. 2004; 279: 42331-42336Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Chong L.D. Traynor-Kaplan A. Bokoch G.M. Schwartz M.A. Cell. 1994; 79: 507-513Abstract Full Text PDF PubMed Scopus (600) Google Scholar), and Arf6 (16Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 17Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1018Crossref PubMed Scopus (368) Google Scholar, 18Aikawa Y. Martin T.F.J. J. Cell Biol. 2003; 162: 647-659Crossref PubMed Scopus (189) Google Scholar, 19Krauss M. 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Chem. 2000; 275: 19389-19394Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 29Park S.J. Itoh T. Takenawa T. J. Biol. Chem. 2001; 276: 4781-4787Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and by tyrosine phosphorylation (12Ling K. Doughman R.L. Firestone A.J. Bunce M.W. Anderson R.A. Nature. 2002; 420: 89-93Crossref PubMed Scopus (388) Google Scholar, 13Di Paolo G. Pellegrini L. Letinic K. Cestra G. Zoncu R. Voronov S. Chang S. Guo J. Wenk M.R. De Camilli P. Nature. 2002; 420: 85-89Crossref PubMed Scopus (382) Google Scholar). Despite the large cadre of potential regulatory mechanisms, nothing is known about the regulation of the PIP5KIs by hypertonic stress. In this paper, we have identified the hypertonic stress regulated PIP5KI and examined the relation between its activation by Ser/Thr dephosphorylation to the hypertonicity-induced increase in PIP2 production and actin remodeling. PIP5KI Overexpression—We use the isoform designation for human PIP5KIs, which is different from that for mouse (30Mejillano M. Yamamoto M. Rozelle A.L. Sun H.Q. Wang X. Yin H.L. J. Biol. Chem. 2001; 276: 1865-1872Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Hemagglutinin (HA)-tagged PIP5KIα,-β,-γ87, and -γ90 were cloned from cDNA provided by other laboratories. In some experiments, recombinant adenovirus vectors expressing β-galactosidase, HA-tagged human PIP5KIβ or myc-tagged human PIP5KIα were used. These were generated using the AdEasyTM adenoviral vector system (Stratagene) (23Yamamoto M. Hilgemann D.H. Feng S. Bito H. Ishihara H. Shibasaki Y. Yin H.L. J. Cell Biol. 2001; 152: 867-876Crossref PubMed Scopus (105) Google Scholar). Hypertonic Treatments—HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. 250 mm sucrose or 100 mm or 150 mm NaCl was added to the isotonic growth medium during hypertonic stimulation. TLC—Cells were labeled for 4 h in phosphate-free Dulbecco's modified Eagle's medium and 40 μCi/ml [32P]PO4 and were then exposed to sucrose or NaCl. Samples were processed for TLC as described previously (23Yamamoto M. Hilgemann D.H. Feng S. Bito H. Ishihara H. Shibasaki Y. Yin H.L. J. Cell Biol. 2001; 152: 867-876Crossref PubMed Scopus (105) Google Scholar). High Performance Liquid Chromatography (HPLC)—Lipids were extracted, deacylated, and analyzed on anion exchange HPLC columns. Negatively charged glycerol head groups were eluted with a NaOH gradient and detected on-line by suppressed conductivity (6Nasuhoglu C. Feng S. Mao Y. Shammat I. Yamamato M. Earnest S. Lemmon M. Hilgemann D.W. Am. J. Physiol. 2002; 283: C223-C234Crossref PubMed Scopus (77) Google Scholar, 31Nasuhoglu C. Feng S. Mao J. Yamamoto M. Yin H.L. Earnest S. Barylko B. Albanesi J.P. Hilgemann D.W. Anal. Biochem. 2002; 301: 243-254Crossref PubMed Scopus (114) Google Scholar). Individual peaks were identified with glycerophosphoryl inositol standards. Peak assignment was validated by spiking some cell samples with purified phospholipids as standards. RNA Interference—The small interfering RNA (siRNA) sequences targeting the three human PIP5KI isoforms individually are as described previously (33Padron D. Wang Y.J. Yamamoto M. Yin H. Roth M.G. J. Cell Biol. 2003; 162: 693-701Crossref PubMed Scopus (125) Google Scholar, 34Wang Y.J. Li W.H. Wang J. Xu K. Dong P. Luo X. Yin H.L. J. Cell Biol. 2004; 167: 1005-1010Crossref PubMed Scopus (80) Google Scholar). Firefly luciferase siRNA (nucleotides 695-715) was used as a negative control. Immunoprecipitation—HeLa cells overexpressing the epitope-tagged PIP5KI isoforms were lysed in buffer containing 25 mm HCl, pH 7.5, 0.15 m NaCl, 5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1 mm dithiothreitol, 1 mm sodium vanadate, 0.5% Nonidet P-40, and protease inhibitors. The overexpressed PIP5KIs were immunoprecipitated with monoclonal anti-epitope tag antibody bound to protein G-Sepharose. Anti-HA and anti-myc are from Convence. In Vitro PIP5KI Kinase Assay—Lipid kinase activity was measured by phosphorylation of PI4P using [γ-32P]ATP as a phosphate donor. Sepharose G beads containing immunoprecipitated epitope-tagged PIP5KI were suspended in a solution containing 25 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm MgCl2,10mm EDTA, 0.1 mm EGTA, 0.4% Nonidet P-40, 10% glycerol, 1 mm dithiothreitol, 1 mm sodium vanadate, 70 μm PI4P (Biomol Inc.) and 35 μm phosphatidylserine (Avanti). Kinase assay was initiated by adding [γ-32P]ATP (1 μCi/50 μl reaction with a final concentration of 0.2 mm ATP, PerkinElmer Life Sciences), and reaction proceeded at room temperature for 15 min. The reactions were stopped by adding CHCl3:MeOH:HCl, and lipids were extracted as described above and separated by TLC. Radioactivity associated with the PIP band that comigrates with a PI4P standard was quantitated by PhosphorImager analysis, and the amount of PIP5KI protein in the equivalent immunoprecipitate was determined by Western blotting. Kinase activity was normalized to the amount of immunoprecipitated protein and expressed as percent of the activity without sucrose stimulation. Under the conditions of our experiments, the kinase activity was linear from 5 to 30 min. Fluorescence Microscopy—Immunofluorescence labeling was as described previously (34Wang Y.J. Li W.H. Wang J. Xu K. Dong P. Luo X. Yin H.L. J. Cell Biol. 2004; 167: 1005-1010Crossref PubMed Scopus (80) Google Scholar, 35Wang Y.J. Wang J. Sun H.Q. Martinez M. Sun Y.X. Macia E. Kirchhausen T. Albanesi J.P. Roth M.G. Yin H.L. Cell. 2003; 114: 299-310Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Anti-PIP2 was from Bio-Assay Systems. Membrane Fractionation—Cell homogenates were centrifuged at 100,000 × g to obtain the cytosol and membrane fractions (33Padron D. Wang Y.J. Yamamoto M. Yin H. Roth M.G. J. Cell Biol. 2003; 162: 693-701Crossref PubMed Scopus (125) Google Scholar). Organelles and organelle membranes were separated according to their density by a multistep centrifugation procedure (36Wei Y.J. Sun H.Q. Yamamoto M. Wlodarski P. Kunii K. Martinez M. Barylko B. Albanesi J.P. Yin H.L. J. Biol. Chem. 2002; 277: 46586-46593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Samples obtained by centrifugation sequentially at 19,000 × g, 41,000 × g (low speed), and 180,000 × g (high speed) were analyzed. Plasma membrane enriched sample was obtained by floatation on a sucrose cushion. The pelleted fractions were adjusted to 100 μl, and equal volumes of membrane fractions were analyzed by Western blotting with anti-HA antibody. Isolation of the Triton X-100-insoluble Actin Cytoskeleton—Cells were lysed in Triton X-100 and centrifuged sequentially at low speed (15,900 × g) to collect cross-linked actin stress fibers and networks (low speed pellet) and at high speed (336,000 × g) to sediment long filaments that were not cross-linked (high speed pellet). Hypertonic Stress Increased the PIP2 Level in Cells—When HeLa cells were exposed to 250 mm sucrose for 10 min, the [32P]PIP2 level increased to a greater extent than [32P]PI4P (Fig. 1A and Table 1). Likewise, addition of 100 mm NaCl to normal culture medium also increased PIP2 levels to a much higher extent than PI4P (Table 1). Therefore, hypertonic stress induced by these two stimuli preferentially increases PIP2 level.TABLE 1Effects of osmotic stress on phosphatidylinositol lipidsTreatmentLipid massaFrom HPLC analyses.32P-LipidsbFrom PhosphorImager analyses of TLC data.PIPI4PPIP2PIPI4PPIP2% of isotonic control% of isotonic control250 mm sucrose105 ± 2 (n = 4)93 ± 17 (n = 4)219 ± 33 (n = 4)113 ± 4 (n = 3)121 ± 10 (n = 8)180 ± 10 (n = 8)100 mm NaClNDNDND128 ± 7 (n = 3)151 ± 18 (n = 3)253 ± 48 (n = 3)a From HPLC analyses.b From PhosphorImager analyses of TLC data. Open table in a new tab The increase in phosphoinositide levels was evident within 2 min of sucrose stimulation and reached a maximum by 10 min (Fig. 1A). While there was a subsequent slow decline, PIP2 was still above prestimulation levels at 20 min. The rapid increase in [32P]PIP2 and [32P]PI4P suggests that the change in phosphoinositide turnover/biosynthesis is an early response to hypertonic stimulation. HPLC, which measures the amount of each lipid and can separate the different types of phosphoinositides better than the TLC method (31Nasuhoglu C. Feng S. Mao J. Yamamoto M. Yin H.L. Earnest S. Barylko B. Albanesi J.P. Hilgemann D.W. Anal. Biochem. 2002; 301: 243-254Crossref PubMed Scopus (114) Google Scholar), confirmed that there was a 2-fold increase in PIP2 (Fig. 1B and Table 1). Unexpectedly, there was almost no change in PI4P mass, despite a modest increase in its labeling by 32P (Fig. 1A). No new peak corresponding to either PI3P, PI(3,4)P2, or PI(3,4,5)P3 was detected (Fig. 1B). The response profile of the HeLA cell establishes that PIP2 is increased selectively. This increase is most likely due to the direct activation of PIP5KIs and/or inactivation of PIP2 phosphatsases and is unlikely to be due to increased availability of the PI4P substrate. The sites of PIP2 increase were identified by immunofluorescence microscopy. As shown previously (34Wang Y.J. Li W.H. Wang J. Xu K. Dong P. Luo X. Yin H.L. J. Cell Biol. 2004; 167: 1005-1010Crossref PubMed Scopus (80) Google Scholar), the PIP2 antibody stained small punctae that lined the plasma membrane (PM pool) and a perinuclear region (internal organelle pool) (Fig. 1C). Hypertonic treatment increased anti-PIP2 staining intensity, especially in large punctae that line the plasma membrane. Some of these punctae were at the tips of retraction fibers that were formed when the cell shrunk (see Fig. 2B). PIP5KIβ Depletion Blunted the Hypertonicity-induced PIP2 and Actin Responses—siRNA oligonucleotides were used to identify the PIP5KI isoform that is primarily responsible for the hypertonic stress-induced PIP2 increase. As reported previously, PIP5KIβ depletion by RNAi (33Padron D. Wang Y.J. Yamamoto M. Yin H. Roth M.G. J. Cell Biol. 2003; 162: 693-701Crossref PubMed Scopus (125) Google Scholar, 34Wang Y.J. Li W.H. Wang J. Xu K. Dong P. Luo X. Yin H.L. J. Cell Biol. 2004; 167: 1005-1010Crossref PubMed Scopus (80) Google Scholar) decreased the basal [32P]PIP2 level to the greatest extent compared with depletion of the other PIP5KI isoforms (Fig. 2A). PIP5KIβ completely blocked the hyprtonicity-induced PIP2 increase, while depletion of the other PIP5KIs had much less effect. These results showed that PIP5KIβ accounts for most of the hypertonicity-induced PIP2 increase. We therefore focused on its behavior for the remainder of this paper. HeLa cells normally have long actin stress fibers and cortical actin filaments. After hypertonic stimulation, the stress fibers became thicker, and retraction fibers were formed at the cell periphery as the cell shrunk (Fig. 2B). An increase in stress fibers and polymerized actin was confirmed biochemically by isolating the Triton X-100-insoluble cytoskeleton (Fig. 2C). The amount of actin in the Triton-insoluble low speed pellet (23Yamamoto M. Hilgemann D.H. Feng S. Bito H. Ishihara H. Shibasaki Y. Yin H.L. J. Cell Biol. 2001; 152: 867-876Crossref PubMed Scopus (105) Google Scholar), which contains cross-linked actin filaments such as stress fibers, and the Triton-insoluble high speed pellet, which contains long actin filaments that are not crosslinked sufficiently to be sedimented by centrifugation are increased, while actin in the high speed supernatant (representing actin monomers and small oligomers) decreased. Therefore, hypertonicity promotes actin polymerization and cross-linking into stress fibers and/or networks. PIP5KIβ depletion dramatically changed the cell shape and decreased the amount of stress fibers (Fig. 2B). Although hypertonic stress shrunk the PIP5KIβ RNAi-treated cells, it did not promote stress fiber formation. Since PIP5KIβ depletion blocked the actin polymerization/reorganization response, we conclude that PIP5KIβ has a major role in orchestrating the hypertonic stress fiber response. PIP5KIβ Was Recruited to Membranes by Hypertonic Stress—Immunofluorescence was used to examine the effect of hypertonic stress on the subcellular distribution of PIP5KIβ. PIP5KIβ was cytosolic and also associated with plasma membrane and endomembranes (Fig. 2D). Hypertonic stress increased the amount of PIP5KIβ at the cell periphery and the retraction fibers also contained PIP5KIβ. The increase in PIP5KIβ membrane association was confirmed by subcellular fractionation using two different methods. Centrifugation at 100,000 × g showed that under isotonic conditions, ∼45% of the kinase was recovered in the pellet (membranes) (Fig. 3A). HA-PIP5KIβ migrated as a doublet in both the supernatant (cytosol) and pellet, which, as will be shown later, was due to a difference in the extent of phosphorylation. Hypertonic stimulation collapsed the doublet into a single band and increased the recovery of PIP5KIβ (70% of total) in the pellet fraction. Multistep fractionation (36Wei Y.J. Sun H.Q. Yamamoto M. Wlodarski P. Kunii K. Martinez M. Barylko B. Albanesi J.P. Yin H.L. J. Biol. Chem. 2002; 277: 46586-46593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) provided additional information about the partitioning of PIP5KIβ among different organelle fractions (Fig. 3B). Under isotonic conditions, slightly less than half of the total PIP5KIβ was membrane associated, and of this, half was recovered in the PM enriched fraction (Fig. 3B). Sucrose stimulation decreased the percentage of PIP5KIβ in the cytosolic fraction by 60% and almost doubled the percentage recovered in the PM and high speed pellet fraction. Therefore, the immunofluorescence and biochemical data both show that there is an increase PIP5KIβ membrane association. Hypertonic Stress Dephosphorylated PIP5KIβ but Not the Other PIP5KIs—Park et al. (29Park S.J. Itoh T. Takenawa T. J. Biol. Chem. 2001; 276: 4781-4787Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) showed that PIP5KI isoforms are constitutively phosphorylated and that they can be activated by Ser/Thr dephosphorylation. Hypertonic stress collapsed the HA-PIP5KIβ doublet into a single band in SDS-polyacrylamide gels, which would be consistent with dephosphorylation (Fig. 3A). Dephosphorylation was confirmed by a decrease in 32P labeling of the upper band in the doublet. Under isotonic conditions, both HA-PIP5KIβ bands were 32P-labeled, and the upper band was more highly phosphorylated (Fig. 4A). The 32P intensity of the upper band decreased dramatically within 3 min of sucrose treatment, while that of the lower band was not decreased to a similar extent. Our results suggest that PIP5KIβ is constitutively phosphorylated at multiple sites and that a subset of these sites is preferentially dephosphorylated as an early response to hypertonic stress. The time course of dephosphorylation of the upper band paralleled the rise in PIP2 level (Fig. 1A), lending further support to the possibility that PIP5KIβ dephosphorylation increases PIP2 levels in cells. Unlike PIP5KIβ, neither PIP5KIα nor PIP5KIγ (both L and S variants) was dephosphorylated by hypertonic stimulation (Fig. 4B). PIP5KIβ is phosphorylated by protein kinase A on its Ser-241 residue (29Park S.J. Itoh T. Takenawa T. J. Biol. Chem. 2001; 276: 4781-4787Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). However, we found that the HA-PIP5KIβS241A mutant was dephosphorylated normally during hypertonic stimulation (data not shown). Therefore, Ser-241 is not a major site of hypertonicity-induced dephosphorylation. Effects of PIP5KIβ Dephosphorylation—The effect of hypertonic stress on the lipid kinase activity of PIP5KIβ was examined using an in vitro kinase assay. HA-PIP5KIβ that was immunoprecipitated from hypertonically stressed cells had three times higher lipid kinase activity than those from the unstimulated control, while the activities of PIP5KIα,-γL, and -γS were not changed significantly (Fig. 4C). Therefore, hypertonic stress selectively activates PIP5KIβ. We next examined the effect of inhibiting Ser/Thr protein phosphatases on the hypertonic response. We tested the following Ser/Thr phosphatase inhibitors: caly A inhibits PP1 and PP2A; okadaic acid inhibits PP2A at 1-10 nm concentrations (IC50 0.51 nm) and PP1 at higher concentrations (IC50 42 nm). Cyclosporine A inhibits PP1B but not PP1A. Caly A increased the intensity of the 32P label in the upper band of the PIP5KIβ doublet under isotonic conditions and blocked the sucrose-induced dephosphorylation (Fig. 5A). Okadaic acid had no effect at 10 nm (data not shown) but did increase HA-PIP5KIβ basal phosphorylation and blocked dephosphorylation at 100 nm. The differential effects of caly A and okadaic acid on PIP5KIβ dephosphorylation suggest that the PP1 phosphatases promote PIP5KIβ dephosphorylation during hypertonic stress. Cyclosporine A did not block dephosphorylation (data not shown), ruling out a PP2B involvement. Caly A was used to evaluate the relationship between the hyperonicity-induced PIP5KIβ dephosphorylation and lipid kinase activation. We found that caly A decreased basal PIP5KIβ activity by 63% and blocked PIP5KIβ activation by sucrose (Fig. 5B). We also used caly A to determine whether PIP5KIβ dephosphorylation is a primary trigger for the hypertonic PIP2 response. Caly A dampened the PIP2 response (Fig. 5C), and this effect was specific for PIP2, because PI4P increased normally. It is curious though that caly A had minimal effect on the PIP2 level of the cell under isotonic condition (Fig. 5C), even though it inhibited PIP5KIβ in vitro (Fig. 5B). It is possible that PIP2 did not decrease in the calyculin A-treated cells because of compensatory changes that restore the ambient isotonic PIP2 level. However, these compensations are not able to raise PIP2 to a sufficiently high level to compensate for the lack of PIP5KIβ activation during hypertonic stress. Taken together, the series of experiments establish that there is a cause and effect relationship between hypertonicity-induced PIP5KIβ dephosphorylation, lipid kinase activation, and PIP2 increase in cells. Effects of PIP5KIβ Dephosphorylation on Its Steady State Membrane Association—Since hypertonicity induces PIP5KIβ dephosphorylation and also promotes its recruitment to membranes (Figs. 2, 3 and 4), we examined the possibility that the more dephosphorylated PIP5KIβ is preferentially membrane associated. However, the ratio of the two bands in the PIP5KIβ doublet in the 100,000 × g supernatant and pellet fractions (Fig. 3A) were similar. Therefore, the more phosphorylated and less phosphorylated PIP5KIβ associate with membranes to a similar extent under the steady state isotonic conditions used here. We conclude that the increase in membrane association during hypertonic stress cannot be sim

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