Signaling by Higher Inositol Polyphosphates
2004; Elsevier BV; Volume: 279; Issue: 42 Linguagem: Inglês
10.1074/jbc.c400286200
ISSN1083-351X
AutoresXavier Pesesse, Kuicheon Choi, Tong Zhang, Stephen B. Shears,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoEvidence has accumulated that inositol pyrophosphates (diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4)) are intracellular signals that regulate many cellular processes including endocytosis, vesicle trafficking, apoptosis, and DNA repair. Yet, in contrast to the situation with all other second messengers, no one studying multicellular organisms has previously described a stimulus that acutely and specifically elevates cellular levels of PP-InsP5 or [PP]2-InsP4. We now show up to 25-fold elevations in [PP]2-InsP4 levels in animal cells. Importantly, this does not involve classical agonists. Instead, we show that this [PP]2-InsP4 response is a novel consequence of the activation of ERK1/2 and p38MAPα/β kinases by hyperosmotic stress. JNK did not participate in regulating [PP]2-InsP4 levels. Identification of [PP]2-InsP4 as a sensor of hyperosmotic stress opens up a new area of research for studies into the cellular activities of higher inositol phosphates. Evidence has accumulated that inositol pyrophosphates (diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4)) are intracellular signals that regulate many cellular processes including endocytosis, vesicle trafficking, apoptosis, and DNA repair. Yet, in contrast to the situation with all other second messengers, no one studying multicellular organisms has previously described a stimulus that acutely and specifically elevates cellular levels of PP-InsP5 or [PP]2-InsP4. We now show up to 25-fold elevations in [PP]2-InsP4 levels in animal cells. Importantly, this does not involve classical agonists. Instead, we show that this [PP]2-InsP4 response is a novel consequence of the activation of ERK1/2 and p38MAPα/β kinases by hyperosmotic stress. JNK did not participate in regulating [PP]2-InsP4 levels. Identification of [PP]2-InsP4 as a sensor of hyperosmotic stress opens up a new area of research for studies into the cellular activities of higher inositol phosphates. Pivotal to the signaling functions of PP-InsP5 1The abbreviations used are: PP-InsP5, diphosphoinositol pentakisphosphate; DN, dominant negative; InsP5, inositol pentakisphosphate; InsP6, inositol hexakisphosphate; PP-InsP4, diphosphoinositol tetrakisphosphate; [PP]2-InsP4, bisdiphosphoinositol tetrakisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PIPKH, polyphosphoinositide kinase homolog; DIPP, diphosphoinositol polyphosphate phosphohydrolase; PH, pleckstrin homology; HEK, human embryonic kidney; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; JNK, c-Jun NH2-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; EGF, epidermal growth factor.1The abbreviations used are: PP-InsP5, diphosphoinositol pentakisphosphate; DN, dominant negative; InsP5, inositol pentakisphosphate; InsP6, inositol hexakisphosphate; PP-InsP4, diphosphoinositol tetrakisphosphate; [PP]2-InsP4, bisdiphosphoinositol tetrakisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PIPKH, polyphosphoinositide kinase homolog; DIPP, diphosphoinositol polyphosphate phosphohydrolase; PH, pleckstrin homology; HEK, human embryonic kidney; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; JNK, c-Jun NH2-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; EGF, epidermal growth factor. and [PP]2-InsP4 are their highly negative electrostatic potential and the considerable free energy of their hydrolysis (for review, see Ref. 1Shears S.B. Biochem. J. 2004; 377: 265-280Crossref PubMed Scopus (159) Google Scholar). The former attribute facilitates their functionally significant binding to PH (2Luo H.R. Huang Y.E. Chen J.C. Sairardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and other protein domains (3Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 4Couchman J.R. Vogt S. Lim S.-T. Lim Y. Oh E.-S. Prestwich G.D. Theibert A. Lee W. Woods A. J. Biol. Chem. 2002; 277: 49296-49303Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Additionally, the high energy phosphate groups may phosphorylate proteins (5Luo H.R. Saiardi A. Yu H. Nagata E. Ye K. Snyder S.H. Biochemistry. 2002; 41: 2509-2515Crossref PubMed Scopus (67) Google Scholar). These effects are believed to underlie the roles of inositol pyrophosphates in regulating clathrin assembly by adaptor proteins (3Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), apoptosis (6Morrison B.H. Bauer J.A. Kalvakolanu D.V. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), vesicle trafficking (7Saiardi A. Sciambi C. McCaffery J.M. Wendland B. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14206-14211Crossref PubMed Scopus (148) Google Scholar), chemotaxis (2Luo H.R. Huang Y.E. Chen J.C. Sairardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), and DNA repair (5Luo H.R. Saiardi A. Yu H. Nagata E. Ye K. Snyder S.H. Biochemistry. 2002; 41: 2509-2515Crossref PubMed Scopus (67) Google Scholar). It has also been pointed out (e.g. Ref. 2Luo H.R. Huang Y.E. Chen J.C. Sairardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) that many of the important cellular functions currently attributed to InsP6 may in vivo be more effectively performed by PP-InsP5 and [PP]2-InsP4. The Dictyostelids have uniquely exploited the physico-chemical properties of the inositol diphosphates by synthesizing near-millimolar levels of these molecules (8Laussmann T. Pikzack C. Thiel U. Mayr G.W. Vogel G. Eur. J. Biochem. 2000; 267: 2447-2451Crossref PubMed Scopus (35) Google Scholar). However, animal cells have 300-fold lower levels of PP-InsP5 and [PP]2-InsP4 (1Shears S.B. Biochem. J. 2004; 377: 265-280Crossref PubMed Scopus (159) Google Scholar). It has therefore generally been anticipated that, in animal cells, there should be substantial, stimulus-dependent increases in cellular levels of PP-InsP5 and [PP]2-InsP4 to elevate levels of these polyphosphates to a functionally significant threshold. However, no such phenomenon has been described previously. Instead, only quite small stimulus-dependent changes in PP-InsP5 and [PP]2-InsP4 turnover have been reported, and in each case the result was a decrease rather than an increase in cellular levels of the pyrophosphates. For example, depletion of endoplasmic reticulum Ca2+ stores by thapsigargin transiently decreases PP-InsP5 levels by 50% (9Glennon M.C. Shears S.B. Biochem. J. 1993; 293: 583-590Crossref PubMed Scopus (97) Google Scholar). Activation of β-adrenergic receptors decrease levels of [PP]2-InsP4 by up to 40% (10Safrany S.T. Shears S.B. EMBO J. 1998; 17: 1710-1716Crossref PubMed Scopus (55) Google Scholar). This absence of a substantial, regulatory context for PP-InsP5 and [PP]2-InsP4 has been addressed in the current study, in which we selected a biological paradigm of fundamental and widespread significance, namely, defense against osmotic stress. The maintenance of osmotic balance across plasma membranes is an ongoing cellular burden in the face of controlled changes in both cell size and intracellular hydration as well as exchange of metabolites and ions across plasma membranes (11Häussinger D. Biochem. J. 1996; 313: 697-710Crossref PubMed Scopus (495) Google Scholar). There are also clinically relevant osmotic challenges that can impose strain upon the cytoskeleton, perturb chromatin structure, damage DNA, and inhibit DNA repair (12Chiasson J.L. Aris-Jilwan N. Belanger R. Bertrand S. Beauregard H. Ekoe J.M. Fournier H. Havrankova J. Can. Med. Assoc. J. 2003; 168: 859-866PubMed Google Scholar, 13Kültz D. Chakravarty D. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 130: 421-428Crossref PubMed Scopus (40) Google Scholar). One way to examine how cells sense and adapt to hyperosmotic stress is to add sorbitol to the medium (11Häussinger D. Biochem. J. 1996; 313: 697-710Crossref PubMed Scopus (495) Google Scholar). In this report, we show that the addition of 0.2 m sorbitol can elevate [PP]2-InsP4 levels up to 25-fold, and we also study the molecular mechanisms that are involved. Cellular levels of inositol phosphates were determined as previously described (10Safrany S.T. Shears S.B. EMBO J. 1998; 17: 1710-1716Crossref PubMed Scopus (55) Google Scholar). InsP6 and PP-InsP5 kinase activities were assayed as described (15Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Several colleagues kindly supplied the following cDNA constructs: DN-p38α and DN-p38β in pCDNA3 (Dr. J. Han, Scripps Research Institute, La Jolla, CA), PIPKH in pCMV2 (Dr. J. Chang, Beth Israel Deaconess Medical Center, Boston, MA), DN-ERK1 and DN-ERK2 in pCMV5 (Dr. C. Der, University of North Carolina). Cells are constantly adapting to the osmotic challenges brought on by normal cellular activities, such as the breakdown of macromolecules, controlled changes in both cell size and intracellular hydration, as well as exchange of metabolites and ions across plasma membranes (11Häussinger D. Biochem. J. 1996; 313: 697-710Crossref PubMed Scopus (495) Google Scholar). We investigated whether inositol diphosphates might be regulated by hyperosmotic stress, which we introduced by the addition of 0.2 m sorbitol. It is notable that many earlier studies into hyperosomotic stress have frequently employed much higher doses of sorbitol (0.5–0.7 m). Cells adapt successfully to 0.2 m sorbitol, since it failed even after 24 h to affect viability of either HEK cells (101 ± 2%) or the DDT1 MF2 vas deferens smooth muscle line (96 ± 1%) compared with non-sorbitol controls. A novel, immediate and selective response of DDT1 MF2 cells and HEK cells to 0.2 m sorbitol is a dramatic elevation of [PP]2-InsP4 levels, in the range of 10–25-fold (Fig. 1). It is worth emphasizing the novelty of this observation; there is no previous report of any extracellular stimulus that can specifically and acutely increase levels of an inositol diphosphate. Both PP-InsP5 and [PP]2-InsP4 are hydrolyzed by DIPPs (14Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (138) Google Scholar, 16Hua L.V. Hidaka K. Pesesse X. Barnes L.D. Shears S.B. Biochem. J. 2003; 373: 81-89Crossref PubMed Google Scholar). Any DIPP inhibition following osmotic stress should elevate both of the diphosphoinositol polyphosphates. On the contrary, a 26 ± 4% (n = 8) drop in PP-InsP5 accompanies the sorbitol-dependent increase in [PP]2-InsP4 levels (Fig. 1A). These effects are consistent with hyperosmotic stress elevating [PP]2-InsP4 levels by activating the PP-InsP5 kinase. However, it is possible that sorbitol stress inhibits an [PP]2-InsP4-specific phosphatase or [PP]2-InsP4-specific phosphorylation of proteins (5Luo H.R. Saiardi A. Yu H. Nagata E. Ye K. Snyder S.H. Biochemistry. 2002; 41: 2509-2515Crossref PubMed Scopus (67) Google Scholar). Time course data indicated that [PP]2-InsP4 levels were elevated within 5–10 min of the initiation of hyperosmotic stress (Fig. 1B). As little as 0.05 m sorbitol promoted a severalfold [PP]2-InsP4 response (Fig. 1C). Within minutes of terminating the hyperosmotic challenge, [PP]2-InsP4 levels declined exponentially, although they did not return to pre-activated levels within the 2-h experimental time frame (Fig. 1D); instead, the cell's memory of their stress history persisted. To assess the mechanisms underlying the sorbitol-dependent increase in [PP]2-InsP4 levels, we considered how this enzyme might interface with other signaling entities. Hyperosomotic stress is known to activate Ser/Thr phosphoprotein phosphatases (17Meier R. Thelen M. Hemmings B.A. EMBO J. 1998; 17: 7294-7303Crossref PubMed Scopus (147) Google Scholar) and Tyr phosphoprotein phosphatases (18Hresko R.C. Mueckler M. J. Biol. Chem. 2000; 275: 18114-18120Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), but inhibitors of either pathway (20 nm okadaic acid or 10 μm phenylarsine oxide, respectively) did not modify the [PP]2-InsP4 response to sorbitol (data not shown). The JNK MAP kinase pathway can be activated by 0.5 m sorbitol (19Bagowski C.P. Besser J. Frey C.R. Ferrell Jr., J.E. Curr. Biol. 2003; 13: 315-320Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In agreement with this earlier study, we also found that 0.5 m sorbitol activated JNK (Fig. 2A). However, JNK was not activated by 0.2 m sorbitol (Fig. 2A), yet this dose of sorbitol was sufficient to maximally elevate [PP]2-InsP4 levels (Fig. 1C). Thus, we conclude that JNK activation is not necessary for [PP]2-InsP4 levels to be increased by hyperosmotic stress. The ERK and p38 MAP kinase pathways are activated by osmotic stress (Fig. 2, B and C, and Refs. 20Cowan K.J. Storey K.B. J. Exp. Biol. 2003; 206: 1107-1115Crossref PubMed Scopus (471) Google Scholar and 21Galcheva-Gargova Z. Derijard B. Wu I.H. Davis R.J. Science. 1994; 265: 806-808Crossref PubMed Scopus (531) Google Scholar). Pretreatment of cells with the MEK inhibitors U0126 (10 μm) or PD098059 (50 μm) inhibited ERK1/2 phosphorylation (Fig. 2, B and C, and Ref. 22English J.M. Cobb M.H. Trends Pharmacol. Sci. 2002; 23: 40-45Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). This was accompanied by a 45–50% reduction of the sorbitol-dependent increase in [PP]2-InsP4 levels (Fig. 3A). The similarity of the results obtained with two structurally unrelated MEK inhibitors makes it unlikely that reversal of the sorbitol effect results from unsuspected nonspecific actions of U0126 or PD098059. Pretreatment of cells with SB203580 inhibited phosphorylation of p38α/β (Fig. 2C and Ref. 23Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (356) Google Scholar) and also elicited a 23% attenuation of the sorbitol-dependent increase in [PP]2-InsP4 levels (Fig. 3A). The relatively mild effect of SB203580 when it was added alone may be in part explained by its ability to non-specifically activate ERK1/2 (Fig. 3A); a similar effect has been noted previously (24Liu Q. Hofmann P.A. Am. J. Physiol. 2004; 286: H2204-H2212Crossref PubMed Scopus (142) Google Scholar). The co-application of a single MEK inhibitor (50 μm PD098059) together with the p38 inhibitor (10 μm SB203580) yielded an approximately additive result (80% reversal of the effect of sorbitol upon [PP]2-InsP4 levels, see Fig. 3A). We eliminated the possibility that the MAP kinase inhibitors might non-specifically block synthesis of the diphosphoinositol polyphosphates: native preparations of PP-InsP5 kinase showed 103 ± 2% activity when incubated with 10 μm SB203580, 105 ± 3% with 10 μm U0126, and 103 ± 2% with 50 μm PD098059 (n = 3), all relative to vehicle controls. The recombinant InsP6 kinase showed 97 ± 6% activity when incubated with 10 μm SB203580, 105 ± 2% with 10 μm U0126 and 96 ± 5% with 50 μm PD098059 (n = 3–4), all relative to vehicle controls. Although MAP kinase inhibitors did not affect PP-InsP5 synthesis in vitro, the level of PP-InsP5 in cells treated with either PD098059 or UO1276 was 25% lower than control cells (Fig. 3B). These data suggest that the ERK activity that is ongoing even in resting cells (Fig. 2B) may weakly enhance InsP6 kinase activity. Consistent with this explanation, the sorbitol-dependent decrease in PP-InsP5 levels was not reversed by the MEK inhibitors (Fig. 3B). The absence of any significant effect of the MAP kinase inhibitors upon PP-InsP5 levels in sorbitol-treated cells (Fig. 3B) is of additional interest; this observation indicates that the attenuation of sorbitol-mediated increases in [PP]2-InsP4 levels by the MAP kinase inhibitors is not a secondary consequence of a reduction in substrate supply for the PP-InsP5 kinase. To substantiate the inhibitor experiments, we next transiently transfected cells with dominant negative (DN) MAP kinase cDNA constructs. It is first important to note that environmental stresses provide "lateral" as well as "top-down" input into MAP kinase responses (25Harrison J.C. Zyla T.R. Bardes E.S. Lew D.J. J. Biol. Chem. 2004; 279: 2616-2622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The DN-ERK constructs block the MAP kinase pathway at a point that is downstream of the site of action of the MEK inhibitors, so these two empirical approaches could yield quantitatively different degrees of attenuation of the sorbitol response if there were any lateral input between MEK and ERK. When co-expressed, DN-ERK1 (K71R) and DN-ERK2 (K52R) (26Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar) reduced the effect of sorbitol upon [PP]2-InsP4 levels by 30% (Fig. 4). Neither DN construct had any significant effect when added alone (data not shown). Also when co-expressed, the TGY-to-AGF DN-p38α and DN-p38β proteins (27Wang X. McGowan C.H. Zhao M. He L. Downey J.S. Fearns C. Wang Y. Huang S. Han J. Mol. Cell. Biol. 2000; 20: 4543-4552Crossref PubMed Scopus (237) Google Scholar, 28Huang S. Jiang Y. Li Z. Nishida E. Mathias P. Lin S. Ulevitch R.J. Nemerow G.R. Han J. Immunity. 1997; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) reduced the effect of sorbitol upon [PP]2-InsP4 levels by 30% (Fig. 4). Again, neither DN construct had any significant effect when added alone (data not shown). Transient transfection of DDT1 MF2 cells with all four DN constructs reduced by 50% the effect of hyperosmotic stress upon [PP]2-InsP4 levels (Fig. 4). This is approximately the maximum effect that could be anticipated, given the limited transfection efficiency (50%; determined by GFP co-transfection). We found that the activity of the native PP-InsP5 kinase was unaffected by recombinant, phosphorylated ERK1/2 (data not shown). It therefore seems likely that another signaling protein that lies downstream of ERK mediates its activation of cellular [PP]2-InsP4 accumulation. Nevertheless, [PP]2-InsP4 signaling offers a new repertoire for MAP kinase actions upon cellular physiology. Its unusual nature is also of interest; MAP kinases do not typically recruit diffusible second messengers. There is a growing appreciation that the inositol diphosphates can bind in a functionally significant manner to certain proteins through largely delocalized (i.e. nonspecific) electrostatic interactions, most notably to the majority of PH domains (2Luo H.R. Huang Y.E. Chen J.C. Sairardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 29Lemmon M.A. Ferguson K.M. Abrams C.S. FEBS Lett. 2002; 513: 71-76Crossref PubMed Scopus (214) Google Scholar), but other protein domains are certainly involved (3Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 4Couchman J.R. Vogt S. Lim S.-T. Lim Y. Oh E.-S. Prestwich G.D. Theibert A. Lee W. Woods A. J. Biol. Chem. 2002; 277: 49296-49303Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In these circumstances, the addition of even just one pyrophosphate group to InsP6 can increase ligand affinity by at least 30–100-fold (2Luo H.R. Huang Y.E. Chen J.C. Sairardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Elevations in [PP]2-InsP4 levels following hyperosmotic stress would therefore be expected to increasingly compete with PtdIns(4,5)P2, another important ligand for PH domains that helps recruit proteins into multimeric signaling complexes (signalosomes) (29Lemmon M.A. Ferguson K.M. Abrams C.S. FEBS Lett. 2002; 513: 71-76Crossref PubMed Scopus (214) Google Scholar). Indeed, dynamic competition between soluble inositol pyrophosphates and membrane-bound inositol lipids has been shown by others to act as a logic gate to determine whether an inositide-binding protein is recruited to signalosomes (2Luo H.R. Huang Y.E. Chen J.C. Sairardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 4Couchman J.R. Vogt S. Lim S.-T. Lim Y. Oh E.-S. Prestwich G.D. Theibert A. Lee W. Woods A. J. Biol. Chem. 2002; 277: 49296-49303Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). If there were autonomous spatio-temporal regulation of the levels of the competing lipid and pyrophosphate ligands, this would facilitate stimulus-specific control over the membership of these multimeric complexes. We have evidence of this metabolic autonomy; [PP]2-InsP4 levels in either control or sorbitol-treated cells were unaffected by inhibition of PtdIns(4,5)P2 hydrolysis by 10 μm U73122, by inhibition of PI 3-kinases with100 nm wortmannin, or by specific activation of PtdIns(3,4,5)P3 synthesis through transient overexpression of PIPKH (data not shown). Thus, [PP]2-InsP4 turnover is completely independent of inositol lipid turnover. Of importance to the subject of stimulus specificity, MAP kinase activation by the inflammatory cytokine tumor necrosis factor α (10 ng/ml for 30 min) had no effect upon [PP]2-InsP4 (data not shown). Furthermore, even though EGF is well known to activate ERK and p38 (Fig. 5 and Ref. 20Cowan K.J. Storey K.B. J. Exp. Biol. 2003; 206: 1107-1115Crossref PubMed Scopus (471) Google Scholar), it barely elevated [PP]2-InsP4 levels (0.7-fold; Fig. 5) and had no effect upon PP-InsP5 levels (Fig. 5). These data indicate that activation of both ERK and p38 are not, by themselves, sufficient to elevate [PP]2-InsP4 levels. Perhaps [PP]2-InsP4 turnover is regulated downstream of yet another signaling pathway that is selectively activated by hyperosmotic stress and not by EGF. Alternately, hyperosmotic stress (but not EGF) may attenuate an inhibitory process that normally constrains [PP]2-InsP4 accumulation in cells. These questions touch on an enigma that pervades MAP kinase research: how does a cell elicit a specific output from the MAP kinase pathway when it is utilized by so many extracellular and intracellular stimulii? Much additional work may be required to solve this problem, but in any case, our data are still significant in their demonstration that it is stress-dependent and not agonist-initiated increases in [PP]2-InsP4 levels that provides a regulatory context for the actions of inositol pyrophosphates. It is intriguing that hyperosmotic shock can damage DNA and affect vesicle trafficking processes (13Kültz D. Chakravarty D. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 130: 421-428Crossref PubMed Scopus (40) Google Scholar), since these are both processes that can be regulated by inositol pyrophosphates (see Introduction). In any case, our demonstration of a stimulus that can specifically elevate one of the higher inositol polyphosphates, in a specific and acute manner, is an unprecedented observation that opens up a new area of research for inositide signaling. Download .pdf (.01 MB) Help with pdf files
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