1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla; and 2MitoKor, San Diego, California
Submitted 17 February 2004 ; accepted in final form 23 September 2004
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ABSTRACT |
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protein tyrosine kinases; Ras; mitogen-activated protein kinase; hyperosmotic shock
One well-characterized signaling pathway is the Ras-MAP kinase cascade (42). Activated receptors bind directly or indirectly to the adaptor protein Grb2. Grb2 is associated with the guanine-nucleotide exchange factor Sos, which is an activator of Ras (44, 45). Activated, GTP-bound Ras recruits Raf to the inner leaflet of the plasma membrane, where Raf is activated. After its activation, Raf activates MEK, and MEK turns on ERK1 and ERK2 (42).
Raf activation is a highly complex process that is not yet completely understood. It involves several protein-protein interactions and protein phosphorylation events (12, 58). Several protein kinases have been implicated in the phosphorylation of Raf, including the p21 (Rac/Cdc42)-activated kinase (PAK), protein kinase C (PKC), and Akt. The phosphorylation of Ser338 by both PAK and PKC correlates with Raf activation.
A second important signaling pathway activated downstream of receptor protein tyrosine kinases is the phosphatidylinositol (PI) 3-kinase-Akt pathway. There is evidence that activation of PI 3-kinase is at least partly dependent on the presence of active Ras (37, 54). PI 3-kinase activation results in the production of PI 3,4,5-trisphosphate, which leads to the recruitment of Akt to the inner leaflet of the plasma membrane, where it is activated by PDK-1 and PDK-2 (1, 2). Akt promotes cell survival by phosphorylating and inactivating a number of proteins that are involved in apoptosis (17).
The cells of the mammalian kidney are frequently presented with hyperosmotic conditions due to the accumulation of both NaCl and urea (5, 6). It is known that several signaling proteins that are commonly activated downstream of growth factor receptors are also turned on by hyperosmotic stress, including phospholipase C (PLC
), PI 3-kinase, and Shc (19, 50, 73). In addition, it was found that hyperosmotic stress turns on the Ras-MAP kinase pathway, resulting in the activation of ERK1 and ERK2 (18, 34, 63, 66). In addition to ERK1 and ERK2, stress-activated MAP kinases, including p38MAPK and c-Jun NH2-terminal kinase (JNK), are known to be activated in response to hyperosmotic stress (26, 31). It remains unclear which molecules in mammalian cells respond directly to hyperosmotic stress.
In cultured cells, hyperosmotic stress can block progression through the cell cycle (40, 47). Blocking the cell cycle is thought to provide the cells with time to adjust to the increasing osmolarity of their environment. Alternatively, hyperosmotic stress can stimulate the cells to initiate apoptosis (47, 72). Apoptosis is a cellular program that results in cell death (28). This program can be initiated by activation of death receptors such as members of the TNF receptor family (28). Alternatively, cellular stress can cause the release of cytochrome c from the mitochondria (29). Cytochrome c, together with the apoptotic protease-activating factor (Apaf) activates caspase-9, which can activate effector caspases (29). Changes in mitochondrial membrane potential are often found to be associated with apoptosis (53). Intermediate levels of osmotic stress appear to induce cell cycle arrest, whereas higher levels induce apoptosis. Exactly how the cell chooses between these two alternatives remains unresolved.
How mammalian cells sense changes in osmolarity in their environment remains poorly understood. Some studies suggest that growth factor receptors themselves are directly involved in sensing osmotic stress (15, 50, 55). Because the signaling in response to hyperosmotic shock resembles that downstream of activated growth factor receptors, we wanted to investigate whether growth factors and hyperosmotic shock can cooperate in the activation of cellular signaling pathways. Surprisingly, we found that activation of MAP kinase downstream of growth factor receptors is inhibited by hyperosmotic conditions and that this inhibition occurs in a transient manner. In addition, we found that hyperosmotic stress inhibits survival signaling downstream of activated growth factor receptors in a sustained manner and that it can lead to the activation of the cellular apoptotic machinery and mitochondrial fragmentation.
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EXPERIMENTAL PROCEDURES |
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Immunoprecipitation.
Cells were rinsed twice with cold PBS and lysed in 1 ml of 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 500 µM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (PLC-lysis buffer) per 10-cm tissue culture dish. Lysates were cleared by centrifugation at 10,000 rpm in a microcentrifuge at 4°C, incubated with 5 µl of polyclonal antiserum or 1 µl of monoclonal antibody for 1 h on ice, and subsequently incubated for 1 h with 100 µl of 10% protein A-Sepharose or anti-mouse IgG-Sepharose for 1 h at 4°C on an agitator. Sepharose beads were collected by centrifugation and washed four times with PLC-lysis buffer. Immunoprecipitates were boiled for 3 min in 62.5 mM Tris-Cl (pH 6.8), 10% glycerol, 5% -mercaptoethanol, 5 mM DTT, 2.3% SDS, and 0.025% bromphenol blue (SDS-sample buffer) and resolved by SDS-PAGE.
Immunoblotting. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes using a Bio-Rad semidry blotting apparatus at 50 mA per gel for 60 min at room temperature. Immunoblotting was performed per the manufacturers protocol. Reactive proteins were visualized using ECL (Amersham Biosciences, Piscataway, NJ).
Ras activation assay.
GST-Ras-binding domain (RBD) was expressed in Escherichia coli and purified on glutathione-Sepharose beads. The beads were washed several times in Tris-buffered saline containing 1% Triton X-100 (TBST) and stored at 4°C in TBST containing 0.02% azide. For affinity precipitation, lysates were incubated with GST-RBD prebound to glutathione-agarose (10 µl beads containing
1020 µg of protein) for 1 h at 4°C on an agitator. Bound proteins were resolved by SDS-PAGE on a 12% acrylamide gel and subjected to Western blot analysis with an anti-Ras monoclonal antibody.
Caspase-3 assay. Vero cells were plated in 96-well plates 24 h before the experiment. Cells were treated with EGF (1.5 nM) in the presence or absence of 150 mM NaCl added to the medium. After 6 h of incubation, the media were aspirated and the cells were incubated with PBS containing 0.03% digitonin and 23.5 µM DEVD-amc (Calbiochem, La Jolla, CA). The appearance of fluorescent cleaved substrate was followed kinetically in an FMAX 96-well fluorescent plate reader (355-nm excitation/460-nm emission). Caspase-3 activity was normalized for cell numbers after staining with 0.01 mg/ml propidium iodide and detection with a 544-nm excitation/612-nm emission filter pair.
Fluorescence microscopy. Vero cells were plated onto polylysine-coated glass coverslips 24 h before treatment. NaCl (150 mM) was added to the growth media for the times indicated. To monitor recovery, the cells were incubated with NaCl for 1 h, washed twice with normal growth medium to remove salt, and incubated with normal growth medium for the times indicated. All incubations were performed in a tissue culture incubator. The cells were then fixed with 3% paraformaldehyde at room temperature for 15 min, washed with PBS, and permeabilized and blocked for 15 min in PBS containing 0.1% Tween 20 (PBST) with 0.3% Triton X-100 and 3% BSA. Coverslips were incubated for 1 h at room temperature with the anti-cytochrome c antibody (clone 6H2.B4; BD Biosciences Pharmingen, San Diego, CA) diluted 1:400 in PBST containing 3% BSA and 2% normal goat serum in a humidified chamber. Coverslips were washed three times for 5 min with PBST at room temperature and incubated for 30 min at room temperature with goat anti-mouse-Alexa 488 (Molecular Probes, Eugene, OR) diluted 1:500 in PBST containing 3% BSA and 2% normal goat serum. Coverslips were washed as before, and nuclei were visualized by staining with Hoechst 33258 during the last wash (Molecular Probes). Coverslips were mounted onto glass slides with Airvol 205 (Air Products and Chemical, Allentown, PA) containing Dabco (Sigma Aldrich) and stored in the dark at 4°C. Immunostained cells were visualized with a Nikon Eclipse E800 microscope and a Hamamatsu camera using Simple PCI imaging software (Compix, Cranberry Township, PA).
To visualize mitochondria using fluorescently labeled adenine nucleotide translocator 3 (ANT3) or cyclophilin D (CypD), Vero cells were transiently transfected with pcDNA3-ANT3-yellow fluorescent protein (YFP) or enhanced cyan fluorescent protein (pECFP)-N1-CypD using FuGENE (Roche Applied Science, Indianapolis, IN) according to the manufacturers protocol. Forty-eight hours after transfection, the cells were treated, fixed, and immunostained for cytochrome c. The samples were further processed and analyzed as described above.
Visualization of mitochondrial membrane potential in intact cells. Vero cells were incubated in 120 mM NaCl, 3.1 mM KCl, 0.4 mM KH2PO4, 5 mM NaHCO3, 1.2 mM Na2SO4, 1.3 mM CaCl2, and 20 mM HEPES (medium) containing 100 nM tetramethylrhodamine methylester (TMRM) for 30 min before being mounted in a perfusion chamber and placed on a 37°C temperature-controlled stage. TMRM (100 nM) was maintained in the medium throughout the experiment. Imaging was performed using a 75-W xenon lamp-based monochromator (Photonics, Martinsried, Germany). The emission light was detected using a charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan), and data acquisition was controlled using Simple PCI imaging software. Cells were excited at 540 nm, and the emission was collected through a 610-nm/75-nm filter. Images were collected every 30 s for a period of 15 min. NaCl (30 mM) was added to the cells after 3 min, and the cells were returned to normal (120 mM) NaCl after 13 min.
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RESULTS |
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Inhibition of survival signaling by hyperosmotic shock is sustained and leads to apoptosis. Because the inhibition of signaling to ERK1, ERK2, and JNK by hyperosmotic stress is transient, we wanted to test whether the same held true for Akt activation. Vero cells were stimulated for 1 min with EGF after preincubation under hyperosmotic conditions for various amounts of time, and whole cell lysates were analyzed by immunoblotting with an antibody that recognizes phosphorylated Akt (Fig. 9A). ERK activation was analyzed in parallel (Fig. 9B). In contrast to what we observed with ERK1 and ERK2, Akt activation was inhibited regardless of the amount of time the cells were present in hyperosmotic medium. NaCl alone did not lead to any detectable Akt phosphorylation (results not shown). Our data show that EGF cannot induce Akt activation, no matter how prolonged the exposure to hypertonic medium. ERK1 and ERK2 were activated in response to EGF after pretreatment under hypertonic conditions for 18 h, although not to the same degree as when the cells were stimulated in isotonic medium. These results indicate that the ability of Vero cells to adapt to the hypertonicity of their environment does not pertain to the activation of survival signaling.
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Hyperosmotic conditions lead to mitochondrial fragmentation in Vero cells. Cellular stress is believed to result in the release of caspase activators, such as cytochrome c, from the mitochondria (29). Because Akt is thought to counteract this process, we tested whether hyperosmotic stress results in the release of cytochrome c from the mitochondria. Vero cells were seeded on coverslips, incubated in growth medium supplemented with 150 mM NaCl for various amounts of time, fixed, and stained with an antibody against cytochrome c. Control Vero cells showed a typical reticular cytochrome c immunostaining pattern (Fig. 10A). Surprisingly, during the incubation in medium supplemented with 150 mM NaCl, the mitochondrial network started to disintegrate. The effects were clearly visible at 5 and 10 min after the onset of hypertonic conditions, and mitochondrial fragmentation was complete within 1 h. To find out whether this response was reversible, we incubated Vero cells in medium supplemented with 150 mM NaCl, and the cells were then washed and allowed to recover for increasing amounts of time in isotonic medium. The results show that mitochondrial fragmentation in response to hyperosmotic stress was completely reversible (Fig. 10B). The network started to recover within 5 min after hypertonic medium was replaced with isotonic medium, and most cells had recovered within 1 h. These results indicate that hyperosmotic stress induces a reversible fragmentation of the mitochondrial network.
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DISCUSSION |
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A variety of signaling proteins have been implicated in the detection of hyperosmotic conditions. These include growth factor receptor protein tyrosine kinases, cytokine receptors, Shc, PI 3-kinase, PLC, PKC, Akt, Ras, Raf, MEK, and several MAP kinase family members (15, 50, 55, 62). On the basis of these observations, it has been speculated that hyperosmotic conditions lead directly to the activation of growth factor receptors and the activation of the pathways normally turned on by these receptors. However, this appears to conflict with the fact that growth factor receptors usually stimulate cell cycle progression and promote cell survival (36). In addition, growth factor receptor activation in response to hyperosmotic shock appears to peak between 15 and 30 min after the onset of hyperosmotic conditions (55). This suggests that growth factor receptor activation may be a secondary response and that these receptors do not directly detect hyperosmolarity in the environment.
Because many of the pathways activated by growth factor receptors are also turned on by hyperosmotic stress, we wanted to know whether growth factors and hyperosmotic shock cooperate in the activation of downstream signaling pathways. In an attempt to learn something about the early events induced by hyperosmotic shock, we stimulated cells with EGF for 1 min immediately after the onset of hyperosmotic conditions. In contrast to what we expected, we observed that hyperosmotic conditions blocked growth factor-induced activation of MAP kinase. This resulted from an apparent block in the ability to turn on Raf. This conclusion is supported by experiments showing normal activation of the EGF receptor and normal levels of GTP-bound Ras but strongly reduced levels of active Raf, active MEK, and active ERK1 and ERK2 in the presence of hyperosmotic concentrations of NaCl. The block in Raf activation occurred within 1 min after the onset of hyperosmotic conditions, suggesting that the cells respond very fast to these conditions. We also observed a block in EGF-induced JNK activation, whereas NaCl-induced p38MAPK activation was normal, peaking when 300 mM NaCl was added to the medium. However, within 10 min, cells recovered their ability to activate these signaling pathways. Our observations suggest strongly that hyperosmotic conditions rapidly and transiently turn on a signaling cascade that interferes with signaling downstream of growth factor receptors.
The activation of Raf is a complex process with several key steps that include the translocation of Raf to the plasma membrane, the interaction of Raf with accessory proteins such as Sur-8, KSR (kinase suppressor of Ras), and CNK (connector enhancer of KSR), and changes in Raf phosphorylation (48, 58). Although it was initially thought that that the interaction with active Ras was both necessary and sufficient for recruitment of Raf to the membrane, recent evidence suggests that CNK may play a role in this process (64). KSR is thought to act as a scaffold for Raf, MEK, and MAP kinase and may also link Raf to upstream activators (3, 38). It is possible that one or more of these proteins is targeted by the osmotic stress-activated pathway.
Several phosphorylation sites have been reported to contribute to Raf activation, including Ser338, Tyr341, and Thr491 and Ser494 in the activation loop (16, 22, 41, 43, 71). Using an anti-serum that is specific for Raf phosphorylated on Ser338, we have shown that the phosphorylation of this site was inhibited in hyperosmotic media. We also observed that Raf normally shifts in mobility during SDS-PAGE after stimulation with EGF. This shift in mobility was blocked when cells were stimulated under hyperosmotic conditions (results not shown). Both PKC and PAK have been implicated as Ser338 kinases, and both are turned on downstream of activated growth factor receptor protein tyrosine kinases (9, 11, 32). We are currently investigating whether activation of either PKC or PAK downstream of the EGF receptor is affected by hyperosmotic shock.
We observed that, in addition to the Ras-MAP kinase pathway, EGF-dependent activation of Akt was also blocked by hyperosmotic shock. Akt is activated downstream of PI 3-kinase, and full PI 3-kinase activation results in part from an interaction with active Ras (37, 54, 68). This observation is consistent with the idea that it is indeed the activation of signaling proteins immediately downstream of Ras that is affected by hyperosmotic shock. However, the inhibition of Akt is sustained, indicating that there are key differences in how hyperosmotic shock affects these signaling pathways. Interestingly, PI 3-kinase signaling pathways have been linked to both upregulating Raf activation through Cdc42, Rac, and Pak signaling (13, 60) and downregulating Raf via Akt (75). Activation of PI 3-kinase signaling itself is regulated by binding both Cdc42 and Rac (7, 10, 74). It will be of interest to analyze growth factor-induced signaling downstream of these other small G proteins for their sensitivity to hyperosmotic shock in an effort to better understand how Raf is affected by PI 3-kinase-regulated signaling pathways.
Activation of both the Ras-MAP kinase pathway and the PI 3-kinase-Akt pathway are thought to be important for cell survival (39, 52). Consistent with sustained inhibition of Akt, we observed that increasing the osmolarity of the medium by adding 200 mM NaCl resulted in cell death by apoptosis. This finding is consistent with previous observations (27, 59).
Our observations have implications for the use of osmotic agents to control cerebral edema and to alleviate intracranial pressure caused by the swelling that often accompanies traumatic brain injury, stroke, and brain tumors (24, 57, 67). Our studies show that, along with causing apoptotic cell death, several signaling pathways are affected under hyperosmotic conditions. If growth factor receptor signaling is important for proper healing, then the use of osmotic agents like mannitol and hypertonic saline may not be the best way to promote full recovery. Our observations support earlier work suggesting that hyperosmotic treatment after brain injury leads to further loss of neurons in the damaged area (25).
In general, there are two different pathways that lead to cell death by apoptosis. Activation of death receptors causes activation of caspase-8, and activated caspase-8 relays the signal to downstream effector caspases (4). Alternatively, cellular stress results in the release of apoptotic activators from the mitochondria. These include cytochrome c, which is involved in the activation of caspase-9, an activator of caspase-3 (29). Surprisingly, when cells that were incubated under hypertonic conditions were stained for cytochrome c, we did not observe cytochrome c release. This suggests that cytochrome c release may not act as the intracellular trigger for hypertonic stress-induced apoptosis.
Instead of cytochrome c release, we observed that the hyperosmotic stress induced the disintegration of the mitochondrial reticulum and mitochondrial dysfunction. This is consistent with previous reports describing disruption of the mitochondrial membrane potential in response to osmotic stress (21, 46). We have demonstrated that this process happens almost immediately and that it is exquisitely sensitive to changes in osmolarity, occurring in as little as 30 mM NaCl.
It is known that mitochondria form a highly dynamic network that is constantly subject to fusion and fission events (69, 70). This dynamic behavior is thought to be important for distribution of mitochondria to daughter cells during cell division, provision of ATP to all parts of the cell, and maintenance of the mitochondrial genome (69, 70). In addition, mitochondrial fusion is essential for spermatogenesis in Drosophila (30). Several dynamin-like and non-dynamin-like GTPases that are involved in mitochondrial fission and fusion, respectively, have been identified (69, 70). However, exactly how this process is regulated remains unresolved. We found that incubation of cells for 1 h in medium supplemented with 150 mM NaCl resulted in mitochondrial fragmentation. The mitochondrial morphology under hypertonic conditions was very similar to that observed in cells lacking mitofusin 1 or mitofusin 2 (14). This suggests that the disintegration of the mitochondrial network in response to hyperosmotic shock may be caused by inhibition of mitofusins. We observed that the cells were able to reestablish a normal-looking mitochondrial network within 1 h after being returned to isotonic medium. This shows that mitochondrial fusion is a relatively fast process. Finally, we observed that hypertonic conditions caused an immediate dissipation of the mitochondrial membrane potential. This suggests that the disintegration of the mitochondrial network could be secondary to the disruption of chemiosmotic energy production and that the mitochondrial membrane potential is exquisitely sensitive to the osmolarity of the extracellular milieu.
In conclusion, we have shown that hyperosmotic conditions, which can be encountered in a variety of situations, block signaling by growth factor receptor protein tyrosine kinases immediately downstream of Ras. This can now be used as a tool for further analysis of both Raf and Akt activation that occurs downstream of Ras. We also have shown that the mitochondrial membrane potential can be turned off quickly and reversibly by hyperosmotic conditions. Finally, our results show that mitochondrial fusion is a rapid process that can be manipulated by the addition of hyperosmotic concentrations of NaCl to the medium.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PR, Reese CB, and Cohen P. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B. Curr Biol 7: 261269, 1997.[ISI][Medline]
3. Anselmo AN, Bumeister R, Thomas JM, and White MA. Critical contribution of linker proteins to Raf kinase activation. J Biol Chem 277: 59405943, 2002.
4. Ashkenazi A and Dixit VM. Death receptors: signaling and modulation. Science 281: 13051308, 1998.
5. Bagnasco SM. How renal cells handle urea. Cell Physiol Biochem 10: 379384, 2000.[CrossRef][ISI][Medline]
6. Beck FX, Guder WG, and Schmolke M. Cellular osmoregulation in kidney medulla. Contrib Nephrol 123: 169184, 1998.[ISI][Medline]
7. Beeton CA, Das P, Waterfield MD, and Shepherd PR. The SH3 and BH domains of the p85 adapter subunit play a critical role in regulating class Ia phosphoinositide 3-kinase function. Mol Cell Biol Res Commun 1: 153157, 1999.[CrossRef][Medline]
8. Belzacq AS, Vieira HL, Kroemer G, and Brenner C. The adenine nucleotide translocator in apoptosis. Biochimie 84: 167176, 2002.[CrossRef][ISI][Medline]
9. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature 361: 315325, 1993.[CrossRef][ISI][Medline]
10. Bokoch GM, Vlahos CJ, Wang Y, Knaus UG, and Traynor-Kaplan AE. Rac GTPase interacts specifically with phosphatidylinositol 3-kinase. Biochem J 315: 775779, 1996.[ISI][Medline]
11. Bokoch GM, Wang Y, Bohl BP, Sells MA, Quilliam LA, and Knaus UG. Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J Biol Chem 271: 2574625749, 1996.
12. Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, and Der CJ. Increasing complexity of Ras signaling. Oncogene 17: 13951413, 1998.[CrossRef][ISI][Medline]
13. Chaudhary A, King WG, Mattaliano MD, Frost JA, Diaz B, Morrison DK, Cobb MH, Marshall MS, and Brugge JS. Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338. Curr Biol 10: 551554, 2000.[CrossRef][ISI][Medline]
14. Chen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, and Chan DC. Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J Cell Biol 160: 189200, 2003.
15. Cheng H, Kartenbeck J, Kabsch K, Mao X, Marques M, and Alonso A. Stress kinase p38 mediates EGFR transactivation by hyperosmolar concentrations of sorbitol. J Cell Physiol 192: 234243, 2002.[CrossRef][ISI][Medline]
16. Chong H, Lee J, and Guan KL. Positive and negative regulation of Raf kinase activity and function by phosphorylation. EMBO J 20: 37163727, 2001.
17. Coffer PJ, Jin J, and Woodgett JR. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 335: 113, 1998.[ISI][Medline]
18. Cohen DM. Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation. Proc Natl Acad Sci USA 93: 1124211247, 1996.
19. Cohen DM, Gullans SR, and Chin WW. Urea signaling in cultured murine inner medullary collecting duct (mIMCD3) cells involves protein kinase C, inositol 1,4,5-trisphosphate (IP3), and a putative receptor tyrosine kinase. J Clin Invest 97: 18841889, 1996.
20. De Rooij J and Bos JL. Minimal Ras-binding domain of Raf1 can be used as an activation specific probe for Ras. Oncogene 14: 623625, 1997.[CrossRef][ISI][Medline]
21. Desai BN, Myers BR, and Schreiber SL. FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction. Proc Natl Acad Sci USA 99: 43194324, 2002.
22. Diaz B, Barnard D, Filson A, MacDonald S, King A, and Marshall M. Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling. Mol Cell Biol 17: 45094516, 1997.[Abstract]
23. Dmitrieva NI, Michea LF, Rocha GM, and Burg MB. Cell cycle delay and apoptosis in response to osmotic stress. Comp Biochem Physiol A Mol Integr Physiol 130: 411420, 2001.[CrossRef][Medline]
24. Doyle JA, Davis DP, and Hoyt DB. The use of hypertonic saline in the treatment of traumatic brain injury. J Trauma 50: 367383, 2001.[ISI][Medline]
25. Famularo G. The puzzle of neuronal death and life: is mannitol the right drug for the treatment of brain oedema associated with ischaemic stroke? Eur J Emerg Med 6: 363368, 1999.[Medline]
26. Galcheva-Gargova Z, Derijard B, Wu IH, and Davis RJ. An osmosensing signal transduction pathway in mammalian cells. Science 265: 806808, 1994.[ISI][Medline]
27. Galvez A, Morales MP, Eltit JM, Ocaranza P, Carrasco L, Campos X, Sapag-Hagar M, Diaz-Araya G, and Lavandero S. A rapid and strong apoptotic process is triggered by hyperosmotic stress in cultured rat cardiac myocytes. Cell Tissue Res 304: 279285, 2001.[CrossRef][ISI][Medline]
28. Green DR. Apoptotic pathways: the roads to ruin. Cell 94: 695698, 1998.[ISI][Medline]
29. Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 13091312, 1998.
30. Hales KG and Fuller MT. Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90: 121129, 1997.[ISI][Medline]
31. Han J, Lee JD, Bibbs L, and Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808811, 1994.[ISI][Medline]
32. He H, Levitzki A, Zhu HJ, Walker F, Burgess A, and Maruta H. Platelet-derived growth factor requires epidermal growth factor receptor to activate p21-activated kinase family kinases. J Biol Chem 276: 2674126744, 2001.
33. Hunter T. Signaling2000 and beyond. Cell 100: 113127, 2000.[ISI][Medline]
34. Itoh T, Yamauchi A, Miyai A, Yokoyama K, Kamada T, Ueda N, and Fujiwara Y. Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells. J Clin Invest 93: 23872392, 1994.[ISI][Medline]
35. Johnson N, Khan A, Virji S, Ward JM, and Crompton M. Import and processing of heart mitochondrial cyclophilin D. Eur J Biochem 263: 353359, 1999.
36. Jones SM and Kazlauskas A. Connecting signaling and cell cycle progression in growth factor-stimulated cells. Oncogene 19: 55585567, 2000.[CrossRef][ISI][Medline]
37. Kodaki T, Woscholski R, Hallberg B, Rodriguez-Viciana P, Downward J, and Parker PJ. The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol 4: 798806, 1994.[ISI][Medline]
38. Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 351: 289305, 2000.[CrossRef][ISI][Medline]
39. Krasilnikov MA. Phosphatidylinositol-3 kinase dependent pathways: the role in control of cell growth, survival, and malignant transformation. Biochemistry (Mosc) 65: 5967, 2000.[ISI][Medline]
40. Kultz D, Madhany S, and Burg MB. Hyperosmolality causes growth arrest of murine kidney cells. Induction of GADD45 and GADD153 by osmosensing via stress-activated protein kinase 2. J Biol Chem 273: 1364513651, 1998.
41. Marais R, Light Y, Paterson HF, and Marshall CJ. Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. EMBO J 14: 31363145, 1995.[Abstract]
42. Marshall CJ. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev 4: 8289, 1994.[Medline]
43. Mason CS, Springer CJ, Cooper RG, Superti-Furga G, Marshall CJ, and Marais R. Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J 18: 21372148, 1999.
44. McCormick F. Activators and effectors of ras p21 proteins. Curr Opin Genet Dev 4: 7176, 1994.[Medline]
45. McCormick F. How receptors turn ras on. Nature 363: 1516, 1993.[CrossRef][ISI][Medline]
46. Michea L, Combs C, Andrews P, Dmitrieva N, and Burg MB. Mitochondrial dysfunction is an early event in high-NaCl-induced apoptosis of mIMCD3 cells. Am J Physiol Renal Physiol 282: F981F990, 2002.
47. Michea L, Ferguson DR, Peters EM, Andrews PM, Kirby MR, and Burg MB. Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells. Am J Physiol Renal Physiol 278: F209F218, 2000.
48. Morrison DK and Cutler RE. The complexity of Raf-1 regulation. Curr Opin Cell Biol 9: 174179, 1997.[CrossRef][ISI][Medline]
49. Nave BT, Ouwens M, Withers DJ, Alessi DR, and Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 344: 427431, 1999.[CrossRef][ISI][Medline]
50. Ouwens DM, Gomes de Mesquita DS, Dekker J, and Maassen JA. Hyperosmotic stress activates the insulin receptor in CHO cells. Biochim Biophys Acta 1540: 97106, 2001.[CrossRef][ISI][Medline]
51. Parrott LA and Templeton DJ. Osmotic stress inhibits p70/85 S6 kinase through activation of a protein phosphatase. J Biol Chem 274: 2473124736, 1999.
52. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, and Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22: 153183, 2001.
53. Petit PX, Susin SA, Zamzami N, Mignotte B, and Kroemer G. Mitochondria and programmed cell death: back to the future. FEBS Lett 396: 713, 1996.[CrossRef][ISI][Medline]
54. Rodriguez-Viciana P, Warne PH, Dhand R, Vanhaesebroeck B, Gout I, Fry MJ, Waterfield MD, and Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370: 527532, 1994.[CrossRef][ISI][Medline]
55. Rosette C and Karin M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science 274: 11941197, 1996.
56. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 103: 211225, 2000.[ISI][Medline]
57. Schwarz S, Georgiadis D, Aschoff A, and Schwab S. Effects of hypertonic (10%) saline in patients with raised intracranial pressure after stroke. Stroke 33: 136140, 2002.
58. Sternberg PW and Alberola-Ila J. Conspiracy theory: RAS and RAF do not act alone. Cell 95: 447450, 1998.[ISI][Medline]
59. Stoothoff WH and Johnson GV. Hyperosmotic stress-induced apoptosis and tau phosphorylation in human neuroblastoma cells. J Neurosci Res 65: 573582, 2001.[CrossRef][ISI][Medline]
60. Sun H, King AJ, Diaz HB, and Marshall MS. Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak. Curr Biol 10: 281284, 2000.[CrossRef][ISI][Medline]
61. Taylor SJ and Shalloway D. Cell cycle-dependent activation of Ras. Curr Biol 6: 16211627, 1996.[ISI][Medline]
62. Terada Y, Inoshita S, Hanada S, Shimamura H, Kuwahara M, Ogawa W, Kasuga M, Sasaki S, and Marumo F. Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells. Kidney Int 60: 553567, 2001.[CrossRef][ISI][Medline]
63. Terada Y, Tomita K, Homma MK, Nonoguchi H, Yang T, Yamada T, Yuasa Y, Krebs EG, Sasaki S, and Marumo F. Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells. J Biol Chem 269: 3129631301, 1994.
64. Therrien M, Wong AM, and Rubin GM. CNK, a RAF-binding multidomain protein required for RAS signaling. Cell 95: 343353, 1998.[ISI][Medline]
65. Thornberry NA and Lazebnik Y. Caspases: enemies within. Science 281: 13121316, 1998.
66. Tian W, Boss GR, and Cohen DM. Ras signaling in the inner medullary cell response to urea and NaCl. Am J Physiol Cell Physiol 278: C372C380, 2000.
67. Toung TJ, Tyler B, Brem H, Traystman RJ, Hurn PD, and Bhardwaj A. Hypertonic saline ameliorates cerebral edema associated with experimental brain tumor. J Neurosurg Anesthesiol 14: 187193, 2002.[CrossRef][ISI][Medline]
68. Vanhaesebroeck B and Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 346: 561576, 2000.[CrossRef][ISI][Medline]
69. Westermann B. Merging mitochondria matters: cellular role and molecular machinery of mitochondrial fusion. EMBO Rep 3: 527531, 2002.
70. Yoon Y and McNiven MA. Mitochondrial division: New partners in membrane pinching. Curr Biol 11: R67R70, 2001.[CrossRef][ISI][Medline]
71. Zhang BH and Guan KL. Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601. EMBO J 19: 54295439, 2000.
72. Zhang Z, Tian W, and Cohen DM. Urea protects from the proapoptotic effect of NaCl in renal medullary cells. Am J Physiol Renal Physiol 279: F345F352, 2000.
73. Zhang Z, Yang XY, Soltoff SP, and Cohen DM. PI3K signaling in the murine kidney inner medullary cell response to urea. Am J Physiol Renal Physiol 278: F155F164, 2000.
74. Zheng Y, Bagrodia S, and Cerione RA. Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. J Biol Chem 269: 1872718730, 1994.
75. Zimmermann S and Moelling K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286: 17411744, 1999.