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【摘要】 The adaptation to hypertonicity in mammalian cells is driven by multiple signaling pathways that include p38 kinase, Fyn, the catalytic subunit of PKA, ATM, and JNK2. In addition to the well-characterized tonicity enhancer (TonE)-TonE binding protein interaction, other transcription factors (and their respective cis elements) can potentially respond to hypertonicity. This review summarizes the current knowledge about the signaling pathways that regulate the adaptive response to osmotic stress and discusses new insights from yeast that could be relevant to the osmostress response in mammals.
【关键词】 osmostress tonicity enhancer/osmotic response element tonicity enhancer binding protein p kinase ERK JNK osmolytes
MANY CELLS AND ORGANISMS CONFRONT an environment that can increase in osmolality. Under this circumstance, the attendant diffusion of water out of the cell induces a variety of adaptive responses, many of which appear to be controlled by a signaling network of protein kinases and transcription factors.
The urinary concentrating mechanism is a fundamental biological process that is essential for water conservation by the kidney. As a result, during dehydration the interstitial osmolality of human kidney medulla may reach 1,200 mosmol/kgH 2 O, whereas that of rat renal medulla may reach 3,500 mosmol/kgH 2 O ( 90 ). Roughly one-half of the prevailing solutes in the kidney medulla consist of urea, whereas the other half is composed of Na, K, and Cl ( 27 ). Although urea easily equilibrates across biological membranes and does not cause water shift between the intracellular and extracellular spaces, the electrolytes are usually compartmentalized, primarily due to the action of the Na-K-ATPase. Thus an increase in extracellular electrolyte content causes dehydration of the intracellular milieu and perturbs macromolecular function. To avoid high intracellular ion concentrations and to balance extracellular and intracellular osmolality, cells typically adapt to sustained hypertonic stress by the preferential accumulation of compatible organic osmolytes ( 109 ). To accomplish this, hypertonically stressed kidney cells induce a group of genes that code for transporters of organic osmolytes [BGT1 for betaine ( 106 ), SMIT for inositol ( 107 ), taurine ( 98 )] and the aldose reductase enzyme [(AR), which catalyzes the conversion of D -glucose to sorbitol ( 28 )]. Other groups of genes are also induced by hypertonicity. Chaperone genes ( 15, 26, 49 ) including heat shock protein (HSP)70 are induced immediately after exposure of cells to hypertonicity and are believed to protect intracellular macromolecules against unfolding and aggregation until the accumulation of organic solutes is complete ( 89 ). Intriguingly, hypertonicity increases the expression of cytokine genes including those for the IL-1 receptor antagonist MIP-2, TNF superfamily ligand member 9, B-cell leukemia/lymphoma 6, and lymphocyte antigen 84 ( 94 ). However, the role of these cytokines in the adaptive response to osmotic stress remains to be determined.
Induction of osmolyte-accumulating genes, such as BGT1 and AR, by hypertonicity correlates with the increase in intracellular Na, K, and Cl concentrations (ionic strength) ( 71, 97 ), although a decrease in cell volume also appears to be a factor ( 38, 71 ). The means by which a change in ionic strength or cell volume is transduced into signals that regulate gene expression in hypertonically stressed cells remain unknown. Promoter analysis of organic osmolytes genes suggests that the signaling "machinery" that drives gene expression in response to hypertonicity appears to be similar ( 105 ). The transcription of SMIT, BGT1, and AR genes under hypertonic conditions is stimulated by the same genomic ci s-element named tonicity enhancer [TonE ( 84 ); otherwise known as osmotic response element ( 24 )] and the transcription factor tonicity enhancer binding protein [TonEBP ( 65 ); otherwise known as NFAT5 ( 57 )]. This is true in most cells examined thus far, including lymphocytes ( 58 ), human liver-derived [HepG2 ( 68 )], Madin-Darby canine kidney [MDCK ( 84 )], rabbit kidney papillary epithelial cells [PAP-HT25 ( 51 )], Chang liver, Cos-7, and HeLa cells ( 47 ). A large number of studies (reviewed in Refs. 36 and 104 ) have demonstrated a central role for TonEBP in the hypertonic response. Most convincing is a recent study by Lopez-Rodriguez et al. ( 56 ) showing that mice with a deletion in the TonEBP gene have an almost complete block in the expression of AR, BGT1, and SMIT genes in the kidney medulla and in hypertonicity-induced expression of AR in fibroblasts. Remarkably, mice lacking TonEBP show atrophy of the renal medulla, which contains smaller cells and displays an increased rate of apoptosis. This is exactly the phenotype that one would expect for cells that cannot adapt to high osmolality; that is, cell shrinkage and subsequent activation of apoptosis. It should be pointed out that TonEBP probably has multiple functions in cell biology in various organs. This transcription factor is expressed not only in the kidney but also in other tissues, including the brain ( 59 ). The underrepresentation of homozygous mouse mutants for NFAT5 after embryonic day 14.5, as shown by Lopez-Rodriguez et al. ( 56 ), is consistent with an important function for this protein outside the kidney as mice can survive past birth without kidney function.
As discussed above, urea does not impose water shift across biological membranes and does not cause hypertonic stress. However, urea exerts complex effects on cellular function. Exposure of cultured cells to an isolated urea stress perturbs cellular function ( 108 ). On the other hand, urea may temper the effects of hypertonicity imposed by NaCl ( 86 ). In addition, urea stress (high osmolality without water shift) has differential effects on gene expression compared with NaCl stress (hypertonic stress with water shift) and appears to activate a growth factor-like gene response ( 94 ), which is distinct from the profile of genes that are induced by NaCl ( 13, 14, 93, 94 ). In this context, Ras-mediated signaling through ERK [and possibly involving PKC ( 14 )] plays a role in the induction of egr-1-mediated transcription in response to urea stress ( 92 ).
p38 KINASE IS A CENTRAL MEDIATOR OF THE OSMOSTRESS RESPONSE
Over the past decade, major strides have been made in the characterization of the transcriptional control of hypertonicity-induced genes and signaling pathways that lead to gene expression by hypertonicity. In yeast, the adaptation to osmotic stress is dependent on the p38 MAPK homolog high-osmolality glycerol (HOG1) kinase ( 9 ).
Although an initial study targeting MAP kinase kinase 3 (MKK3), an activator of p38 kinase, suggested that p38 was not required in rabbit kidney papillary epithelial cells for induction of osmoprotective genes ( 51 ), more recent studies show that p38 is an essential component of the hyperosmotic signaling response pathway in mammals. The major subfamilies of MAP kinases include ERK, JNK, and p38 kinase. In each case, the MAP kinase is activated by an upstream MKK on phosphorylation of threonine/tyrosine residues, which in turn is regulated by distinct upstream MKKK. Diverse extracellular stimuli, including UV light, -irradiation, heat shock, osmotic stress, proinflammatory cytokines, and certain mitogens, trigger the activation of p38 MAPK ( 43 ). p38 Kinase plays a major role in apoptosis, cytokine production, transcriptional regulation, and cytoskeletal reorganization and has been implicated in a variety of diseases such as sepsis, arthritis, human immunodeficiency virus infection, cardiac ischemia, and Alzheimer's ( 74 ). To date four different p38 Kinase isoforms have been described, p38 ( 6, 30, 33, 62 ), p38 ( 91 ), p38 ( 16, 54, 55 ), and p38 ( 30, 44 ). p38 Is expressed exclusively in muscle ( 30, 55 ), whereas p38 is expressed predominantly in the lungs and glomeruli ( 44 ). p38 And p38 are expressed in many tissues including the kidneys ( 44, 55 ) and are equally inhibited by the imidazole compounds (SB-203580 as a prototype). Both display near identical responses to TNF-, IL-1, EGF, PMA, UV light, H 2 O 2, osmotic stress, and arsenate and appear to be redundant ( 43 ). However, p38 and p38 differ in their upstream activators; p38 is activated by MKK3, MKK4, and MKK6, whereas p38 is preferentially activated by MKK6 ( 34 ). Thus the expression of more than one p38 isoform and upstream MKKs (for p38 kinase) in the kidney may provide redundancy in the pathway, and inhibition of one of the upstream activators of p38 kinase as shown by Kultz et al. ( 51 ) may not have been sufficient to block p38-dependent gene expression. Indeed, using specific pharmacological inhibitors of p38 kinases in macrophages, kidney cells, and liver cells, we and others have shown that these inhibitors block the induction of mRNAs for SMIT, BGT1, AR, and HSP70 by hypertonicity, as well as the expression of TonE-mediated gene expression and the binding of trans -acting factors to TonE/ORE ( 21, 68, 88 ). Ko et al. ( 46 ) have also demonstrated inhibition of hypertonicity-induced TonE-mediated reporter gene expression by mutated p38 MAPK. In addition, they brought to light the involvement of Fyn, a non-receptor tyrosine kinase related to Src (which is activated by cell shrinkage), in the expression of genes by hypertonicity and suggested parallel yet complementary and cooperative action of Fyn with p38 kinase in TonE induction ( 46 ). Of note, the glucocorticoid-inducible kinase sgk-1 is induced at the mRNA level by hypertonic stress through sp1-responsive elements and in a manner that is p38 kinase dependent ( 4 ). These findings are consistent with previous observations in Saccharomyces cerevisiae, in which the Msn family of transcription factors are targeted by the yeast p38 homolog HOG1 and bind to a DNA sequence that is similar to the Sp1 binding sequence ( 61, 87 ). However, the role of this kinase in the adaptation to osmotic stress remains unclear. Collectively, these recent findings, plus others discussed below, start to unravel the complex topknot of signaling proteins in the adaptation to osmotic stress.
At the MKKK level [see review by Widmann et al. ( 101 )], activation of p38 may be accomplished through multiple protein kinase families including the MEKKs ( 53 ), thousand and one kinases (TAOs) ( 39 ), apoptosis signal-regulating kinase-1 (ASK1) ( 40 ), and TGF- activating kinase-1 (TAK1) ( 67 ). Recent data from Uhlik et al. ( 99 ) suggest the involvement of MEKK3 in the activation of p38 kinase in the context of hypertonicity. MEKKs are the most diverse of eukaryotic MKKKs, and except for the catalytic domain, which is conserved compared with the S. cerevisiae equivalent STE11 (which regulates yeast mating pheromone and osmosensing pathways), the proteins are dissimilar. Accordingly, these enzymes can catalyze different MAP kinase pathways and may interact with a wide array of regulatory proteins ( 53 ). MEKK3 apparently resides in an actin-bound scaffold of proteins that also includes MKK3, the Rac GTPase, and a newly discovered protein, osmosensing scaffold for MEKK3 (OSM), which appears to be required for hypertonicity-mediated p38 kinase activation ( 99 ). Depletion of MEKK3 using RNA interference (RNAi) attenuated p38 kinase activation in hypertonically stressed cells. The authors concluded that the Rac-OSM-MEKK3-MKK3 complex is the mammalian counterpart of the osmosensing CDC42-STE50-STE11-PBS2 complex of S. cerevisiae and is required for p38 kinase activation in response to hypertonicity in mammalian cells ( 99 ). Consistent with these findings, activation of MEKK3 enhances the expression of BGT1 and TonE-mediated reporter gene expression under isotonic or hypertonic conditions (unpublished observations from our laboratory). Thus our data support and complement the report by Uhlik et al. ( 99 ) and suggest signaling from Rac-OSM-MEKK3 p38 TonE/TonEBP in mammalian cells. There are still many unresolved issues, particularly at the beginning and end of the signaling pathways involved. For example, the nature of the sensor proteins that activate kinase pathways in response to cell volume and intracellular tonicity changes in response to hypertonic stress is still unknown. In addition, there is yet no clear molecular link between TonEBP and protein kinases, including p38.
A COMPLEX NETWORK OF PROTEIN KINASE SIGNALS MEDIATES THE OSMOSTRESS RESPONSE
In mammalian cells, hypertonicity activates not only p38 kinase but other MAP kinases such as ERK, JNK, the non-receptor tyrosine kinases Fyn and Syk, PKC, the catalytic subunit of PKA (PKAc), p21-activated serine/threonine protein kinase (PAK2), and DNA damage-inducible kinase ( 5, 23, 41, 45, 46, 63, 82, 85, 114 ). In the following paragraphs, we will briefly summarize the contribution of these kinases to the adaptation to hypertonicity.
In addition to p38 kinase and Fyn, PKAc appears to have a role in TonE-mediated gene expression. PKA exists as a tetramer consisting of two regulatory subunits and two catalytic subunits. In many instances, PKA is regulated by intracellular cAMP levels; cAMP binds to the regulatory subunits of PKA, resulting in the release of the catalytic subunits, which in turn interact with transcription factors such as cAMP-reponse element binding protein (CREB) ( 96 ). PKA is regulated by a broad range of cellular processes, and the specificity of PKA activation is conferred through variations in the combination of catalytic and regulatory subunits assembly and through subcellular compartmentalization of the enzyme by kinase-anchoring-proteins ( 96, 115 ). Inhibition of PKAc using pharmacological inhibitors (H89) reduces tonicity-dependent TonE/ORE-mediated gene expression, and a dominant-negative PKAc reduces the activity of an TonE/ORE-driven reporter gene ( 23 ). PKAc associates with TonEBP and contributes to its activation; however, activation of TonEBP by PKA appears to be cAMP independent ( 23 ).
A recent publication by Irarrazabal et al. ( 41 ) suggests an important role for ATM, a DNA damage-inducible kinase in the activation of TonE/TonEBP-mediated transcription, in response to hypertonicity. The observations made in this publication are particularly intriguing. Hypertonicity (NaCl) causes persistent DNA damage in cell culture ( 50 ) and in vivo ( 22 ), and thus DNA damage in hypertonicity-stressed cells could function as a sensor for transcriptional activation of specific genes required for the adaptation to osmotic stress, i.e., transporters for organic solutes and HSPs. Hence, these findings could explain the correlation between intracellular ionic strength and gene expression in the context of hypertonic stress. However, whereas ATM can activate TonE/TonEBP-mediated transcription in response to hypertonicity, it is insufficient to induce maximal response, and as concluded by Irarrazabal et al. ( 41 ), maximal expression and induction of TonE/TonEBP-mediated transcription result from the convergence of input from multiple pathways that include, but may not necesesarily be limited to, Fyn ( 46 ), ATM ( 41 ), PKAc ( 23 ), and p38 kinase ( 46, 68, 88 ).
Early studies suggested that ERK activity was not essential for transcriptional regulation of BGT1 and SMIT ( 52 ) or the stimulation of inositol uptake ( 5 ). Intriguingly, inhibition of MEK1, an upstream activator of ERK, using pharmacological inhibition downregulated TonE-mediated reporter gene expression and the binding of trans -acting factors to TonE in HepG2 cells ( 68 ). However, it did so in a manner that was ERK independent ( 68 ). ERKs are responsible for the phosphorylation and activation of intracellular targets such as c-Myc, c-Jun, c-Fos, and the ternary complex factors ( 18 ). Activation of ERKs requires sequential action of the Raf family of protein kinases, which phosphorylate and activate MEKs. These, in turn, lead to dual phosphorylation of ERK on threonine/tyrosine residues, resulting in activation of the intrinsic protein serine/threonine kinase activity of ERKs. ERKs are activated by growth factors and are thought to be involved in cell proliferation and survival. Because ERK activation during exposure of cells to hypertonicity does not appear to affect TonE-mediated gene expression ( 52, 102 ), it is conceivable that activation of the ERK pathway in hypertonicity-stressed cells serves as a cell survival signal that opposes the apoptotic signals triggered by hypertonicity ( 64 ).
As for PKC, pharmacological inhibition of the enzyme (using rothelrin, phorbol ester, or GF-109203X) blocks TonE-mediated gene expression in hypertonically stressed kidney cells. However, the effects of these inhibitors on TonE appear to be independent of PKC, as transfection of cells with mutated PKC or - was devoid of any effect ( 114 ). PKC, however, appears to play a role in urea-mediated gene expression ( 14 ).
The role of JNK in the adaptive response to hypertonicity is complex. Although data in hypertonically stressed (by glucose or mannitol) vascular smooth muscle cells suggested upregulation of HB-EGF in a Pyk2/JNK1/activator protein (AP)-1-dependent manner ( 48 ), data in kidney cells suggested the involvement of JNK2, but not JNK1 or JNK3, in cell survival and expression of HSP70, COX-2, and Na-K-ATPase under hypertonic conditions ( 10, 102, 103, 110 ). JNKs are activated after phosphorylation on threonine/tyrosine residues by well-defined dual-specificity MAPK kinases (MKK4 and MKK7), which are in turn regulated by distinct upstream MKKKs ( 17 ). In addition to G protein-coupled receptors, JNKs are activated by cytokine receptors, growth factor-coupled receptor tyrosine kinases ( 12 ), and a host of cellular stresses, such as osmotic stress, UV light, ischemia, heat shock, and cytotoxic drugs ( 12 ). Of note, cell stress often selectively activates the JNK pathway with little activation of the ERK pathway, a setting that generally favors apoptosis. This stands in contrast to JNK activation that follows the stimulation of receptor tyrosine kinases and G protein-coupled receptors, which is typically accompanied by ERK activation and is associated with cell growth and proliferation. Thus the role of JNK in a particular cellular context may depend on the cell type and the profile of stress and growth signals to which the cell is exposed. JNKs are alternatively spliced at 2 sites (internal and COOH terminal), yielding 10 different isoforms ( 31 ). COOH-terminal splicing defines the 46- and 54-kDa forms of the enzymes, whereas internal splicing determines the affinity of the kinase to its substrate, c-Jun. Although the various isoforms of JNK are considered a single group, it is likely that individual isoforms have distinct cellular functions. Data from our laboratory ( 88 ) and others ( 102 ) do not support a role for JNK1 in TonE-mediated gene expression, as JNK1 is actually upregulated in MDCK cells on p38 kinase inhibition, a setting in which TonE-mediated gene expression is inhibited ( 88 ). As discussed above, data from Wojtaszek et al. ( 102 ) demonstrated that inhibition of JNK2 reduced mouse inner medullary collecting duct cell survival under hypertonic conditions; however, it did not affect inositol uptake or volume regulation, suggesting that the protective effects afforded by JNK2 in hypertonically stressed kidney cells are not related to the expression/function of organic osmolytes genes or volume regulation but rather to the induction of cytoprotective genes [COX-2 ( 110 ) and HSP70 ( 103 )] and enhancement of Na-K-ATPase activity ( 10 ), a crucial enzyme responsible for maintenance of normal electrolyte distribution between the intracellular and extracellular spaces. However, it is unclear at present whether the induction of HSP70, COX-2, and Na-K-ATPase by JNK2 is TonE/TonEBP mediated.
The non-receptor tyrosine kinases Fyn ( 45, 82, 85 ), Yes and Lck ( 82 ), and Syk ( 63 ) also appear to be activated by hyperosmotic stress. This is not a general property for all members of this protein kinase class, as Src is actually inhibited by hyperosmotic stress ( 45 ). As discussed above, Fyn appears to act upstream of TonEBP, whereas Yes and Syk kinases appear to regulate JNK under hyperosmotic conditions ( 63, 82 ). The target of Lck activation remains unknown. The mechanism of activation of these non-receptor tyrosine kinases by hypertonic stress is unclear. A possible pathway may involve the p21-activated kinases (PAKs). A recent study ( 63 ) suggests that Syk activation by hypertonic stress (sorbitol) requires interaction with Pak2, which is activated by CDC42 in a cascade that proceeds from CDC42 PAK2 Syk Jnk, and we speculate that activation of other non-receptor tyrosine kinases by hypertonicity may follow a similar pattern.
The transcription factor HSF-1 drives the expression of chaperones (HSPs) in response to environmental stresses (thermal stress, heavy metals, amino acid analogs, and arsenate) and also responds to osmotic stress ( 3, 11 ). Thus it could confer responsiveness of various HSPs to hypertonicity, independently of the presence of TonE in their respective promoters. Of note, HSF-1 is activated by shifts in ambient tonicity in both hypo- and hypertonicity ( 11 ). Consistent with that, HSP70 is upregulated by hypertonicity ( 89 ) and hypotonicity ( 113 ).
NF- B is closely related to TonEBP ( 58 ). Recent data in intestinal epithelial cells suggest activation of NF- B and stimulation of NF- B-mediated transcription by osmotic stress ( 70 ). Similarly, data from Hao et al. ( 35 ) suggest that COX-2 expression in hypertonically stressed cultured renal medullary interstitial cells is NF- B dependent and that water deprivation increases renal NF- B-driven reporter gene expression in transgenic mice. Intriguingly, data from Iwata et al. ( 42 ) suggest induction of the AR gene (an organic osmolyte gene whose promoter contains TonE elements and is induced by TonEBP) by TNF- and IF- in human liver cells in a manner that is TonE and NF- B dependent. The NF- B consensus sequence and TonE differ by one base, and NF- B binds TonE ( 42 ). Thus NF- B- and TonEBP-mediated signaling may converge or complement each other in the regulation of some stress genes (AR and possibly COX-2) by cytokines and osmotic stress.
Environmental stress (e.g., anisosmolarity and UV light) activates signal transducer and activator of transcription (STAT) in a manner that is ligand independent, and inhibition of p38 diminishes STAT activity in osmotically stressed cells. Conversely, overexpression of wild-type p38 mitogen or its upstream activator MKK6 enhances STAT tyrosine phosphorylation on osmotic shock ( 7 ). Numerous cytokines, growth, and differentiation factors elicit their intracellular responses via STAT. Osmotic stress phosphorylates STAT and leads to nuclear translocation of this transcription factor. Experiments using a diffusible solute suggest that activation of STAT by hypertonicity is triggered by cell shrinkage and does not result from the increase in intracellular ionic strength ( 29 ). Thus STAT activation appears to be p38 kinase dependent and may contribute to gene expression triggered by cell shrinkage, which normally occurs in hypertonically stressed cells.
As stated above, CREB-mediated gene expression classically results from activation of PKA by increases in intracellular cAMP. In cells of the hypothalamus, cAMP regulates vasopressin gene expression in response to variations in serum osmolality in a manner that is dependent on the cAMP response element ( 75 ). Similarly, expression of the proenkephalin gene in the paraventricular nucleus is regulated by CREB in response to saline infusion. These data suggest cAMP/CREB-dependent vasopressin and proenkephalin expression in the hypothalamus in response to hypertonicity ( 8 ). On the other hand, data from Ferraris et al. ( 23 ) suggest that PKAc associates with TonEBP and enhances TonE-mediated gene expression in hypertonically stressed cells in a manner that is cAMP independent, suggesting that the effects of PKAc on TonE-mediated gene expression are CREB independent.
Last, immediate early gene products (c-Fos, c-Jun, and their cognates) act as transcription factors coupling physiologically relevant stimuli to long-term responses by binding to the AP-1 site in the promoter region of target genes. Hyperosmolality increases the binding of c-Fos and c-Jun to AP-1 consensus sequences in cells of the supraoptic nuclei of the brain, suggesting that hyperosmolality leads to a selective and specific increase in AP-1 DNA binding activity, which may be responsible for regulating secondary target gene expression in the hypothalamic supraoptic nuclei ( 111 ).
In summary, the adaptation to osmotic stress in mammalian cells, vis-à-vis the activation of TonE/TonEBP, is driven by multiple signaling pathways that include p38 kinase, Fyn, PKAc, ATM, and possibly JNK2 ( Fig. 1 ). However, in addition to the well-characterized TonE-TonEBP interaction, other transcription factors (and their respective cis -elements) can potentially regulate gene expression in the context of hypertonicity. These transcription factors include HSF-1 ( 3, 11 ), NF- B ( 35, 70 ), CREB ( 8 ), STAT ( 7 ), and AP1 ( 111 ).
Fig. 1. Scheme representing signaling molecules known to regulate tonicity enhancer (TonE). Fyn and p38 kinase appear to operate in parallel and complementary fashion. Although input from Fyn to the catalytic subunit of PKC (PKAc) is possible (Steve Chung, personal communication), the signaling intermediates that link Fyn to transcriptional regulation of TonE are currently unknown.
INSIGHTS FROM THE YEAST HOG PATHWAY FOR UNDERSTANDING p38 FUNCTION IN KIDNEY CELLS
Many of the signaling pathways that exist in yeast have their equivalent systems in mammalian cells, and to a great extent the mammalian counterparts demonstrate conservation of function. So is the case with the p38 kinase pathway. The yeast HOG pathway is an osmosensing signal transduction pathway whose components have almost all been identified from osmosensors to gene targets ( Fig. 2 and Refs. 32 and 73 ). The conservation of function between the HOG1 MAPK in yeast and p38 kinase in mammals is not surprising, but it should be noted that p38 MAPK (in mammals) and HOG1 (in yeast) function as signal relay proteins in their respective pathways and do not appear to directly sense osmotic stress. Although both kinases have been co-opted for other uses in their respective systems, the similar osmostress response function suggests that other structural features of the yeast signaling pathway have relevance to the ongoing exploration of similar signaling systems in mammals. However, there are some differences between yeast and mammals; the JNK pathway, for instance, does not exist in yeast, yet it appears to play a role in the expression of some genes in osmotically stressed mammalian cells. In the following paragraphs, we summarize two new insights regarding the yeast HOG pathway that may be relevant to the mammalian p38 MAPK.
Fig. 2. Scheme representing the yeast high-osmolality glycerol (HOG) pathway. The proteins in the 2 upstream branches of the osmosensing yeast Hog1/p38 MAPK pathway are shown. These two branches are completely separate and converge on a common MKK Pbs2 and MAPK Hog1. The right branch contains the scaffold protein and osmosensor Sho1, which appears to be most similar to the mammalian pathway in that both have a small G protein (Cdc42 in yeast, Rac in mammals) upstream of the MAPK cascade. This branch also contains the p21-activated kinase Ste20, the MKKK Ste11, and its regulatory subunit Ste50. The left branch of the yeast HOG pathway contains the osmosensor Sln1, whose histidine kinase domain places it in a gene family found in bacteria and plants but not mammals. The Sln1-Ypd1-Ssk1 phosphorelay system regulates a pair of redundant MKKKs Ssk2 and Ssk22 that converge on Pbs2. More details on downstream targets of Hog1 can be found in Refs. 37 and 73.
The first of these important findings is that yeast Hog1 kinase is directly involved in transcription regulation. Like p38 in mammals, Hog1 directly phosphorylates transcription factors. Phosphorylation of these factors [the CREB homolog Sko1 ( 78 ) and the MEF2-like Smp1 ( 19 )] is required for subsequent activation of osmostress response genes in yeast. Just as for other kiss-and-run kinases, these results show that the actions of Hog1 on transcription are mediated by downstream transcription factors. Like p38, Hog1 moves into the nucleus after its phosphorylation ( 25, 83 ). Apart from the regulation of transcription factors, new insight suggests that Hog1 itself is part of the transcription activation complex for osmostress-regulated genes ( 1, 2, 79 ). Even more significant is that Hog1 induces expression of a large subset of target genes through direct interaction with the histone deacetylase Rpd3 and recruits a complex containing this chromatin modifier to the promoter ( 20 ). Although histone deacetylation usually represses gene expression, here Rpd3 is an activator. However, whereas p38 directly interacts with the transcription apparatus in mammalian cells ( 1 ), the interaction partners of p38 MAPK (e.g., histone modifying enzymes) have yet to be defined.
A second new insight from recent studies of the yeast HOG pathway is the discovery of a mechanism to switch off the signaling pathway through competing protein-protein interactions. As discussed previously, the yeast and mammalian pathways share a similar scaffold-supported organization of signaling proteins upstream of the MAP kinase cascade. The yeast HOG pathway has two upstream branches that connect the MAP kinase Hog1 and the MKK Pbs2 to different plasma membrane osmosensor proteins. One of these sensors is Sho1, an integral membrane protein that contains a COOH-terminal SH3 domain ( 60 ). Interaction between this domain and a proline-rich motif in the NH 2 terminus of Pbs2 is necessary for Sho1-mediated activation of the MAP kinase cascade by osmostress. Other components of this branch include the Cdc42 GTPase, the p21-activated protein kinase Ste20, the MKKK Ste11, and its adapter Ste50 ( 72, 76, 77, 80, 81 ). Although Pbs2 was originally thought to act as the scaffold for the Sho1 branch of the pathway ( 76 ), activation of Ste11 by Sho1 can take place in the absence of Pbs2 ( 72, 112 ). In addition, Ste11 binds to Sho1 independently of Pbs2 ( 112 ). Thus Sho1 and Pbs2 appear to cooperate in organizing the upstream components of one branch of the HOG pathway acting as co-scaffolds. As described earlier, a similar cascade of regulatory proteins (Rac GTPase and OSM serving as scaffold for MEKK3) has been shown to act upstream of p38 in the mammalian osmostress response pathway ( 99 ). Although unique scaffold proteins (Pbs2, OSM) hold these signaling pathways together and are important for their recruitment to the membrane ( 76, 100 ) or actin cytoskeleton ( 99 ), the manner in which these complexes are turned off or on remains a mystery. The discovery of the OSM scaffold is important because there are still many missing gaps at the top of the mammalian pathway, and finding other proteins that interact with the OSM scaffold is one promising and possible way to fill these gaps. For example, a recent analysis of proteins that interact with the Sho1 scaffold has revealed a new way to regulate the yeast HOG pathway. The new finding is that the plasma membrane Fus1 competes with Pbs2 for interaction with the SH3 domain of Sho1 ( 69, 95 ). Both Pbs2 and Fus1 bind to the SH3 domain of Sho1 through similar short proline-rich motifs. During mating of yeast, cells switch off the Sho1-dependent HOG pathway by expressing Fus1. Fus1 itself appears to be a scaffold for formation of proteins needed for cell-cell fusion during mating ( 69 ), and cell fusion is further enhanced by Fus1 inhibition of the HOG pathway. Thus competition between different pathways for control of cell response is a general theme, particularly with the switch from one task to another as the cells deal with stress. The manner in which different osmostress pathways activate or inhibit their MAP kinase pathways, however, remains a large unsolved problem.
GRANTS
This work was supported by Renal Section funds provided by the Baylor College of Medicine to D. Sheikh-Hamad and National Science Foundation Grant MCB91236 to M. C. Gustin.
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作者单位：1 Renal Section, Department of Medicine, Baylor College of Medicine, and 2 Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77030