Stress-Activated Protein Kinases in Cardiovascular Disease
Cells respond to extracellular stimuli by activating signal transduction pathways, which culminate in changes in gene expression. The particular genetic program activated determines, in large part, the response of the cell (eg, growth versus growth arrest versus apoptosis; differentiation versus dedifferentiation). A critical component of eukaryotic signal transduction is the activation of protein kinases, which phosphorylate a host of cellular substrates, including transcription factors controlling the induction of various genes. For example, the Ras/ERK-1 and ERK-2 (or MAP kinase) pathway transduces critical components of the growth factor–induced mitogenic response to the nucleus. Expression of inactive or interfering mutants of components of the pathway disrupts, and expression of constitutively active mutants activates, mitogenesis.1
Recently, protein serine/threonine kinases related to ERK-1 and -2 have been identified; these kinases transduce signals to the nucleus not in response to growth factors and other mitogens but in response to cellular stresses such as inflammatory cytokines (IL-1β and TNF-α), ischemia, reversible ATP depletion, heat shock, endotoxin, and genotoxic stress. These kinases, called the SAPKs2 or, alternatively, c-Jun N-terminal (amino-terminal) kinases (JNKs, named after one of their physiological substrates),3 and p38,4 likely play critical roles in the genetic response of many components of the cardiovascular system to disease processes (Table⇓). In this review, we will discuss these stress-activated kinases, how they are regulated, and the evidence suggesting roles they may play in cardiovascular disease.
The SAPKs were first described in 1990 as the dominant microtubule-associated protein 2 kinase activated in rat liver in response to systemic administration of the protein synthesis inhibitor cycloheximide.5 The kinase shared two major characteristics with the mitogen-activated p42 and p44 MAP kinases (later renamed ERK-2 and ERK-1, respectively). The first was proline-directed substrate specificity. Serine or threonine residues in substrates were phosphorylated only if followed by a proline residue. The second common characteristic was a requirement for dual phosphorylation (on both a threonine and a tyrosine residue) of the kinase for activation. To reflect its relationship to the p42/44 MAP kinases, the kinase was named p54 MAP kinase.5 Interest in the kinase heightened when it was found to readily phosphorylate c-Jun, a component of the AP-1 transcription factor (see below).6 After the isolation of cDNAs, the kinases were found to be activated in response to inflammatory cytokines (TNF-α and IL-1β), heat shock, and several metabolic inhibitors in addition to cycloheximide, and the kinases were renamed the SAPKs.2 Currently, three genes have been identified, and with two different forms of alternative splicing demonstrated, up to 12 isoforms may exist. One of the alternative splice sites accounts for their apparent Mr of 46 kD and 54 kD on SDS-PAGE.2
p38 is a second ERK family member activated by cellular stress. p38 was initially cloned on the basis of its homology to a yeast kinase, HOG1, which allows yeast to respond to osmolar stress by increasing glycerol synthesis. In mammalian cells, p38 is activated by many of the same stimuli as the SAPKs, including osmolar stress and heat shock.4 7 8 More relevant to cardiovascular disease, p38, like the SAPKs,9 is markedly activated by TNF-α and IL-1β and also by lipopolysaccharide and the ATP-depleting agent, sodium arsenite.8 10
The Signaling Cascades
Like the ERK-1/-2 signaling cascade, which is activated primarily by stimulation with growth factors or other mitogens, the SAPK and p38 cascades consist of three-tiered modules of protein serine/threonine kinases (Fig 1⇓). In these cascades, an ERK family member (ERK-1/-2, SAPK, or p38) is activated by an MEK, which is, in turn, activated by an MEKK. This cassette arrangement has been remarkably conserved over millions of years of evolution, and strikingly similar cascades exist in yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe11 (Fig 1⇓). Not only is the three-tiered module conserved, but within each tier, there are marked homologies between yeast and human kinases. For example, the kinase domains of ERK-1 and -2 are nearly 50% identical to Fus3 and Kss1, two protein kinases that mediate cell cycle arrest in yeast as part of the mating response to pheromone (Fig 1⇓). Activators upstream of the MEKK tier in both the SAPK and p38 cascades have recently been identified, and again, evolutionary conservation appears to be maintained. Specifically, the SAPK cascade can be activated by GCK,12 and both the p38 and SAPK cascades can be activated by Pak1.13 GCK and Pak1 are human homologues of yeast Ste20 (so named because mutations in the STE20 gene prevent yeast from mating properly in response to pheromone).11 Further upstream in the SAPK and p38 cascades are two members of the Rho family of small GTP binding proteins, Rac and Cdc42Hs,13 14 15 which regulate actin cytoskeleton rearrangements, resulting in the formation of lamellipodia, filopodia, and membrane ruffles. When complexed to GTP, Rac and Cdc42Hs activate the cascades by binding to Pak1, which stimulates the autophosphorylation and activation of the kinase. It is not clear, as yet, how GCK is activated, since it lacks the Rac binding domain of Pak1. It is presumed that GCK and Pak1 activate the cascade via direct activation of MEKK-1 or a related MEKK, but this has not been demonstrated for these kinases or for Ste20 and Ste11 in yeast.
Despite the fact that many stimuli activate both the SAPKs and p38, the pathways of activation appear to be relatively insulated from one another (Fig 1⇑). Unless markedly overexpressed, upstream activators of the SAPKs (MEKK-1 or SEK-1) minimally activate p38 and ERK-1/-2, and another MEK, MKK3, specifically activates p38 but not the SAPKs or ERK-1/-2.16 17 18 This is somewhat surprising given the sequence similarities between components of the different cascades and the fact that SEK-1 can activate p38 when incubated in vitro. This segregation of the kinase cascades into distinct pathways may be accomplished by a tethering protein, such as Ste5 in yeast, which forms a complex with and may sequester Ste11 (the MEKK yeast homologue), Ste7 (the MEK homologue), and Fus3 (the ERK homologue).11 Under this scenario, SEK-1, which could activate p38, never gains access to it because SEK-1 and p38 are bound to different Ste5 homologues. Segregation is probably critical to the cell in order to minimize cross talk among and inappropriate activation of the ERK family cascades.
Stress-activated kinase substrates that have been identified include the transcription factors c-Jun, ATF-2, and Elk-1, which will be discussed below. p38 also phosphorylates and activates MAPKAP kinase-2,8 a kinase that phosphorylates and presumably plays a role in the regulation of the small heat shock protein Hsp25/HSP27. The function of Hsp25/HSP27 is unclear, but it is abundantly expressed in the heart, and its overexpression in cells confers resistance to heat shock. Hsp25/HSP27 is phosphorylated in response to inflammatory cytokines and other cellular stresses, and phosphorylation coincides with a number of stress responses, including growth arrest and inhibition of actin polymerization.
Inactivation of ERKs
ERK family members are inactivated by phosphatases. Since phosphorylation at the conserved Tyr and Thr residues activates the ERK family, it is not surprising that dephosphorylation in vitro by serine/threonine or tyrosine phosphatases inactivates them. In the cell, ERK-1 and ERK-2 activity appears to be regulated by MKP-1 or PAC1, dual-specificity phosphatases (dephosphorylating both Ser/Thr and Tyr residues) that are rapidly and transiently expressed in response to mitogenic stimuli. It is likely that the SAPKs and possibly p38 are regulated in a similar fashion by MKP-1 or related phosphatases, since overexpression of MKP-1 inhibits SAPK activity.19
Regulation of Transcription Factors
One clue to the role of the SAPKs in cardiovascular disease comes from the identification of one of their major substrates, c-Jun. c-Jun is the product of one of the immediate-early genes and is induced in response to a number of stresses, including ischemia and inflammatory cytokines. c-Jun is a member of the bZip (basic leucine zipper) family of transcription factors. c-Jun functions primarily as a heterodimer with c-Fos (or family members) or ATF-2 (a member of the CREB family). When complexed with c-Fos, the dimer is targeted to promoters, such as that of the collagenase gene, containing canonical AP-1 elements (TGAC/GTCA). When complexed with ATF-2, however, the dimer appears to prefer CRE sequences (TGACGTCA) and AP-1 variants such as that contained in the c-jun promoter (the jun2 TRE [TTACCTCA]), which controls induction of c-jun in response to a variety of stimuli.
The transcriptional activating activity of c-Jun is regulated at the posttranslational level by phosphorylation of c-Jun. c-Jun is phosphorylated at two residues within the amino-terminal transactivation domain, serines 63 and 73, in response to a variety of cellular stresses.20 Phosphorylation of these two residues is critical for the transcriptional activating activity of c-Jun, since mutation of them markedly decreases this activity. The SAPKs readily phosphorylate c-Jun at Ser 63/73 and do so at a rate 10 times faster than ERK-1 and -2.6 SAPKs avidly bind to a region adjacent to Ser 63/73 called the δ domain (residues 31 to 57), a region deleted from the oncogene v-jun (to which the SAPKs cannot bind). The SAPKs are the predominant c-Jun kinases activated by many types of cellular stress. For example, after TNF-α activation, at least 70% of c-Jun N-terminal kinase activity is accounted for by the SAPKs.2 This figure is derived from experiments in which the SAPKs are immunodepleted from cellular extracts and then remaining c-Jun kinase activity is assayed.
ATF-2, unlike the related CREB, is not activated by agents that increase cAMP. ATF-2 is activated by adenoviral E1a protein–induced transformation and by stimuli that activate the SAPKs. ATF-2 is regulated by the SAPKs and probably p38,21 and the mechanism of regulation of transcriptional activating activity appears to be remarkably similar to that of c-Jun. ATF-2, like c-Jun, contains an N-terminal transcriptional activation domain, and the SAPKs and p38 phosphorylate two residues within this domain (Thr 69 and 71), which are critical for transcriptional activating activity. ATF-2 also contains a C-terminal DNA binding domain, and phosphorylation of one or more residues near this domain, also readily catalyzed by the SAPKs, enhances DNA binding activity.22 23 ATF-2 can dimerize not only with c-Jun but also with itself and some other members of the ATF family, including ATF-3, CREB, and the closely related ATFa, and with NF-κB. These interactions greatly expand the list of genes that may be regulated by ATF-2 and, in turn, the SAPKs and p38.
Recent evidence suggests that Elk-1, a TCF, may also be a substrate of the SAPKs.24 Elk-1, together with serum response factor, controls transcription from the serum response element. The serum response element mediates the expression of many immediate-early genes, including c-fos and Egr-1. SAPKs appear to enhance DNA binding activity of Elk-1, formation of the ternary complex, and transcriptional activating activity of Elk-1. Since Elk-1 is also activated by the ERKs, it appears that the mitogen-activated ERK cascade and the stress-activated SAPK cascade converge at Elk-1, accounting in part for the observed induction of c-fos by a host of widely divergent stimuli.
Examples of genes relevant to cardiovascular disease that have promoters with sites either known or suspected to bind the SAPK- or p38-regulated transcription factors include c-jun, the adhesion molecule, E-selectin, c-fos, MMPs, and possibly inducible NO synthase, IL-8, and proliferating cell nuclear antigen. The mechanisms of induction of specific genes will be discussed below in the context of disease states characterized by their expression.
Role of Stress-Activated Kinases in Specific Disease States
Despite the fact that it is only recently that recombinant SAPKs and p38 and antibodies to them have become available, a great deal has been learned about their potential roles in various disease processes. The sine qua non of demonstrating that one or more pathways are involved in a cellular response to a stimulus is demonstrating activation of the kinases by the stimulus. This usually involves immunoprecipitation of the kinase with specific antibodies from cells before and after the stimulus. Kinase activity in the immunoprecipitate is then quantified by measuring phosphorylation of an added substrate after addition of [γ-32P]ATP. This approach allows one to conclude that the kinase of interest either is or is not activated in a certain situation but does not allow one to determine the precise role the kinase plays in the physiological response. Using homologous recombination to “knock out” a particular gene in order to study its physiological role may have little utility for studying the SAPKs, since there are multiple genes that in all likelihood have overlapping functions. Furthermore, there may be significant overlap of function by the SAPKs and p38, since both are activated by similar stimuli and they share common substrates (eg, ATF-2). Determination of the physiological role played by these kinases may have to await the creation of transgenic animals expressing, for example, a dominant inhibitory mutant of one component of the cascade that suppresses signal transduction down the stress-activated kinase pathway. In addition, the use of pharmacological inhibitors of individual components of stress-activated kinase pathways holds great promise for dissecting out specific functions of those components. Short of that, much can be learned by examining those cardiovascular disease states that are clearly associated with activation of the SAPKs or p38 or that may be associated with enhanced production of mediators, such as TNF or IL-1, for which activation of the kinases is an important component of the cell’s response.
The response to the extreme stress of reperfusion of ischemic tissue is one of the clearest examples of a potentially vitally important role for the SAPKs in cardiovascular disease. We have found that ischemia alone does not activate the SAPKs, but after reperfusion for as little as 5 minutes after 40 minutes of unilateral renal ischemia, there is a marked increase in SAPK activity, which is sustained for at least 90 minutes.25 Similarly, partial restoration of cellular ATP stores in MDCK or LLC-PK1 renal tubular epithelial cells after chemical anoxia induced by cyanide and 2-deoxyglucose is associated with marked activation of the SAPKs.25 Clearly this is not a generalized or nonspecific activation of protein kinases, since even the closely related ERK-1 and ERK-2 are only minimally activated by either reperfusion or reversible ATP depletion.
The SAPKs account for the majority of c-Jun transactivation domain (Ser 63/73) kinase activity after reperfusion,25 suggesting that they trigger part of the kidney’s very early genetic response to ischemia by enhancing the transcriptional activating activity of c-Jun. Since induction of c-jun is autoregulated by c-Jun, it is likely that activation of the SAPKs is, at least in part, responsible for the induction of c-jun following myocardial or renal ischemia.
After ischemia, the role of SAPKs in the control of gene expression extends well beyond the regulation of c-Jun. First, the SAPKs are the predominant ATF-2 C-terminal (DNA binding and dimerization domain) kinases activated by reperfusion.23 This enhances the DNA binding activity of ATF-2. After ischemia and reperfusion, ATF-2 and c-Jun are targeted as a heterodimer to both ATF/CRE motifs and the jun2 TRE from the c-jun promoter. The SAPKs are also the predominant ATF-2 transactivation domain kinase after reperfusion.23 These data, taken together, suggest that after the reperfusion of ischemic tissue, the SAPKs target ATF-2/c-Jun dimers to various promoters, including the c-jun promoter, and also enhance transcriptional activating activity of both components of the c-Jun/ATF-2 dimer. This would provide a potent mechanism for the induction of a large number of genes regulated by promoters containing ATF/CRE sites or AP-1 variants to which the heterodimer binds (Fig 2⇓).
The role of p38 in the response to ischemia is less well defined but probably no less important than that of the SAPKs. We have found that p38 is not an important ATF-2 kinase after reperfusion of the ischemic kidney or reversible ATP depletion in renal tubular epithelial cells, since virtually all of the ATF-2 kinase activity coelutes with the SAPKs on Mono Q (Pharmacia LKB) anion exchange chromatography.23 However, p38 appears to play a critical role in inflammatory cytokine production and release (see below), which occurs in postischemic tissue and is believed to enhance postischemic injury, in part by triggering the expression of adhesion molecules.26 27
It is not clear where ischemia (or the metabolic inhibitors) “feed in” to the SAPK cascade (ie, what protein in the cascade is activated first). For example, anisomycin-induced activation of the SAPKs, as opposed to IL-1, is not prevented by dominant inhibitory mutants of Rac, suggesting that anisomycin may activate a more distal component. It is also not clear precisely what ischemia-induced signal activates the cascade component. Clearly, partial repletion of intracellular ATP stores is necessary for activation, but it remains to be demonstrated that this alone is sufficient. Oxidant stress may be critical to activation, since N-acetylcysteine, which is converted to the free radical scavenger GSH in the cell, prevents induction of c-jun and activation of the SAPKs in response to UV irradiation of cells28 and in response to reperfusion of the ischemic kidney (R. Safirstein, personal communication, 1995).
Cells that are exposed to an ischemic insult may die if the insult is severe enough; if the insult is less severe and the cells are not terminally differentiated, the cells may dedifferentiate and enter the cell cycle to replace irreversibly injured cells. In some cases, postischemic cells may undergo programmed cell death or apoptosis. In theory, the SAPKs and p38 could play a role in any of these processes. However, since activation of this pathway by expression of a constitutively active MEKK-1, the upstream activator of SEK-1 and SAPK (Fig 1⇑), is growth inhibitory,17 it is unlikely that the SAPKs or p38 play a direct role in triggering the entry of the cells into the cell cycle and much more likely that they modulate growth arrest and, in susceptible cells, apoptosis.1 29 Finally, all cells, including cardiac myocytes, which are terminally differentiated, activate adaptive responses to stress, which are designed to help the cell survive future insults. The heat shock response is the clearest example of an adaptive response that is modulated in part by a stress-activated kinase, p38. The role of stress-activated kinases in other adaptive responses, including the phenomenon of ischemic preconditioning, is currently under investigation.
The inflammatory cytokines IL-1β and TNFα, acting upon macrophages and vascular smooth muscle cells within the atherosclerotic plaque, are believed to play critical roles in both the progression of the plaque and its susceptibility to rupture. When monocytes or macrophages are activated, they secrete IL-1β and TNF-α, which, in turn, activate adjacent macrophages. A novel class of anti-inflammatory agents, pyridinyl-imadazole compounds, have been found to inhibit cytokine production in response to inflammatory stimuli and were termed CSAIDs. The CSAIDs block IL-1β and TNF-α production at the translational level.30 Recently, the intracellular targets of these potentially extremely important agents were identified as two isoforms of p38.30 The CSAIDs bind directly to p38 and inhibit its kinase activity. These studies define a critical role for p38 in the production of inflammatory cytokines and indicate that p38 stimulates translation of cytokine mRNA. In the inflammatory response, TNF-α and IL-1β activate p38, which then triggers production of more TNF-α and IL-1β. This autoamplification of the inflammatory response is, in part, responsible for the syndrome of septic shock but probably also plays an important role in the low-grade inflammation characteristic of the atherosclerotic plaque.
The stress-activated kinases also play an integral role in regulation of gene expression in response to inflammatory cytokines. Induction of MMPs, including collagenases, stromelysin, and gelatinases, is one of the hallmarks of the response to inflammatory cytokines. MMPs are involved in the turnover of the extracellular matrix and appear to play an important role in the breakdown of collagen in the fibrous caps of atherosclerotic plaques. This process may lead to weakening of the fibrous cap and eventual plaque rupture. The promoter of the gene encoding the MMP, collagenase, was one of the first identified that was regulated by AP-1.31 Regulation of transcription from AP-1 sites is complex,31 but typically, a c-Jun/c-Fos dimer controls transcription. The SAPKs can be expected to play a role in the induction of collagenase by increasing the transcriptional activating activity of c-Jun, acting at the collagenase promoter. c-Jun also acts at its own promoter to increase transcription of c-jun. The resulting increase in the amount of c-Jun will further increase cellular AP-1 activity. Finally, SAPK-induced activation of Elk-1 will increase the amount of c-Fos available to bind at the collagenase promoter (Fig 2⇑).
As described previously, SAPKs phosphorylate the DNA binding/dimerization domain of ATF-2, which increases ATF-2 DNA binding activity. In addition to the jun2 TRE of the c-jun promoter, to which ATF-2 binds as a dimer with c-Jun,23 31 two target control regions for ATF-2 have been identified. These are the virus-inducible enhancer of the human interferon beta gene and the promoter of the gene encoding the adhesion molecule E-selectin (ELAM-1).32 E-selectin expression is upregulated on endothelial cells in response to IL-1β, TNF-α, and lipopolysaccharide, potent activators of the SAPKs and p38. Induction of the E-selectin gene by cytokines is regulated in part by a promoter element (NF-ELAM1 [TGACATCA]), which is a variant of a CRE/ATF element (TGACGTCA). After TNF-α, there is a change in the composition of the transcription factors binding at NF-ELAM1 from ATF-2 homodimers to ATF-2/c-Jun heterodimers.32 Although a direct link between stress-activated kinases and E-selectin expression has not been demonstrated, it is likely that such expression is regulated, at least in part, by phosphorylation of ATF-2 and c-Jun by these kinases and subsequent enhanced transcriptional activating activity and possibly by a change in the DNA binding activity of the ATF-2/c-Jun dimer similar to that seen after ischemia (Fig 2⇑). Demonstration of a role for the stress-activated kinases in the expression of adhesion molecules could implicate them not only in the inflammatory response but also in the initiation and progression of the atherosclerotic plaque and in postischemic injury in the heart and kidney.
Components of the kinase cascades other than the stress-activated kinases themselves may also modulate the inflammatory response. Pak1, the Rac- and Cdc42-regulated kinase that activates both the p38 and SAPK cascades (Fig 1⇑), may play a direct role in the neutrophil oxidative burst in response to chemoattractants by phosphorylating p47phox, which regulates NADPH oxidase.33
TNF-α has recently been postulated to play a role in the progression of heart failure. Levels of TNF-α rise in advanced stages of heart failure, and increasing evidence suggests that this may not be an epiphenomenon. As noted, TNF-α production in response to inflammatory stimuli is blocked by the CSAIDs,30 raising the possibility that p38 is involved in the rise in TNF-α levels and the progression of heart failure. The role played by TNF-α in heart failure is not clear, but one intriguing effect of TNF-α is the induction of programmed cell death or apoptosis in some types of susceptible cells. Apoptosis is an active suicidal response of the cell to a stimulus (or withdrawal of a stimulus) and is regulated by induction of a specific set of genes. It is characterized by blebbing of the plasma membrane, condensation of the nucleus, and endonucleolytic cleavage of DNA.34 TNF-α potently activates the SAPKs and p38, fueling speculation that these kinases may transduce a component of the apoptotic signal to the nucleus of myocytes as has recently been demonstrated in PC-12 pheochromocytoma cells.29 Withdrawal of nerve growth factor from PC-12 cells markedly activated the SAPKs and p38 and induced apoptosis. Expression of dominant-negative MKK3, the kinase immediately upstream from p38 (Fig 1⇑) inhibited the apoptotic response. Apoptosis in response to withdrawal of nerve growth factor was also prevented by the expression of constitutively active MEK-1 (Fig 1⇑), which activates ERK-1/-2, indicating that the balance between SAPK/p38 activity and ERK-1/-2 activity may determine whether or not a cell will activate the programmed cell death response after a cellular stress.29
MAP kinase pathways in yeast evolved to help the organism respond to physical or nutritional stresses.11 For example, HOG-1, the yeast homologue of p38, allows the yeast to proliferate despite conditions of high osmolarity by producing glycerol, which increases internal osmolarity. Another MAP kinase, Mpk1, which is downstream from yeast protein kinase C, allows the yeast to maintain cell wall integrity at high temperatures and low osmolarity.11 Not surprisingly, mammalian stress-activated kinases also respond to the physical stresses of osmolar stress and heat shock.2 4 7 8 In yeast, activation of the HOG-1 cascade is controlled by osmosensors, but in mammalian cells, the trigger is not known. It is not clear whether an osmosensor controls activation when mammalian cells are exposed to high or low osmolarity (both of which activate the SAPKs and p38) or, as suspected for Mpk1, a mechanosensor is also operative. If a mechanosensor is involved in kinase activation, a similar mechanism may play a role in the complex genetic response of cells exposed to mechanical stretch and to shear stress.
The release of growth factors is thought to play an important role in many disease processes, including the hypertrophic response, progression of atherosclerotic plaques, and restenosis following balloon injury. Many of the intracellular signals generated in response to growth factors with receptors having intrinsic tyrosine kinase activity (eg, platelet-derived growth factor, epidermal growth factor, and colony stimulating factor-1) are triggered by the activation of Ras.1 Most important for this discussion, Ras controls activation of the c-Raf-1 protein kinase cascade, culminating in the activation of the Erk family members, ERK-1 and ERK-2 (Fig 1⇑). In most cells, growth factors only weakly and inconsistently activate the SAPKs, and the activation appears to proceed via Ras, since a dominant inhibitory mutant of Ras (which avidly binds GDP and sequesters the guanine nucleotide exchange factor necessary for growth factor–induced activation of Ras) blocks SAPK activation.31 In contrast, the marked activation of the SAPKs by cytokines is Ras independent and, as noted above, may proceed via Rac and/or Cdc42Hs.14 15
It is not clear how Ras activates the SAPK cascade, but Ras is capable of activating MEKK-1 (Fig 1⇑), suggesting a mechanism.31 Since activation of the SAPK cascade appears to be growth inhibitory,17 SAPK activation by growth factors could provide a negative modulatory signal to limit the mitogenic response.
Activation by G Protein–Coupled Receptors
Seven transmembrane-spanning receptors linked to all subclasses of heterotrimeric G proteins except Gs have recently been shown to activate the SAPK pathway. Persistent activation of the m1 muscarinic receptor with carbachol activates the SAPKs and leads to induction of c-jun.35 Constitutively active forms of α12 and α13, G proteins with less well-defined signaling pathways, also activate the SAPK cascade.36 Thrombin activates p38 in platelets, and the kinase may then activate cytosolic phospholipase A2 by phosphorylating Ser 505, the same site phosphorylated by ERK-1/-2 in response to growth factors.37
It is not clear how agonists with heterotrimeric G protein–linked receptors activate the SAPKs. The chemotactic peptide, fMLP, which has a receptor linked to a pertussis toxin–sensitive G protein, activates Pak1 via Rac.33 These data raise the possibility that Rac and Pak1 may mediate activation of the SAPKs and p38 not only in response to inflammatory cytokines but also by some agonists with G protein–linked receptors.
Angiotensin II, which is linked via its receptor to Gq, markedly activates the SAPKs.38 Activation of the SAPKs is Ca2+ dependent, but protein kinase C independent, and appears to require activation of a novel 115- to 120-kD tyrosine kinase.38 It remains to be determined what role SAPK activation plays in the response of the cell to angiotensin II. The ERK-1/-2 cascade and, more recently, p70 ribosomal S6 kinase have been shown to play important roles in the angiotensin II–induced hypertrophic adaptation of myocytes (Reference 3939 and references therein). Given the marked activation of the SAPKs and the induction of c-jun after angiotensin II, it is likely that the SAPKs also modulate aspects of the hypertrophic response.
In a remarkably short period of time, skeletons of the signal transduction cascades of the stress-activated kinases, the SAPKs and p38, have been assembled. We now understand the general types of stimuli that activate these kinases and, in some cases, how the stimuli accomplish activation of the cascade. Great progress has also been made in identifying some of the transcription factors regulated by these kinases and how their transcriptional activating activity is modulated. The major challenge now is twofold: (1) at the basic science level, to define the role of the kinases in the complex genetic response to various cellular stresses by expanding the list of transcription factors regulated by these kinases and identifying pathophysiologically relevant genes that are regulated by those transcription factors and (2) at the integrated physiology level, to define the consequences of activation of the stress-activated kinases in various pathophysiological states. Accomplishing these two goals will lay the groundwork for designing rational therapies directed at modulating the activity of this critically important class of protein kinases.
Selected Abbreviations and Acronyms
|ATF-2||=||activating transcription factor-2|
|CREB||=||cAMP response element binding protein|
|CSAID||=||cytokine-suppressive antiinflammatory drug|
|ELAM||=||endothelial leukocyte adhesion molecule|
|ERK||=||extracellular signal–regulated kinase|
|GCK||=||germinal center kinase|
|HOG||=||high osmolarity glycerol response|
|MAPKAP||=||MAP kinase–activated protein kinase|
|MEK, MKK||=||MAPK/ERK kinase|
|MKP||=||MAP kinase phosphatase|
|SAPK||=||stress-activated protein kinase|
|Ste7, Ste11, Ste20||=||sterile 7, 11, and 20|
|TCF||=||ternary complex factor|
|TNF||=||tumor necrosis factor|
|TRE||=||tetradecanoyl phorbol ester response element|
This study was supported by a Grant-in-Aid from the American Heart Association, a grant from the National Kidney Foundation of Massachusetts and Rhode Island, the James A. Shannon Director’s Award from the National Institute of Diabetes and Digestive and Kidney Diseases to Dr Force, and US Public Health Service grants DK-39773, NS-10828, and DK-38452 to Dr Bonventre and grants DK-41513 and GM-46577 to Dr Kyriakis. We apologize to investigators whose work could not be cited because of space constraints.
- Received November 14, 1995.
- Accepted January 4, 1996.
- © 1996 American Heart Association, Inc.
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