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Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast
Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast Andrew Jan Waskiewicz Fred Hutchinson
and Jonathan A Cooper
Cancer Research Center, Seattle, USA and University
of Washington,
Seattle, USA
Evolutionarily conserved from yeast to man, mitogen-activated protein kinase (MAPK) pathways respond to a variety of disparate signals which induce differentiation, proliferation, or changes in intracellular enzyme regulation. Recent advances have identified two new mammalian MAPK relatives, JNKl and ~38, and the pathways which are responsible for their activation.
Current Opinion in Cell Biology 1995, 7:798-805 Introduction cells rely on both internal and external signals to determine when and whether to divide. In vertebrates, the actions of many growth stimulators converge at the cellular kinase cascade that activates ERKl and ERK2 (extracellular signal regulated kinases 1 and 2). These are mitogen-activated protein kinases (MAP&) of 44kDa and 42 kDa respectively, which upon activation translocate to the nucleus and activate immediate early gene transcription [l]. Recent advances have demonstrated what yeast researchers have known for a long time: that the mammalian MAPK cascade is far horn unique. JNKl (Jun amino-terminal kinase 1) and ~38, two MAPK relatives, are activated independently of ERKs, and have slightly different substrate specificities [2,3*,4*]. These cascades, in combination with other signaling pathways, can differentially alter the phosphorylation status of the transcription machinery such that it reflects the particular stimulus on the outside of the cell. Each pathway is downregulated by a variety of phosphatases, which also ensure a low basal kinase activity in unstimulated cells. In this review, we will examine the specificity of activation of both the kinases and the phosphatases which have been implicated in MAPK signal transduction. Eukaryotic
Mammalian MAPK
signaling
cascade
Most stimuli which membrane anchored into its GTP-bound to the seryl/threonyl membrane-anchoring
activate ERKs convert plasma Ras &om its GDP-bound state state. Ras-GTP binds directly kinase Raf, forming a transient signal [5]. Localization of Raf to
the membrane by addition of a constitutive membrane localization tag or by binding to Ras in vitro is not sunicient for full activation of Raf [6*,7*]. For example, membrane-localized Raf is activated &IO-fold by epidermal growth Victor (EGF) in a Ras-dependent manner. This implies the existence of a second molecule which converts Raf into its fully active state. Active Raf can phosphorylate the dual-specificity kinase MEK (MAPK or ERK kinase) on Ser218 and Ser222 [8-l 11, allowing MEK to phosphorylate its only known substrates, ERKs 1 and 2. MEK mutants which mimic the effect of phosphorylation of residues 218 and ‘222 (by the substitution of serines with either aspartic acids or glutamic acids) have constitutive activity in vitro and are oncogenic in vivo [12*,13’]. Once phosphorylated on Thr183 and Tyr185 (known as the TEY phosphorylation site) [14,15], ERKsl and 2 phosphorylate numerous cytoplasmic proteins, including another kinase (pp90 ribosomal S6 kinase [Rsk]), cytosolic Phospholipase A2, and the tail of the EGF receptor [16-191. In addition, ERKs translocate to the nucleus, where they can phosphorylate Elk-l (see Fig. 1) (a TCF, or ternary complex factor) on five residues near its carboxyl terminus [20,21]. Elk-l and SRF (serum response factor) form a ternary complex which binds the serum response element in the c-Fos promoter. Phosphorylation of Elk-l leads to increased transcription of c-Fos mRNA, presumably by increasing the transcriptional activation strength of the TCF/SRF complex [22].
Stress response
In response to extracellular stresses, including cycloheximide treatment and ultraviolet radiation (UV), the
Abbreviations CREB-cyclic AMP response element binding protein; EGF-epidermal growth factor; ERK-extracellular signal regulated kinase; JNK-Jun amino-terminal kinase; MAP-mitogen-activated protein; MAPK-MAP kinase; MAPKAPK-MAPK-activated protein kinase; MKP-MAPK phosphatase; NCF-nerve growth factor; PAC-phosphatase of activated cells; Rsk-pp90 ribosomal 5.6 kinase; SAPK-stress-activated protein kinase; SEK-SAPK or ERK kinase; SRF-serum response factor; TCF-ternary complex factor.
798
0 Current
Biology Ltd ISSN 0955-0674
Mitogen and stress response pathways Waskiewicz
and Cooper
Fig. 1. Extracellular stimuli, represented here as mitogen or stress, result in the intracellular activation of MAPK-type cascades. Ras/Raf/MEK/ERK activation (+) ultimately results in a pool of ERKl/Z translocating (dotted path) to the nucleus (Rsk is phosphorylated by ERKs 1 and 2). Similarly, Racl/PAKl/Znd signal/MEKKl/SEKl/JNKl and X/MKK3/p38 activation are likely to result in translocation of the MAPK homologs JNKl and p38 (MAPKAPK-2 is phosphorylated by ~38). Nucleus-localized ERK/JNK/p38 have been shown to phosphorylate and activate the SRF/Elk-1 and JUN/ATF-2 transcription complexes. These cascades are inhibited (4 by the phosphatases PPZA and MKP-1 (black).
transcription factor Jun becomes phosphorylated on Ser63 and Ser73 [23]. A family of MAPK relatives known as JNKs (also known as SAPKs or stress-activated protein kinases) phosphorylate the activation domain of Jun. Between the JNKs and the stress-related stimulus lies another kinase cascade, in many ways very similar to the MAPK cascade (see Fig. 1). JNKs are doubly phosphorylated in a TPY site by a dual-specificity kinase, SEKl (SAPK or ERK kinase 1, also known as MKK4 and JNKK). This protein shares 45% identity with the ERK activator, MEK [24,25*,26*]. SEKl is activated
by MEKKl, which is the product of a gene that was originally cloned on the basis of its similarity to the yeast mating pathway kinase Stell [27]. Although MEKKl was first thought to encode an activator of MEK, inducible expression of MEKKl in NIH3T3 cells activates JNKl activity at least fourfold, whereas ERK2 activity remains mostly unchanged [28*]. Thus MEKKl would not appear to activate MEK (see Fig. 1). How is MEKKl cascade, MEKKl
regulated? By analogy with the MAPK should be regulated by a low molecular
800
Cell multiplication
weight GTP-binding protein comparable to Ras. It now seems clear that JNK activation is downstream of the activation of Racl and CddHs, which are two small G-proteins. Expressing activated versions of Racl or Cdc42Hs mutants in COS-7 cells is sufficient to increase JNKl activity five- to tenfold without increasing MAPK activity [29**,30”]. Similarly, expression of Dbl or Ost, which are proteins that function in vitro as exchange proteins for RhoA and Cdc42Hs, results in increased JNKl activity [29**]. Membrane localized Ras exchange protein (SOS) stimulates ERKs 1 and 2, but does not affect JNKl activity. Thus, JNKs are likely to be activated by a pathway of kinases which are downstream of the. Rho-like GTPases Cdc42Hs and Racl. If this is true, stress stimuli, such as treatment with protein synthesis inhibitors and W, should increase the levels of GTP bound to Cdc42Hs and Racl within the cell. The p65P*Kl protein has been identified as a kinase which interacts only with the GTP-bound forms of Racl and Cdc42Hs [31-331. Sequence analysis has shown that the nearest relatives of p65P*Kt are the budding yeast pheromone signaling kinase Ste20 and its fission yeast homolog, Shkl. Ste20 and Shkl interact with yeast Cdc42Hs [34]. By analogy with yeast, where Ste20 is thought to be an activator of Stell (a MEKKl homolog) [35], p65P*Kl may encode the activator of MEKKl. Alternatively, Racl and Cdc42Hs could activate MEKKl directly or via another kinase. Also activated by stress (W or increased extracellular osmolarity), but differing in substrate specificity, is another MAPK relative, known as ~38, RK (reactivating kinase) or MPK2. Th’is is a vertebrate homolog of the yeast HOG? gene [2,36,37]. Like its homologs, ~38 is activated by phosphorylation on both threonine and tyrosine, this time in a TGY site. Two p38 activators have been characterized: SEKl (the activator of JNKl) and MKK3, a protein which is apparently specific for ~38 [24,25*,26’] (Fig. I). Overexpression of MKK3 or SEKl results in increased p38 activity. Cotransfection of both MEKKl and SEKl results in raising the activity ofJNK1 to above the levels obtained by expressing either MEKKl or SEKl, but does not potentiate ~38 activity [25*,26*]. Thus, although it is likely to be upstream of JNKl and SEKl, MEKKl may not be the physiological upstream activator of ~38. Also, in these experiments MEKKl should have resulted in increased SEKl activity SEKl may activate ~38 only when overexpressed. Perhaps an independent pathway upstream of MKK3 results in ~38 activation in viva. The known downstream targets of ~38 are a seryl/threonyl kinase, MAPK-activated protein kinase (MAPKAPK) 2, and the transcription factor ATF-2 [36-381.
Orphan pathways
It seems unlikely that ERK1/2, JNKl, and ~38 are the only MAPK-related cascades in mammals (Fig. 2). Rhodependent activation of SRF activity is independent of JNK, ERK, or ~38 activation. Thus, by analogy with
other small G proteins, Rho may activate a novel cascade [39]. Similarly, Ras-dependent-phosphorylation of Fos Thr232 is catalyzed by a novel 88 kDa proline-directed kinase, Fos-regulating kinase [40]. Also, nerve growth factor (NGF)-induced phosphorylation of CREB (cyclic AMP response element binding protein) at Ser133 is distinct fi-om that by known MAPK relatives and the known CREB kinase, Rsk [41]. Finally, using the polymerase chain reaction and the two-hybrid system, Zhou et al. [42] have isolated MEK5 and ERK5, which function on an unknown pathway. These and other undiscovered pathways will add to our mechanistic understanding of signal-mediated alterations in transcription.
Specificity of signal propagation
When mammalian cells are treated with mitogenic agents (growth factors or phorbol esters), ERKs become strongly activated but JNKl is poorly activated. Conversely, when cells are challenged with stress (protein synthesis inhibitors or W), JNKl is potently activated and ERK activity is only weakly increased. It would seem from this information that ERK1/2 and JNKl activation are biologically distinct. Yet ~38, JNKl, and ERK1/2 are all proline-directed kinases, sharing numerous in vitro substrates. For example, ATF-2 is phosphorylated by all three kinases [38,43]. Also, Jun and Elk-l are known to be phosphorylated by both ERK2 and JNKl [23,44,45]. Yet these pathways must result in unique transcriptional activity, because stress, mitogens, and differentiation factors should not elicit identical changes in transcription machinery. As a partial explanation of in viva specificity, many members of these kinase cascades have been shown to exist as members of multiprotein complexes. Indeed, JNKl binds tightly to its substrates Jun and ATF-2; also, ERK2 co-immunoprecipitates with both Elk-l and Rsk [4*,38,46,47]. It seems likely that these protein-protein interactions are crucial in generating distinct outputs.
Downstream kinases
Some of the specificity of these pathways is also contributed by the downstream kinases which each MAPK homolog activates. ERKs 1 and 2 phosphorylate and activate the seryl/threonyl kinase Rsk, which translocates to the nucleus where it phosphorylates numerous transcription factors, including CREB, Fos, and SRF [41,48,49]. Similarly, ~38 activates MAPKAPK-2, which phosphorylates heat shock protein 25/27 [36,50]. As the substrate specificities of Rsk and MAPKAPK-2 are somewhat different, the combined outputs of ERK/Rsk and p38/MAPKAPK-2 generate an even greater level of distinction between the pathways. Rsk and MAPKAPK-2 are not the only kinases that are activated by MAPK-type signaling, so some of the complexity is yet to be discovered (AJ Waskiewicz, unpublished data).
Mitogen
Yeast signaling
addition, Fus3 phosphorylates of Cdc28/cyclin [51].
From the above descriptions of mammalian signaling, it is clear that many members of these pathways have homologs in the budding yeast, Sartharomyces cerevisiae. In fact the genetic dissection of fundamental cellular processes including mating, adaptation to nitrogen starvation and changes in external osmolarity, polarized growth, and sporulation - has revealed the existence of at least six independent MAPK-related cascades in yeast [35]. For example, in response to mating pheromone the kinases Ste20, Stell, Ste7, Fus3 and Kssl are activated. Each of these kinases represents a prototype for a signaling molecule in a MAPK cascade. Although our discussion has started with mammalian pathways, the yeast kinases were cloned first. As the genes encoding the mammalian MAPK cascade enzymes were cloned, sequence analysis and functional complementation revealed the amazing evolutionary conservation between yeasts and higher eukaryotes (see Fig. 2). The biological similarities with mammalian signaling cascades are striking. The MAPK homologs Fus3 and Kssl phosphorylate Ste12, a transcription factor which induces transcription of mating-specific genes [51]. In
Racl
Farl,
and Cooper
a negative
regulator
Additional pathways regulate processes involving nitrogen starvation of diploid cells (pseudohyphal growth) (Ste20+Stell+Ste7) [52], haploid invasive growth (Ste20+Stel l+Ste7) [53], increases in extracellular osmolarity (Ssk2/22+Pbs2+Hogl), polarized cell growth (Bckl-+Mkkl/2+Mpkl), and sporulation (Spsl+ Smkl) [35]. Each of the proteins listed in parentheses is encoded by a homolog of either p65P*Kl, MEKK, MEK, or MAPK. For a precise listing of these similarities and members of MAPK pathways in fission yeast, Schizosaccharomyces pombe, see Figure 2. By expressing mammalian MAPK cascade enzymes at high levels in yeast, researchers are able to functionally replace individual yeast kinases. Although these experiments have confirmed that these kinases function in a certain tier, they cannot predict whether a kinase will be stress activated or growth activated. ERK2 will complement a deletion of the yeast MPKl gene [54,55]. Similarly, JNKl will complement a deletion in yeast HOG1 [56]. SEKl will complement a deletion in PBS2, which encodes the activator of Hog1 [24]. In
S. cerevisiae
S. pombe
Vertebrate
Ras
and stress response pathways Waskiewicz
Ras
PAKI
Ste20
Ste20
Pkcl
Stell
Stell
Bckl
Ste7
Ste7
Spsl
? # Raf
MEKKl
MEK
SEKI
MKK3
MEK5
Byrl
ERK1/2
INK1
~38
ERK5
Spkl
Bvr2
MKP-1 PPZA > 1995 Current Opinm
Wisl
Fus3/Kssl
Ptcl,2,3
Msg5
Ssk2l22
Mkkl/MkkZ
Pbs2
Mpkl
Hog1
Smkl
ptc1,3 Ptp2
rn Cell Biology
Fig. 2. A diagram demonstrating the evolutionary conservation among the MAPK signal transduction pathways. With the exception of Raf, members of each tier display considerable amino acid similarity. As many of these cascades have been discovered independently there are more names than we have shown. Bars indicate inhibition of the cascade. A partial list of synonyms: MEK = MKK, ERKl = MAPK = p44MAPx, ERK2 = MAPK = Mpkl (Xenopus) = Xp42 = p42 MAPK, SEK = MKK4 =JNKK, JNK = SAPK = p54M,+‘K, MKK3 = RKK, ~38 = Mpk2 = RK.
801
802
Cell multidication
contrast, XMEK2, thought to be the Xenopus homolog of SEKl, has been shown to functionally replace Mkkl/2 in the polarized cell growth pathway, but cannot replace Pbs2 as might have been expected [57]. Finally, MEKKl will complement a deletion in BCKl, the gene encoding an activator of Mkkl/2 [58]. Thus, the yeast complementarity patterns of JNKl, XMEK2, and MEKKl would suggest they operate on different, rather than the same, pathways. Similarly, MEKKl and ERK should fimction on the same instead of distinct pathways. Perhaps protein-protein interactions that limit specificity have not been conserved throughout evolution.
Checkpoint
regulation
Most MAPK pathways seem to be designed to respond to external stimuli rather than to the time on the internal cell cycle clock. Injection of MAPK activators, however, such as Mos, Ras or thiophosphorylated MAPK into early embryonic cells causes mitotic arrest [59-611. It has now become evident that ERKs are physiologically activated at M phase if the mitotic spindle is damaged. This activation arrests the cell cycle [62**]. This ‘spindle assembly checkpoint’ ensures that incomplete spindles do not prematurely start to pull chromosomes to their respective poles; this action would probably cause non-disjunction of one or more chromosomes. Under these arrested conditions, ERK2 is activated. Moreover, blocking the activation of ERK2 prevents the checkpoint arrest [62**]. Thus MAPKs are activated as a response to microtubule depolymerization. They then function to arrest mitosis at metaphase, preventing non-disjunction.
Phosphatase control
of signaling
In contrast to kinases, which can be very selective in choosing substrates, phosphatases seem to possess a broader specificity. The tyrosyl phosphatase CD45, the dual-specificity MAPK phosphatase MKP-1 (also known as 3CH134 or CLlOO), and the seryl/threonyl phosphatase PP2A (protein phosphatase 2A) are capable of dephosphorylating and inactivating ERKs 1 and 2 in vitro [15,63,64]. Figuring out which of these phosphatases is responsible in vivo for maintaining low basal kinase activity and inactivating MAPK after a signal has ceased has proven quite difficult. MKP-1 was initially identied as a murine seryl/threonyl/ tyrosyl phosphatase which specifically dephosphorylates both regulatory threonyl and tyrosyl residues on ERKs 1 and 2 in vitro. There is good evidence that MKP-1, and
the related phosphatase PAC @hosphatase of activated cells)-1 can function iti vivo to dephosphorylate MAPK. Ectopic expression of a catalytically inactive mutant MKP-1 (in which Cys258 is replaced with serine) activates ERKl/2, presumably by ‘trapping’ these ERKs in the nucleus [63]. Overexpression of MKP-1 blocks Ras-dependent DNA replication [65*]. Also, in S. cerevisiae, a phosphotyrosyl phosphatase, Msg5, which is related to mammalian MKP-1, genetically opposes the function of Fus3 and has been shown to inactivate Fus3 in vitro [66]. Is MKP-1 the only phosphatase which dephosphorylates MAPKs? MKP-1 and its relatives are immediate early gene products and are strictly localized to the nucleus [64,67-69]. Thus the induction of MKP-l/PAC-1 in response to external stimuli may explain the slow dephosphorylation of nuclear ERKl/%. However, the action of MKP-1 cannot account for the rapid dephosphorylation of cytoplasmic ERKl/2 [70,71].
The other leading candidate for a MAPK phosphatase is PP2A, one member of a Emily of enzymes which can also dephosphorylate MEK. The carboxyl terminus of SV40 small tumor antigen binds to PP2A, inhibiting its ability to dephosphorylate its substrates [72,73]. Removal of this interactive domain blocks both small tumor antigen induced MAPK activation and cell proliferation [73]. Both cytoplasmic PP2A and MAPK have been shown to localize to microtubules [74,75]. Following growth factor stimulation, PP2A becomes tyrosyl phosphorylated (possibly by Src); this temporarily inhibits its ability to dephosphorylate substrates [76]. Temporally this coincides with MAPK activation. Thus it appears that PP2A is appropriately regulated and positioned to maintain low basal MAPK activity and to rapidly downregulate MAPK following activation.
In yeast several seryl/threonyl and tyrosyl phosphatases have been genetically shown to participate in MAPK signaling. Perhaps the most convincing results support the idea of roles for PP2C in regulating MAPK pathways in both budding and fission yeasts. In fission yeast, deletion of the three phosphatase 2C isoforms is lethal [77]; however, mutation of the MEK homolog Wisl suppresses this lethality [78]. This suggests that PP2C normally downregulates the Wisl cascade. In budding yeast, mutation of an inhibitor (SLN1) of the Hog1 MAPK pathway is lethal [79**]. This defect is suppressed by overexpression of either SLNl, PTP2 (which encodes a phosphotyrosine phosphatase) or the PP2C genes PTCl and PTC3 [79**]. This and other genetic data suggest that Ptp2 dephosphorylates Hog1 and that Ptcl and Ptc3 also inactivate the pathway. The exact biochemical function of the PP2Cs in yeast, and whether they inactivate MAPK or one of the upstream kinases, still remains to be elucidated. The role of mammalian phosphatase 2C in regulating MAPK signaling also needs to be examined.
Mitogen and stressresponsepathways Waskiewicz
and Cooper
Conclusions
type Raf, implying the existence of a second mitogen-regulated signal for Raf activation.
In this review, we have described the positive and negative regulation of MAPK signaling cascades in higher eukaryotes and yeast. Clearly, there is much still to be learned. For example, although ERK, JNK, and ~38 activation are apparently distinct, oncogenic forms of Ras have been shown to activate all three pathways. Whether this activation occurs via an autocrine/paracrine loop or is the result of Ras activating the Rho family of GTPases is still unclear. In addition, do the same phosphatases regulate the ERK, JNK, and p38 pathways? MKP-1 does not inactivate JNKl, implying the existence of a JNK-specific phosphatase [65*]. Another remaining mystery is the existence of gene families. ERKs, RSKs, JNKs, MEKs and MEKKs are all encoded by members of large multigene families, which often have very precise tissue specific expression. Whether there is any biochemical difference among the isoforms has remained largely unstudied. Finally, although the activity of phosphatases in vitro is now ofien correlated with a biological effect, a careful examination of colocalization between the MAPK homologs and their putative phosphatases would add greatly to the existing evidence. Although it may not seem helpful to end a review with a series of questions, in the MAPK signaling field it seems appropriate.
7. .
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26. .
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79. ..
AJ Waskiewicz and JA Cooper, Fred Hutchinson Cancer Research Center, A2-025, Weintraub Basic Sciences Building, 1100 Fairview Avenue, Seattle, WA 98109, USA. AJ Waskiewicz E-mail: [email protected]
805
and Jonathan A Cooper
Cancer Research Center, Seattle, USA and University
of Washington,
Seattle, USA
Evolutionarily conserved from yeast to man, mitogen-activated protein kinase (MAPK) pathways respond to a variety of disparate signals which induce differentiation, proliferation, or changes in intracellular enzyme regulation. Recent advances have identified two new mammalian MAPK relatives, JNKl and ~38, and the pathways which are responsible for their activation.
Current Opinion in Cell Biology 1995, 7:798-805 Introduction cells rely on both internal and external signals to determine when and whether to divide. In vertebrates, the actions of many growth stimulators converge at the cellular kinase cascade that activates ERKl and ERK2 (extracellular signal regulated kinases 1 and 2). These are mitogen-activated protein kinases (MAP&) of 44kDa and 42 kDa respectively, which upon activation translocate to the nucleus and activate immediate early gene transcription [l]. Recent advances have demonstrated what yeast researchers have known for a long time: that the mammalian MAPK cascade is far horn unique. JNKl (Jun amino-terminal kinase 1) and ~38, two MAPK relatives, are activated independently of ERKs, and have slightly different substrate specificities [2,3*,4*]. These cascades, in combination with other signaling pathways, can differentially alter the phosphorylation status of the transcription machinery such that it reflects the particular stimulus on the outside of the cell. Each pathway is downregulated by a variety of phosphatases, which also ensure a low basal kinase activity in unstimulated cells. In this review, we will examine the specificity of activation of both the kinases and the phosphatases which have been implicated in MAPK signal transduction. Eukaryotic
Mammalian MAPK
signaling
cascade
Most stimuli which membrane anchored into its GTP-bound to the seryl/threonyl membrane-anchoring
activate ERKs convert plasma Ras &om its GDP-bound state state. Ras-GTP binds directly kinase Raf, forming a transient signal [5]. Localization of Raf to
the membrane by addition of a constitutive membrane localization tag or by binding to Ras in vitro is not sunicient for full activation of Raf [6*,7*]. For example, membrane-localized Raf is activated &IO-fold by epidermal growth Victor (EGF) in a Ras-dependent manner. This implies the existence of a second molecule which converts Raf into its fully active state. Active Raf can phosphorylate the dual-specificity kinase MEK (MAPK or ERK kinase) on Ser218 and Ser222 [8-l 11, allowing MEK to phosphorylate its only known substrates, ERKs 1 and 2. MEK mutants which mimic the effect of phosphorylation of residues 218 and ‘222 (by the substitution of serines with either aspartic acids or glutamic acids) have constitutive activity in vitro and are oncogenic in vivo [12*,13’]. Once phosphorylated on Thr183 and Tyr185 (known as the TEY phosphorylation site) [14,15], ERKsl and 2 phosphorylate numerous cytoplasmic proteins, including another kinase (pp90 ribosomal S6 kinase [Rsk]), cytosolic Phospholipase A2, and the tail of the EGF receptor [16-191. In addition, ERKs translocate to the nucleus, where they can phosphorylate Elk-l (see Fig. 1) (a TCF, or ternary complex factor) on five residues near its carboxyl terminus [20,21]. Elk-l and SRF (serum response factor) form a ternary complex which binds the serum response element in the c-Fos promoter. Phosphorylation of Elk-l leads to increased transcription of c-Fos mRNA, presumably by increasing the transcriptional activation strength of the TCF/SRF complex [22].
Stress response
In response to extracellular stresses, including cycloheximide treatment and ultraviolet radiation (UV), the
Abbreviations CREB-cyclic AMP response element binding protein; EGF-epidermal growth factor; ERK-extracellular signal regulated kinase; JNK-Jun amino-terminal kinase; MAP-mitogen-activated protein; MAPK-MAP kinase; MAPKAPK-MAPK-activated protein kinase; MKP-MAPK phosphatase; NCF-nerve growth factor; PAC-phosphatase of activated cells; Rsk-pp90 ribosomal 5.6 kinase; SAPK-stress-activated protein kinase; SEK-SAPK or ERK kinase; SRF-serum response factor; TCF-ternary complex factor.
798
0 Current
Biology Ltd ISSN 0955-0674
Mitogen and stress response pathways Waskiewicz
and Cooper
Fig. 1. Extracellular stimuli, represented here as mitogen or stress, result in the intracellular activation of MAPK-type cascades. Ras/Raf/MEK/ERK activation (+) ultimately results in a pool of ERKl/Z translocating (dotted path) to the nucleus (Rsk is phosphorylated by ERKs 1 and 2). Similarly, Racl/PAKl/Znd signal/MEKKl/SEKl/JNKl and X/MKK3/p38 activation are likely to result in translocation of the MAPK homologs JNKl and p38 (MAPKAPK-2 is phosphorylated by ~38). Nucleus-localized ERK/JNK/p38 have been shown to phosphorylate and activate the SRF/Elk-1 and JUN/ATF-2 transcription complexes. These cascades are inhibited (4 by the phosphatases PPZA and MKP-1 (black).
transcription factor Jun becomes phosphorylated on Ser63 and Ser73 [23]. A family of MAPK relatives known as JNKs (also known as SAPKs or stress-activated protein kinases) phosphorylate the activation domain of Jun. Between the JNKs and the stress-related stimulus lies another kinase cascade, in many ways very similar to the MAPK cascade (see Fig. 1). JNKs are doubly phosphorylated in a TPY site by a dual-specificity kinase, SEKl (SAPK or ERK kinase 1, also known as MKK4 and JNKK). This protein shares 45% identity with the ERK activator, MEK [24,25*,26*]. SEKl is activated
by MEKKl, which is the product of a gene that was originally cloned on the basis of its similarity to the yeast mating pathway kinase Stell [27]. Although MEKKl was first thought to encode an activator of MEK, inducible expression of MEKKl in NIH3T3 cells activates JNKl activity at least fourfold, whereas ERK2 activity remains mostly unchanged [28*]. Thus MEKKl would not appear to activate MEK (see Fig. 1). How is MEKKl cascade, MEKKl
regulated? By analogy with the MAPK should be regulated by a low molecular
800
Cell multiplication
weight GTP-binding protein comparable to Ras. It now seems clear that JNK activation is downstream of the activation of Racl and CddHs, which are two small G-proteins. Expressing activated versions of Racl or Cdc42Hs mutants in COS-7 cells is sufficient to increase JNKl activity five- to tenfold without increasing MAPK activity [29**,30”]. Similarly, expression of Dbl or Ost, which are proteins that function in vitro as exchange proteins for RhoA and Cdc42Hs, results in increased JNKl activity [29**]. Membrane localized Ras exchange protein (SOS) stimulates ERKs 1 and 2, but does not affect JNKl activity. Thus, JNKs are likely to be activated by a pathway of kinases which are downstream of the. Rho-like GTPases Cdc42Hs and Racl. If this is true, stress stimuli, such as treatment with protein synthesis inhibitors and W, should increase the levels of GTP bound to Cdc42Hs and Racl within the cell. The p65P*Kl protein has been identified as a kinase which interacts only with the GTP-bound forms of Racl and Cdc42Hs [31-331. Sequence analysis has shown that the nearest relatives of p65P*Kt are the budding yeast pheromone signaling kinase Ste20 and its fission yeast homolog, Shkl. Ste20 and Shkl interact with yeast Cdc42Hs [34]. By analogy with yeast, where Ste20 is thought to be an activator of Stell (a MEKKl homolog) [35], p65P*Kl may encode the activator of MEKKl. Alternatively, Racl and Cdc42Hs could activate MEKKl directly or via another kinase. Also activated by stress (W or increased extracellular osmolarity), but differing in substrate specificity, is another MAPK relative, known as ~38, RK (reactivating kinase) or MPK2. Th’is is a vertebrate homolog of the yeast HOG? gene [2,36,37]. Like its homologs, ~38 is activated by phosphorylation on both threonine and tyrosine, this time in a TGY site. Two p38 activators have been characterized: SEKl (the activator of JNKl) and MKK3, a protein which is apparently specific for ~38 [24,25*,26’] (Fig. I). Overexpression of MKK3 or SEKl results in increased p38 activity. Cotransfection of both MEKKl and SEKl results in raising the activity ofJNK1 to above the levels obtained by expressing either MEKKl or SEKl, but does not potentiate ~38 activity [25*,26*]. Thus, although it is likely to be upstream of JNKl and SEKl, MEKKl may not be the physiological upstream activator of ~38. Also, in these experiments MEKKl should have resulted in increased SEKl activity SEKl may activate ~38 only when overexpressed. Perhaps an independent pathway upstream of MKK3 results in ~38 activation in viva. The known downstream targets of ~38 are a seryl/threonyl kinase, MAPK-activated protein kinase (MAPKAPK) 2, and the transcription factor ATF-2 [36-381.
Orphan pathways
It seems unlikely that ERK1/2, JNKl, and ~38 are the only MAPK-related cascades in mammals (Fig. 2). Rhodependent activation of SRF activity is independent of JNK, ERK, or ~38 activation. Thus, by analogy with
other small G proteins, Rho may activate a novel cascade [39]. Similarly, Ras-dependent-phosphorylation of Fos Thr232 is catalyzed by a novel 88 kDa proline-directed kinase, Fos-regulating kinase [40]. Also, nerve growth factor (NGF)-induced phosphorylation of CREB (cyclic AMP response element binding protein) at Ser133 is distinct fi-om that by known MAPK relatives and the known CREB kinase, Rsk [41]. Finally, using the polymerase chain reaction and the two-hybrid system, Zhou et al. [42] have isolated MEK5 and ERK5, which function on an unknown pathway. These and other undiscovered pathways will add to our mechanistic understanding of signal-mediated alterations in transcription.
Specificity of signal propagation
When mammalian cells are treated with mitogenic agents (growth factors or phorbol esters), ERKs become strongly activated but JNKl is poorly activated. Conversely, when cells are challenged with stress (protein synthesis inhibitors or W), JNKl is potently activated and ERK activity is only weakly increased. It would seem from this information that ERK1/2 and JNKl activation are biologically distinct. Yet ~38, JNKl, and ERK1/2 are all proline-directed kinases, sharing numerous in vitro substrates. For example, ATF-2 is phosphorylated by all three kinases [38,43]. Also, Jun and Elk-l are known to be phosphorylated by both ERK2 and JNKl [23,44,45]. Yet these pathways must result in unique transcriptional activity, because stress, mitogens, and differentiation factors should not elicit identical changes in transcription machinery. As a partial explanation of in viva specificity, many members of these kinase cascades have been shown to exist as members of multiprotein complexes. Indeed, JNKl binds tightly to its substrates Jun and ATF-2; also, ERK2 co-immunoprecipitates with both Elk-l and Rsk [4*,38,46,47]. It seems likely that these protein-protein interactions are crucial in generating distinct outputs.
Downstream kinases
Some of the specificity of these pathways is also contributed by the downstream kinases which each MAPK homolog activates. ERKs 1 and 2 phosphorylate and activate the seryl/threonyl kinase Rsk, which translocates to the nucleus where it phosphorylates numerous transcription factors, including CREB, Fos, and SRF [41,48,49]. Similarly, ~38 activates MAPKAPK-2, which phosphorylates heat shock protein 25/27 [36,50]. As the substrate specificities of Rsk and MAPKAPK-2 are somewhat different, the combined outputs of ERK/Rsk and p38/MAPKAPK-2 generate an even greater level of distinction between the pathways. Rsk and MAPKAPK-2 are not the only kinases that are activated by MAPK-type signaling, so some of the complexity is yet to be discovered (AJ Waskiewicz, unpublished data).
Mitogen
Yeast signaling
addition, Fus3 phosphorylates of Cdc28/cyclin [51].
From the above descriptions of mammalian signaling, it is clear that many members of these pathways have homologs in the budding yeast, Sartharomyces cerevisiae. In fact the genetic dissection of fundamental cellular processes including mating, adaptation to nitrogen starvation and changes in external osmolarity, polarized growth, and sporulation - has revealed the existence of at least six independent MAPK-related cascades in yeast [35]. For example, in response to mating pheromone the kinases Ste20, Stell, Ste7, Fus3 and Kssl are activated. Each of these kinases represents a prototype for a signaling molecule in a MAPK cascade. Although our discussion has started with mammalian pathways, the yeast kinases were cloned first. As the genes encoding the mammalian MAPK cascade enzymes were cloned, sequence analysis and functional complementation revealed the amazing evolutionary conservation between yeasts and higher eukaryotes (see Fig. 2). The biological similarities with mammalian signaling cascades are striking. The MAPK homologs Fus3 and Kssl phosphorylate Ste12, a transcription factor which induces transcription of mating-specific genes [51]. In
Racl
Farl,
and Cooper
a negative
regulator
Additional pathways regulate processes involving nitrogen starvation of diploid cells (pseudohyphal growth) (Ste20+Stell+Ste7) [52], haploid invasive growth (Ste20+Stel l+Ste7) [53], increases in extracellular osmolarity (Ssk2/22+Pbs2+Hogl), polarized cell growth (Bckl-+Mkkl/2+Mpkl), and sporulation (Spsl+ Smkl) [35]. Each of the proteins listed in parentheses is encoded by a homolog of either p65P*Kl, MEKK, MEK, or MAPK. For a precise listing of these similarities and members of MAPK pathways in fission yeast, Schizosaccharomyces pombe, see Figure 2. By expressing mammalian MAPK cascade enzymes at high levels in yeast, researchers are able to functionally replace individual yeast kinases. Although these experiments have confirmed that these kinases function in a certain tier, they cannot predict whether a kinase will be stress activated or growth activated. ERK2 will complement a deletion of the yeast MPKl gene [54,55]. Similarly, JNKl will complement a deletion in yeast HOG1 [56]. SEKl will complement a deletion in PBS2, which encodes the activator of Hog1 [24]. In
S. cerevisiae
S. pombe
Vertebrate
Ras
and stress response pathways Waskiewicz
Ras
PAKI
Ste20
Ste20
Pkcl
Stell
Stell
Bckl
Ste7
Ste7
Spsl
? # Raf
MEKKl
MEK
SEKI
MKK3
MEK5
Byrl
ERK1/2
INK1
~38
ERK5
Spkl
Bvr2
MKP-1 PPZA > 1995 Current Opinm
Wisl
Fus3/Kssl
Ptcl,2,3
Msg5
Ssk2l22
Mkkl/MkkZ
Pbs2
Mpkl
Hog1
Smkl
ptc1,3 Ptp2
rn Cell Biology
Fig. 2. A diagram demonstrating the evolutionary conservation among the MAPK signal transduction pathways. With the exception of Raf, members of each tier display considerable amino acid similarity. As many of these cascades have been discovered independently there are more names than we have shown. Bars indicate inhibition of the cascade. A partial list of synonyms: MEK = MKK, ERKl = MAPK = p44MAPx, ERK2 = MAPK = Mpkl (Xenopus) = Xp42 = p42 MAPK, SEK = MKK4 =JNKK, JNK = SAPK = p54M,+‘K, MKK3 = RKK, ~38 = Mpk2 = RK.
801
802
Cell multidication
contrast, XMEK2, thought to be the Xenopus homolog of SEKl, has been shown to functionally replace Mkkl/2 in the polarized cell growth pathway, but cannot replace Pbs2 as might have been expected [57]. Finally, MEKKl will complement a deletion in BCKl, the gene encoding an activator of Mkkl/2 [58]. Thus, the yeast complementarity patterns of JNKl, XMEK2, and MEKKl would suggest they operate on different, rather than the same, pathways. Similarly, MEKKl and ERK should fimction on the same instead of distinct pathways. Perhaps protein-protein interactions that limit specificity have not been conserved throughout evolution.
Checkpoint
regulation
Most MAPK pathways seem to be designed to respond to external stimuli rather than to the time on the internal cell cycle clock. Injection of MAPK activators, however, such as Mos, Ras or thiophosphorylated MAPK into early embryonic cells causes mitotic arrest [59-611. It has now become evident that ERKs are physiologically activated at M phase if the mitotic spindle is damaged. This activation arrests the cell cycle [62**]. This ‘spindle assembly checkpoint’ ensures that incomplete spindles do not prematurely start to pull chromosomes to their respective poles; this action would probably cause non-disjunction of one or more chromosomes. Under these arrested conditions, ERK2 is activated. Moreover, blocking the activation of ERK2 prevents the checkpoint arrest [62**]. Thus MAPKs are activated as a response to microtubule depolymerization. They then function to arrest mitosis at metaphase, preventing non-disjunction.
Phosphatase control
of signaling
In contrast to kinases, which can be very selective in choosing substrates, phosphatases seem to possess a broader specificity. The tyrosyl phosphatase CD45, the dual-specificity MAPK phosphatase MKP-1 (also known as 3CH134 or CLlOO), and the seryl/threonyl phosphatase PP2A (protein phosphatase 2A) are capable of dephosphorylating and inactivating ERKs 1 and 2 in vitro [15,63,64]. Figuring out which of these phosphatases is responsible in vivo for maintaining low basal kinase activity and inactivating MAPK after a signal has ceased has proven quite difficult. MKP-1 was initially identied as a murine seryl/threonyl/ tyrosyl phosphatase which specifically dephosphorylates both regulatory threonyl and tyrosyl residues on ERKs 1 and 2 in vitro. There is good evidence that MKP-1, and
the related phosphatase PAC @hosphatase of activated cells)-1 can function iti vivo to dephosphorylate MAPK. Ectopic expression of a catalytically inactive mutant MKP-1 (in which Cys258 is replaced with serine) activates ERKl/2, presumably by ‘trapping’ these ERKs in the nucleus [63]. Overexpression of MKP-1 blocks Ras-dependent DNA replication [65*]. Also, in S. cerevisiae, a phosphotyrosyl phosphatase, Msg5, which is related to mammalian MKP-1, genetically opposes the function of Fus3 and has been shown to inactivate Fus3 in vitro [66]. Is MKP-1 the only phosphatase which dephosphorylates MAPKs? MKP-1 and its relatives are immediate early gene products and are strictly localized to the nucleus [64,67-69]. Thus the induction of MKP-l/PAC-1 in response to external stimuli may explain the slow dephosphorylation of nuclear ERKl/%. However, the action of MKP-1 cannot account for the rapid dephosphorylation of cytoplasmic ERKl/2 [70,71].
The other leading candidate for a MAPK phosphatase is PP2A, one member of a Emily of enzymes which can also dephosphorylate MEK. The carboxyl terminus of SV40 small tumor antigen binds to PP2A, inhibiting its ability to dephosphorylate its substrates [72,73]. Removal of this interactive domain blocks both small tumor antigen induced MAPK activation and cell proliferation [73]. Both cytoplasmic PP2A and MAPK have been shown to localize to microtubules [74,75]. Following growth factor stimulation, PP2A becomes tyrosyl phosphorylated (possibly by Src); this temporarily inhibits its ability to dephosphorylate substrates [76]. Temporally this coincides with MAPK activation. Thus it appears that PP2A is appropriately regulated and positioned to maintain low basal MAPK activity and to rapidly downregulate MAPK following activation.
In yeast several seryl/threonyl and tyrosyl phosphatases have been genetically shown to participate in MAPK signaling. Perhaps the most convincing results support the idea of roles for PP2C in regulating MAPK pathways in both budding and fission yeasts. In fission yeast, deletion of the three phosphatase 2C isoforms is lethal [77]; however, mutation of the MEK homolog Wisl suppresses this lethality [78]. This suggests that PP2C normally downregulates the Wisl cascade. In budding yeast, mutation of an inhibitor (SLN1) of the Hog1 MAPK pathway is lethal [79**]. This defect is suppressed by overexpression of either SLNl, PTP2 (which encodes a phosphotyrosine phosphatase) or the PP2C genes PTCl and PTC3 [79**]. This and other genetic data suggest that Ptp2 dephosphorylates Hog1 and that Ptcl and Ptc3 also inactivate the pathway. The exact biochemical function of the PP2Cs in yeast, and whether they inactivate MAPK or one of the upstream kinases, still remains to be elucidated. The role of mammalian phosphatase 2C in regulating MAPK signaling also needs to be examined.
Mitogen and stressresponsepathways Waskiewicz
and Cooper
Conclusions
type Raf, implying the existence of a second mitogen-regulated signal for Raf activation.
In this review, we have described the positive and negative regulation of MAPK signaling cascades in higher eukaryotes and yeast. Clearly, there is much still to be learned. For example, although ERK, JNK, and ~38 activation are apparently distinct, oncogenic forms of Ras have been shown to activate all three pathways. Whether this activation occurs via an autocrine/paracrine loop or is the result of Ras activating the Rho family of GTPases is still unclear. In addition, do the same phosphatases regulate the ERK, JNK, and p38 pathways? MKP-1 does not inactivate JNKl, implying the existence of a JNK-specific phosphatase [65*]. Another remaining mystery is the existence of gene families. ERKs, RSKs, JNKs, MEKs and MEKKs are all encoded by members of large multigene families, which often have very precise tissue specific expression. Whether there is any biochemical difference among the isoforms has remained largely unstudied. Finally, although the activity of phosphatases in vitro is now ofien correlated with a biological effect, a careful examination of colocalization between the MAPK homologs and their putative phosphatases would add greatly to the existing evidence. Although it may not seem helpful to end a review with a series of questions, in the MAPK signaling field it seems appropriate.
7. .
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