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MINIREVIEW Regulation of stress-activated protein kinase signaling pathways by protein phosphatases Shinri Tamura, Masahito Hanada, Motoko Ohnishi, Koji Katsura, Masato Sasaki and Takayasu Kobayashi Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Japan Stress-activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines. SAPK pathways are composed of three successive protein kinase reactions. The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems. Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specif- icity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function. Differences in substrate specificities and regulatory mechanisms for these phos- phatases form the molecular basis for the complex regulation of SAPK signaling. Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways. Keywords: stress response; stress-activated protein kinase; protein phosphatase. INTRODUCTION Stress-activated p rotein kinases (SAPKs), a subfamily of the mitogen-activated protein kinase (MAPK) superfamily, are highly conserved from yeast to mammals. SAPKs relay signals in response to various e xtracellular stimuli, including environmental stresses and proinflammatory cytokines. In mammalian cells, two distinct classes of SAPKs have been identified, the c-Jun N-terminal kinases (JNK) and the p38 MAPKs [1,2] (Fig. 1). The activation of SAPKs requires phosphorylation of conserved tyrosine and threonine residues within the catalytic domain. This phosphorylation is mediated by dual specificity protein kinases, members of the MAPK kinase (MKK) family. MKK3 and MKK6 are specific for p38, MKK7 selectively phosphorylates JNK, and MKK4 recognizes either class of the stress actived kinases (Fig. 1). The MKKs are also activated by the phosphorylation of conserved serine and threonine residues [1,2]. Several MKK-activating MKK kinases (MKKKs) have been identified, some of which are activated again by phosphory- lation [3,4]. In the absence of a signal, the constituents of t he SAPK cascade return to their inactive, dephosphorylated state, suggesting an essential role for phosphatases in SAPK regulation. Protein p hosphatases are classifi ed into three groups, Ser/Thr phosphatases, Ser/Thr/Tyr phosphatases and Tyr phosphatases, depending on their phosphoamino-acid specificity. The dephosphorylation of SAPK signal p athway components on either Ser/Thr or Thr/Tyr residues requires the participation of various p hosphatases. In t his article, we first review the roles of protein phosphatases in the regulation of yeast SAPK pathways, then fo cus on the properties of the protein phosphatases implicated in the mammalian SAPK systems. REGULATION OF SAPK SIGNAL PATHWAYS BY PROTEIN PHOSPHATASES IN YEAST CELLS A molecular g enetic analysis of ye ast cells indicated that two distinct protein phosphatase groups, protein Tyr phospha- tases (PTP) and protein Ser/Thr phosp hatases of type 2C (PP2C), act as negative regulators of SAPK pathways [5,6]. In the budding yeast, Saccharomyces cerevisiae,hyper- osmotic shock activates the SSK2/SSK22 (MKKK)-Pbs2 (MKK)-Hog1 (SAPK) kinases. In the fission yeast, Schizosaccharomyces pombe, heat shock, oxidative stress, nutrient stress and osmotic shock all induce the Wik1 (MKKK)-Wis1 (MKK)-Spc1 (SAPK) pathway; the activa- ted Spc1 in turn changes gene expression through the activation of the Atf1 transcription factor [7–10]. The PTPs of S. cerevisiae (Ptp2 and Ptp3) and S. pombe (Pyp1 and Pyp2) suppress the SAPK pathways, as demon- strated by molecular genetic studies [5,8,10–12]. In S. pombe, Pyp2 dephosphorylates the tyrosine residue of Spc1 both in v ivo and in vitro [8,12]. Extracellular stress induces expres- sion of the pyp2 gene in an Spc1-Atf1-dependent manner Correspondence to S. Tamura, Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku University 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan. Fax: + 81 2 2 717 8476, Tel.: + 81 22 717 8471, E-mail: tamura@idac.tohoku.ac.jp Abbreviations: SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; MKK, MAPK kinase; MKKK, MKK kinase; PTP, protein Tyr phosphatase; PP, protein Ser/Thr phosphatase; DSP, dual specificity protein phosphatase; MKP, MAPK phosphatase; ERK, extracellu- lar signal-regulated kinase; TPA, 12-O-tetradecanoylphorbol 13-acetate; TCR, T cell receptor; TGF-b, transforming growth factor-b;TAK1,TGF-b-activated kinase 1; IL-1, interleukin-1; KIM, kinase interaction motif; PA, 1,2-dioleoyl-sn-glycero- 3-phosphate. (Received 6 August 2001, accepted 20 September 2001) Eur. J. Biochem. 269, 1060–1066 (2002) Ó FEBS 2002 [10,11]. In addition, PP2C (Ptc1 and Ptc3 of S. cerevisiae and Ptc1 and Ptc3 of S. pombe) acts as a negative regulator of SAPK pathways [13–15]. In S. pombe, Ptc1 acts upon a target downstream of SAPK (Spc1) [6]. When Spc1 enhances the expression of Atf1, this up-regulation induces Ptc1 expression, suppressing Atf1 function. Ptc1 and Ptc3 directly dephosphorylate the threonine of Spc1, but not th e tyrosine [16]. In a ddition, Ptc1 dephosphorylates Hog1 in S. cerevisiae both in vivo and in vitro [15]. REGULATION OF SAPK SIGNAL PATHWAYS BY PROTEIN PHOSPHATASES IN MAMMALIAN CELLS In mammalian cells, like yeast cells, both PTP and PP2C regulate the SAPK signal pathways [17–23]. Mammalian cells are unique in several r espects as, in addition to PTP and PP2C, they contain a large family of dual specificity protein phosphatases (DSP) that negatively influence the SAPK pathways [24]. Although the participation of a DSP, MSG5, in the negative regulation of mating hormone-induced MAPK (Fus3p) activation is well documented [25], the parti- cipation of such DSPs in the regulation of the yeast SAPK system has not been observed. In addition, protein phospha- tase 2A (PP2A) may also function in the regulation of the mammalian SAPK pathway [26]. In this section, we describ e the properties of mammalian protein phosphatase mole- cules involved in the regulation of SAPK signal pathways. Dual specificity protein phosphatases The gene products of at least 10 distinct DSP genes share two unique structural features; they contain a common active site sequence motif [VXVHCXXGXSRSXTXXX AY(L/I)M] and two N-terminal CH2 domains, homologous to the cell cycle regulator Cdc25 [27]. DSP substrate studies indicate that MAPK phosphatase-3 (MKP-3) specifically dephosph- orylates extracellular signal-regulated kinase (ERK) but not JNK or p38 [27,28]. In contrast, both MKP-5 and M3/6 dephosphorylate both JNK and p38 but not ERK (Table 1) [27,29,30]. The high specificity of MKP-2 for ERK and JNK (but not for p38) and that of PAC-1 for ERK and p38 (but not for JNK) has been reported (Table 1) [31]. On the other hand, MKP-1 and MKP-4 were found to dephosphorylate ERK, JNK and p38 [31,32]. These facts indicate an unexpected complexity for the negative regulation of the MAP kinase signaling. In the forthcoming paragraphs we present a detailed description of the mammalian DSPs involved in the regulation of SAPK signaling pathways. MKP-1 (CL100). MKP-1, a protein of 39.5 kDa, is expressed upon oxidative stress and heat shock in human skin cells [33]. MKP-1 mRNA is ubiquitously expressed in various tissu es, with the protein product localized preferen- tially to the cell nucleus [34]. This enz yme acts as a DSP, dephosphorylating both threonine and tyrosine residues of ERK, JNK and p38 [31,35]. In addition to oxidative stress and heat shock, MKP-1 is induced by various stimuli such as, o smotic shock, anisomycin, g rowth factors, UV, 12-O- tetradecanoylphorbol 13-acetate (TPA), Ca 2+ ionophores and lipopolysaccharide [33–42]. MKP-1 expression is part of a feed back mechanism: the activation of MAPKs induces MKP-1; that in turn inactivates MAPKs. The details of the regulatory mechanism depend on the cell lineage. In vascular smooth muscle cells, mesangial cells and U937 cells, the activation of either ERK, JNK or p38 induces MKP-1; in NIH3T3 cells, the activation of JNK but not ERK up-regulates MKP-1 expression [35,37,40,43–45]. In addition, activation of p38 but not ERK o r JNK enhances MKP-1 induction in H4IIE hepatoma cells [36]. In Rat1 fibroblasts, MKP-1 is induced by Ca 2+ signaling, independently of MAPK activation [41]. In this context, Ca 2+ /calmodulin-activated protein phosphatase (PP2B) participates in the induction of MKP-1 in cardiac myocytes Fig. 1 . SAPK signaling modules. The p rotein kinase cascades of SAPK signaling pathways and the points where the ph osphatases can interfere with the signals are shown. MKKK, MKK kinase; MKK, MAPK kinase; MAPK, MAP kinase; TAK1, T GF-b-activated k inase 1;MEKK,MEKkinase;MLK,mixedlineage kinase; ASK1, apoptosis signal-regulating kinase 1; JNK, c-Jun N-terminal kinase. Ó FEBS 2002 Regulation of SAPK signaling pathways (Eur. J. Biochem. 269) 1061 [46]. MKP-1 binds to C-terminal region of p38, that results in its activation [34]. The stability of MKP-1 is regulated by ERK-mediated phosphorylation of two C-terminal serine residues [47]. This phosphorylation, while not modifying the intrinsic activity of MKP-1, stabilizes the protein. MKP-2 (hVH2). MKP-2, a 42.6-kDa nuclear DSP, is widely expressed in various tissues [48]. This phosphatase is highly specific f or ERK and JNK, but not p38 [31]. MKP-2 is induced by nerve growth factor, TPA and hepatocyte growth factor in PC12 cells, peripheral blood T cells and MDCK cells, respectively [31,49,50]. In MDCK cells, hepatocyte growth factor-activated ERK induces MKP-2 expression; that inactivates JNK, which has also been activated by GF, by dephosphorylation [50]. Overexpres- sion of v-Jun, a constitutively active form of c-Jun, enhances the expression of MKP-2 mRNA in chick embryo fibro- blasts [51]. Therefore, the activation of JNK may also influence in MKP-2 expression. MKP-4. MKP-4 is a DSP of 41.8 kDa displaying moderate substrate specificity f or ERK over JNK or p38 [32]. Immunostaining of MKP-4 expressed in either NIH3T3 cells or COS7 cells revealed that MKP-4 is localized mainly to the cytoplasm; a subset of cells, however, also displays a punctuate nuclear staining [ 32]. Expression of MKP-4 mRNA is highly restricted to the placenta, kidney a nd embryonic liver [32]. Phosp hatase activation is mediated by substrate binding [52]. MKP-5. MKP-5, a widely expressed 52.6-kDa protein, preferentially dephosphorylates both JNK and p38, and demonstrates extremely low activity against ERK [29,30]. This enzyme is evenly localized throughout the cytoplasm and nucleus [29]. In cultured cells, the expression of MKP-5 is elevated by stress stimuli such as an isomycin and osmotic stress but not by UV irradiation [29]. MKP-5 binds to p38 and JNK, but not ERK [29,30]. MKP-6. MKP-6 (25 kDa) was found as a CD28 (T cell costimulatory r eceptor) binding protein [53]. In vitro, MKP- 6 d ephosphorylates ERK, JNK and p38. However, expres- sion of a dominant negative form of MKP-6 in T cells further stimulates the T cell receptor (TCR)/CD28- enhanced phosphorylation of both ERK and J NK but not p38, suggesting that ERK and JNK are the preferred substrates of MKP-6 in the cells. MKP-6 expression is up-regulated by CD28 costimulation of T cells. Binding of the expressed MKP-6 to CD28 is required for the feed back regulation of ERK and JNK by MKP-6 [53]. M3/6 (hVH5). M3/6 was the first DSP found to selectively inhibit stress-induc ed activation of JNK and p38; M3/6 does not, however, affect growth factor- induced activation of ERK in mammalian cells [27]. In K562 human leukemia cells, hVH5 (human orthologue of mouse M3/6) mRNA levels are rapidly enhanced by TPA treatment [54]. The induction of exogenous M3/6 inhibited TPA-stimulated phosphorylation of JNK and p38, sug- gesting a feedback loop governing SAPK activity. The activation of JNK stimulates the phosphorylation of M3/6; unlike MKP-1, however, the phosphorylation of M3/6 does not regulate its half life [54]. An internal motif, XILPXL(Y/F)LG, homologous to the SAPK binding site of c-Jun (delta domain), is important for M3/6 activity [54]. PAC-1. PAC-1 is a DSP of 32 kDa, originally found to be expressed predominantly in hematopoietic cells [55]. Subse- quently, induction of PAC-1 mRNA in hippocampus neurons following forebrain ischemia or kainic acid-induced seizure has been reported [56,57]. PAC-1 dephosphorylates both ERK and p38 but not JNK [31]. Activation of ERK induces the enhanced-expression of PAC-1 and the expressed PAC-1 the n inactivates ERK in T cells [58]. Protein phosphatase 2C Protein phosphatase 2C (PP2C) is one of the four major protein serine/threonine phosphatases (PP1, PP2A, PP2B and PP2C) in eukaryotes. At least six distinct PP2C gene products (2Ca,2Cb,2Cc,2Cd,Wip1andCa 2+ /calmodu- lin-dependent protein kinase phosphatase) operate in mammalian cells [59–65]. Studies of mammalian PP2C function indicated that P P2Ca, PP2Cb and Wip1 a re involved in the negative regulation o f SAPK cascades [20– 23]. In addition, PP2Ca and PP2Cb may regulate cell cycle progression [66]. PP2Ca is implicated in Wnt signaling regulation [67]. Here, we describe the properties of PP2C isoforms regulating the SAPK s ignal pathways. PP2Ca. PP2Ca, a 42-kDa phosphatase, was first cloned from a rat kidney c DNA library [59]. The existence of two distinct human PP2Ca isoforms (a-1 and a-2), differing at their C-terminal regions, was subsequently reported [20,68]. A cDNA clone encoding PP2Ca-2 was isolated in the screening of a human cDNA library for genes down- regulating the yeast Hog1 MAPK pathway [20]. When expressed in mammalian cells, PP2Ca-2 inhibits stress- induced activation of p38 and JNK, but does not affect mitogen-induced activation of ERK. Mouse PP2Ca,cor- responding to human PP2Ca-1, exhibited a similar inhibi- tion pattern [21]. PP2Ca-2 dephosphorylates and inactivates MKK4, MKK6 and p38 both in vivo and in vitro [20]. Table 1. Protein phosphatases involved in regulation of SAPK signal pathways. Phosphatase Substrate References DSP family MKP-1 (CL100, 3CH134) JNK, p38, ERK [31,35] MKP-2 (hVH2, Typ-1) JNK, ERK [31] MKP-4 JNK, p38, ERK [32] MKP-5 JNK, p38 [29,30] MKP-6 JNK, ERK [55] M3/6 (hVH5) JNK, p38 [27] PAC-1 p38, ERK [31] PP2C family PP2Ca-2 MKK4, MKK6, p38 [20] PP2Cb-1 TAK1 [23] Wip1 p38 [22] PTP family HePTP/LC-PTP p38, ERK [17,18] PTP-SL/STEP p38, ERK [19,78] PP2A family PP2A JNK [26] 1062 S. Tamura et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Furthermore PP2Ca-2 specifically associates with phos- phorylated p38. PP2Cb. The PP2Cb gene encodes at least six distinct isoforms (43 kDa), which are splicing variants of a single premRNA [60,69–71]. These isoforms differ only at the C-terminal regions. PP2Cb-1 is expressed ubiquitously in various tissues, while PP2Cb-2 expression is restricted to th e brain a nd heart. PP2Cb-3, -4 and -5 transcripts were detec- ted predominantly in the liver, testes and intestine [69,70]. In mammalian cells, PP2Cb-1 selectively supp resses the stress- induced activation of p38 and JNK but has no effect on the mitogen-induced activation of ERK [21]. Investigation of the PP2Cb-1-mediated suppression of the SAPK pathway revealed that PP2Cb-1 dephosphorylates and inactivates transforming growth factor- b (TGF-b)-activated k inase (TAK1), a MKKK activated either by stress, TGF-b treat- ment or interleukin-1 (IL-1) stimulation [23]. In addition, PP2Cb-1 selectively associates with TAK1 in a stable com- plex. Expression of a dominant-negative form of PP2Cb-1 enhances the IL-1-induced activation of AP-1 reporter gene, suggesting PP2Cb-1 ne gatively regulates TAK1 signaling through the depho sphorylation of TAK1 in vivo [23]. Wip1. Wip1, a 61-kDa Mg 2+ -dependent protein phospha- tase, is induced by ionizing radiation in a p53-de pendent manner [64]. It is localized to the nucleus, the nuclear levels of Wip1 increase in response to the ionizing irradiation. The expression of Wip1 is also induced by treatment with methyl methane sulfonate, H 2 O 2 or anisomycin [22]. Functional studies of Wip1 revealed its role in the down- regulation of p38/p53-induced signaling during the recovery of damaged cells [22]. Thus, t he induction of Wip1 b y stress selectively blocks the activation of p 38, and suppresses subsequent p53 activation. In vitro, Wip1 inactivates p38 by the specific dephosphorylation of a conserved threonine residue; however, it does not accept ERK, JNK, MKK4 or MKK6 as a substrate [22]. Other protein phosphatases Recently e vidence has emerged suggesting the participation of okadaic acid-sensitive protein phosphatases and PTPs in the regulation of mammalian SAPK pathways [17–19,26]. In this section, we describe the roles of protein phosphatase 2A (PP2A) and tyrosine phosphatases, HePTP/LC-PTP and PTP-SL/STEP, in SAPK signaling. PP2A. Addition of okadaic acid to the culture medium enhanced the lipopolysaccharide-induced activation of JNK in THP-1 cells (a human acute monocytic leukemia ce ll line) [26]. In addition the regulatory subunit of PP2A, PP2A-Aa, coprecipitates with JNK [26]. JNK activity was unaffected by specific pharmacological inhibition of protein phospha- tase 1 by 1 ,2-dioleoyl- sn-glycero-3-phosphate (PA); the activation of PP2A by high doses of PA, however, d ecreased JNK activity [26]. These results suggest that PP2A may suppress the lipopolysaccharide-induced JNK th rough the direct dephosphorylation of JNK. HePTP/LC-PTP. HePTP and LC-PTP are closely related human cytosolic PTPs, predominantly expressed in hem- opoietic cells [17,18]. In T lymphocytes, the transcription of HePTP is enhanced by IL-2 treatment [72]. When expressed in Jurkat T cells, HePTP/LC-PTP inhibits the TCR-induced activation of both ERK and p38, but not JNK [17,18]. Both ERK a nd p38 (but not JNK) associate with the kinase interaction motif (KIM) in the N-terminal segment of HePTP/LC-PTP. The phosphorylation of HePTP by PKA inhibits its association with ERK a nd p38 [73]. Conse- quently the PKA-mediated r elease o f the phosphatase activates both ERK and p38. PTP-SL/STEP. PTP-SL and STEP are non-nuclear PTPs, which exist in transmembrane and cytosolic forms and are mainly expressed in n euronal cells [74–77]. PTP-SL dephosphorylates both ERK and p38 [19,78]. Like HePTP, PTP-SL associates with ERK and p38 but not with JNK through its KIM located in the juxtamembrane region [78]. The phosphorylation of PTP-SL by PKA was found to inhibit its association with ERK and p38, and the subsequent tyrosine dephosphorylation of these MAPKs [19]. CONCLUSIONS AND PERSPECTIVES Numerous phosphatase molecules are capable of negatively regulating SAPK signaling pathways (summarized in Table 1 and Fig. 1) including the members of four distinct groups: DSP, PP2C, PP2A and PTP. Regulation of a single substrate by multiple protein phosphatases suggests redundancy. Alternatively, the level of phosp horylation in each protein component of t he SAPK pathway may be regulated by multiple upstream s ignals functioning via distinct protein phosphatases. We conclude that at least two distinct mechanisms can operate in the regulation. The expression of phosphatases, such as MKP-1, MKP-2, MKP-5, M3/6, PTC-1, Wip1 and HePTP, is positively regulated through the activation of MAP kinases. In addition, some phosphatases are regulated by direct association with MAPKs. For example both MKP-1 and MKP-4 are activated via binding to their MAPK substrates [34,52]. Direct association was also observed between MKP-5 and p38 or JNK [29,30]. Interestingly, a sequence motif, XILPXL(Y/F)LG, which is similar to a delta domain consensus motif critical for binding to JNK and ERK in other proteins, is converved in all of these DSPs. The delta-like domain is located N-terminal to the c atalytic consensus sequence of t he DSPs. The delta-like domain is also conserved in M3/6; deletion of this sequence bloc ks the ability of M3/6 to dephosphorylate JNK [54]. These results suggest that the delta-like domain is involved in the association of phosphatases with MAPKs. Another association between MAPKs a nd HePTP/LC-PTP or PTP-SL/STEP phos- phatases and regulation of this association by PKA is also of great importance [19,73]. Substrate specificity studies indicated that s everal mem- bers of the DSP and PTP families dephosp horylate both ERK and JNK/p38 (Table 1). This suggests that phospha- tases may mediate the signaling between the ERK and SAPK pathways. Future studies will certainly clarify the significance of s uch cross-talk between the ERK and SAPK pathways via protein phosphatases. The protein phosphatases that dephosphorylate MKKs and MKKKs have not been well investigated. The PP2C Ó FEBS 2002 Regulation of SAPK signaling pathways (Eur. J. Biochem. 269) 1063 family may play a central role in the regulation of these kinases as PP2Ca-2 dephosphorylates both MKK4 and MKK6 [20]. In addition, PP2Cb-1 dephosphorylates TAK1, but not MKK6 [23]. These results suggest that each isoform of PP2C may have a distinct specificity for substrates in SAPK pathways. Future studies are required for identification of phosphatases responsible for dephos- phorylation of other MKK and MKKK members. ACKNOWLEDGEMENT The au thors are gr ateful to Dr Masato Ogata (Osaka University) for critically revi ewing this a rticle. REFERENCES 1. Ip, Y.T. & Davis, R.J. 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