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

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: [email protected]
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

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

[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].

[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-

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

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.

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. (1998) Signal transduction by the c-Jun
N-terminal kinase (JNK) – from i nflammation to development.
Curr. Opin. Cell Biol. 10, 205–219.
2. Garrington, T.P. & Johnson, G.L. (1999) Organization and reg-
ulation of mitogen-activated protein k inase signaling pathways.
Curr. Opin. Cell Biol. 11, 211–218.
3. Davis, R.J. (2000) Signal transduction by the JNK group of MAP
kinases. Cell 103, 239–252.
4. Kishimoto, K., Matsumoto, K. & Ninomiya-Tsuji, J. (2000)
TAK1 mitogen-activated protein kinase kinase kinase is activated
by autophosphorylation within its activation loop. J. Biol. Chem.
275, 7359–7364.
5. Wurgler-Murphy, S.M. & Saito, H. (1997) Two-component signal
transducers and MAPK cascades. Trends Biochem. Sci. 22,172–
176.
6. Gaits, F., Shiozaki, K. & Russell, P. (1997) Protein phosphatase

15. Warmka, J., Hanneman, J., Lee, J., Amin, D. & Ota, I . (2001)
Ptc1, a type 2C Ser/Thr phosphatase, inactivates the HOG path-
way by d ephosphorylating the mitogen-activated protein k inase
Hog1. Mol. Cell. Biol. 21, 51–60.
16. Nguyen, A.N. & Shiozaki, K. (1999) Heat-shock-induced activa-
tion of stress MAP kinase is regulated by threonine- and tyrosine-
specific pho spha tases. Genes D ev. 13, 1653–1663.
17. Saxena, M., Williams, S., Brockdorff, J., Gilman, J. & Mustelin, T.
(1999) Inhibition of T cell signaling by mitogen-activated protein
kinase-targeted h ematopoietic tyrosine pho sphatase (HePTP).
J. Biol. Chem. 274, 11693–11700.
18. Oh-hora, M., Ogata, M., Mori, Y., Adachi, M., Imai, K., Kosugi,
A. & Hamaoka, T. (1999) Direct suppression of TCR-mediated
activation of extracellular signal-regulated kinase by leukocyte
protein tyrosine p hosphatase, a tyrosine-specific phosphatase.
J. Immunol. 163, 1282–1288.
19. Blanco -Aparicio, C., Torres, J. & P ulido, R. (1999) A novel reg-
ulatory mechanism of MAP kinases activation and nuclear
translocation mediated by PKA and the PTP-SL tyrosine phos-
phatase. J. Cell. Biol. 147, 1129–1136.
20. Takekawa, M., Maeda, T. & Saito, H. (1998) Protein phosphatase
2Ca inhibits the human stress-responsive p38 and JNK MAPK
pathways. EMBO J. 17, 4744–4752.
21. Hanada, M., Ko bayashi, T., Ohnishi, M., Ikeda, S., Wang, H.,
Katsura, K., Yanagawa, Y., Hiraga, A., Kanamaru, R. &
Tamura, S. (1998) Selective suppression of stress-activated protein
kinase pathway by protein phosphatase 2C in mammalian cells.
FEBS Lett. 437, 172–176.
22. Takekawa, M., Adachi, M., Nakahata, A., Nakayama, I., Itoh,
F., Tsukuda, H., Taya, Y. & Imai, K. (2000) p53-inducible wip1

worth, A. (1999) MKP5, a new member of the MAP kinase
phosphatase family, which selectively dephosphorylates stress-
activated kinas es. Oncogene 18, 6981–6988.
31. Chu, Y., Solski, P.A., Khosravi-Far, R., C.J. & Kelly, K. (1996)
The mitogen-activated protein kinase phosphatases PAC1, M KP-
1, and MKP-2 have unique substrate specificities and reduced
activity in vivo toward the ERK2 sevenmak er mutation. J. Biol.
Chem. 271, 6497–6501.
32. Muda, M., Boschert, U., S mith, A., Antonsson, B., Gillieron, C.,
Chabert, C., Camps, M. , M artinou, I ., Ashworth, A . &
Arkinstall, S. (1997) Molecular cloning and functional charac-
1064 S. Tamura et al. (Eur. J. Biochem. 269) Ó FEBS 2002
terization o f a novel mitogen-activated protein kinase phospha-
tase, MKP-4. J. Biol. Chem. 272, 5141–5151.
33. Keyse, S.M. & Emslie, E.A. (1992) Oxidative stress and heat shock
induce a human gene encoding a protein-tyrosine phosphatase.
Nature 359, 644–647.
34. Hutter, D., Chen, P., Barnes, J. & Liu, Y. (2000) C atalytic acti-
vation of m itogen-activated protein (MAP) kinase phosph atase-1
by binding to p38 MAP kinase: critical role of the p38 C-terminal
domain in its negative regulation. Biochem. J. 352, 155–163.
35. Li, C., Hu, Y., Mayr, M. & Xu, Q. ( 1999) Cyclic strain stress-
induced mitogen-activated protein kinase (MAPK) p hosphatase 1
expression in vascular smooth muscle cells is regulated by Ras/
Rac-MAPK pathways. J. Biol. Chem. 274, 25273–25280.
36. Schliess, F., Heinrich, S. & Haussinger, D. (1998) Hyperosmotic
induction of the mitogen-activated protein kinase phosphatase
MKP-1 in H4IIE rat hepatoma cells. Arch. Biochem. Biophys. 351,
35–40.
37. Bokemeyer, D., Sorokin, A. & Dunn, M.J. (1997) Differential

smooth muscle cells. Hypertension 32, 661–667.
45. B okemeyer, D., Sorokin, A., Yan , M., Ahn, N .G., Templeton,
D.J. & Dunn, M.J. (1996) Induction of mitogen-activated protein
kinase phosphatase 1 by the stress-activated protein kinase sig-
naling p athway but not by extracellular signal-regulated kinase i n
fibroblasts. J. Biol. Chem. 271, 639–642.
46. L im, H.W., New, L., Han, J. & Molkentin, J.D. (2001) Essential
role of calcium in the regulation of MAP kinase phosphatase-1
expression. J. Biol. Chem. 276, 15913–15919.
47. Brondello, J.M., Pouyss egur, J. & McKenzie, F .R. (1999)
Reduced M AP kinase phosphatase-1 degradation after p42/
p44MAPK-dependent phosphorylation. Science 286, 2514–2517.
48. Misra-Press, A., Rim, C.S., Yao, H., Roberson, M.S. & Stork, P.J.
(1995) A novel mitogen-activated protein kinase phosphatase.
Structure, expression, and regulation. J. Biol. Chem. 270, 1 4587–
14596.
49. Hirsch, D.D. & Stork, P.J. (1997) Mitogen-activated protein
kinase phosphatases inactivate stress-activated protein kinase
pathways in vivo. J. Biol. Chem. 272, 4568–4575.
50. Paumelle, R., Tulasne, D., Leroy, C., Coll, J., Vandenbunder, B.
& Fafeur, V. (2000) Sequential activation of ERK and repression
of JNK by scatter factor/hepatocyte growth factor in madin-darby
canine kidney epithelial cells. Mol. Biol. Cell 11, 3751–3763.
51. Fu, S.L., Waha, A. & Vogt, P.K. (2000) Identification and char-
acterization of genes upregulated in cells transformed by v -Jun.
Oncogene 19, 3537–3545.
52. C amps, M., Nich ols, A., G illieron, C., Ant onsson, B., M uda, M.,
Chabert, C., Boschert, U. & Arkinstall, S. (1998) Catalytic acti-
vation of the phosphatase MKP-3 by E RK2 mitogen-activated
protein kinase. Science 280, 1262–1265.

phosphatase witha uniqueacidic domain.FEBS Lett. 412, 415–419.
62. Guthridge, M .A., Bellosta, P., Tavoloni, N. & Basilico, C. (1997)
FIN13, a novel growth factor-inducible serine-threonine phos-
phatase which can inhibit cell cycle progression. Mol. Cell. Biol.
17, 5485–5498.
63. T ong, Y., Quirion, R. & Shen, S H. (1998) Clo ning and char-
acterization of a novel mammalian PP2C isozyme. J. Biol. Chem.
273, 35282–35290.
64. F iscella, M., Zhang, H., Fan, S., Sakaguchi, K., Shen, S ., Mercer,
W.E., Vande Woude, G.F., O’Connor, P.M. & Appella, E. (1997)
Wip1, a novel human protein phosphatase that is induced in
response to io nizing radiation in a p53-dependent m anner. Proc.
Natl Acad. Sci. USA 94, 6048–6053.
65. K itani, T., Ishida, A., Ok uno, S., Takeuchi, M ., Kameshita, I. &
Fujisawa, H. (1999) Molecular cloning of Ca
2+
/calmodulin-
dependent proteinkinase phosphatase.J. Biochem. 125, 1022–1028.
66. Cheng, A., Kaldis, P. & Solomon, M.J. (2000) Dephosphorylation
of human cyclin-dependent kinases by protein phosphatase type
2Ca and b2 isoforms. J. Biol. Chem. 275, 34744–34749.
67. Strovel, E.T., Wu, D. & Sussman, D.J. (2000) Protein phosphatase
2Ca dephosphorylates axin and activates LEF-1-dependent tran-
scription. J. Biol. Chem. 275, 2399–2403.
68. M ann, D.J., C ampbell, D.G., McGowan, C.H. & Cohen, P.T.
(1992) Mammalian protein serine/threonine phosphatase 2C:
cDNA cloning and comparative analysis of amino acid sequences.
Biochim. Biophys. Acta 1130, 100–104.
Ó FEBS 2002 Regulation of SAPK signaling pathways (Eur. J. Biochem. 269) 1065
69. Terasawa, T., Kobayashi, T., Murakami, T., Ohnishi, M.,

Chem. 270, 2337–2343.
76. Shiozuka, K., Watanabe, Y., Ikeda, T., Hashimoto, S. & Kawa-
shima, H. (1995) Cloning and expression of PCPTP1 encoding
protein tyrosine phosphatase. Gene 162, 279–284.
77. Ogata, M., Oh-hora, M., Kosugi, A. & Hamaoka, T. (1999)
Inactivation of mitogen-activated protein kinases by a mammalian
tyrosine-specific phosphatase, PTPBR7. Biochem. Biophys. Res.
Commun. 256, 52–56.
78. Zuniga, A., Torres, J., Ubeda, J. & Pulido, R. (1999) Interaction of
mitogen-activated protein kinases with the kinase interaction
motif of the tyrosine phosphatase PTP-SL provides substrate
specificity and r etains ERK2 in the cytoplasm. J. Biol. Chem. 274,
21900–21907.
1066 S. Tamura et al. (Eur. J. Biochem. 269) Ó FEBS 2002