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Cell cycle regulation by p38 MAP kinases
Biology of the Cell 93 (2001) 47−51 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0248490001011248/REV
Review
Cell cycle regulation by p38 MAP kinases Concetta Ambrosino*, Angel R. Nebreda European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Received 30 June 2001; revised 1 August 2001; accepted 15 August 2001
1. INTRODUCTION MAP kinases are signaling intermediaries that allow eukaryotic cells to interpret and respond to many different stimuli. Three major MAP kinase pathways have been identified. These include the extracellular signal-regulated kinases (ERK1 and ERK2), which are mainly activated by mitogens and serum stimulation. In contrast, the c-Jun N-terminal kinases (JNK) and the p38 MAP kinases are strongly activated by environmental and genotoxic stresses. The p38 MAP kinases are also implicated in several aspects of the immune response. The first member of this family to be isolated was a protein (p38), which was rapidly phosphorylated on tyrosine residues upon lipopolysaccharide stimulation (Han et al., 1994). It was also found as an stress-activated protein kinase (Reactivating Kinase or RK) that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock protein Hsp27 (Rouse et al., 1994). Finally, the same protein was identified as a target of pyridinylimidazole drugs (Cytokine-Suppressive antiinflammatory drug Binding Protein or CSBP) that inhibit the production of the pro-inflammatory cytokines interleukin-1 and tumor necrosis factor (Lee et al., 1994). Analysis of the cDNA sequence revealed that this protein (p38/RK/CSBP) was the vertebrate homologue of the Saccharomyces cerevisiae protein kinase Hog1. Other p38 isoforms were subsequently cloned and named p38β, p38γ and p38δ or SAPK2b, SAPK3 and SAPK4, respectively (Jiang et al., 1996, Lechner et al., 1996; Mertens et al., 1996; Goedert et al., 1997; Jiang et al., 1997; Enslen et al., 1998). These four p38 MAP * Correspondence and reprints: fax: [49] 6221 387166. E-mail addresses: [email protected] (C. Ambrosino), [email protected] (A.R. Nebreda).
Cell cycle regulation by p38 MAP kinases
kinase isoforms are 57-73% identical in their amino acid sequences but differ in their expression patterns and sensitivities to chemical inhibitors such as SB203580 and SB202190, which only interacts with p38α and p38β (reviewed by Cohen, 1997). In addition, the p38 MAP kinase isoforms are activated with differential specificity by the MAP kinase kinases MKK3, MKK4 and MKK6 (Enslen et al., 1998; Alonso et al., 2000) and they also have differential substrate specificities, albeit with some overlap (reviewed by Cohen, 1997). The p38 MAP kinase pathway has been traditionally associated with the stress and immune response and more recently with the regulation of apoptosis and some differentiation processes (reviewed by Nebreda and Porras, 2000). Here we will focus on the emergent role of p38 MAP kinases (p38α and p38β, unless otherwise indicated) in the regulation of cell proliferation and cell cycle checkpoints (figure 1).
2. CELL CYCLE ENTRY AND THE G1/S TRANSITION In normally growing mammalian cells, the mitogenic regulation of cell proliferation occurs at the transition from the quiescent state G0 to the G1 phase and during G1 progression until the restriction point (R), where the cells become growth factor independent and committed to enter the S phase. In recent years, it has been shown that p38 MAP kinases can regulate cellular growth in different ways depending on the cell type and the stimulus. For example, the activation of p38 MAP kinases plays an important role in the proliferation of T lymphocytes in response to stimulation with the mitogenic cytokines interleukin-2 and interleukin-7 (Crawley et al., 1997). In addition, mitogenic stimuli for B lymphocytic cell lines and human tonsillar B cells, such as the antibody Ambrosino and Nebreda
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engagement of either CD40 (a receptor that mediates the interaction with CD4+ lymphocytes) or IgM, specifically induce p38 MAP kinase activation (Craxton et al., 1998). However, blocking p38 MAP kinase activity by treatment with SB203580, resulted in the inhibition of the CD40 induced proliferation but not of proliferation induced by stimulation with an anti-IgM antibody. These results illustrate that mitogenic activation of p38 MAP kinases does not necessarily correlate with cell proliferation. JNK and p38 MAP kinase activation also has an important role in the regulation of hematopoietic cell proliferation induced by granulocyte colony stimulating factor (Rausch and Marshall, 1999). In these studies, p38 MAP kinases have been shown to regulate the activity of different transcriptional factors, such as NF-kB, ATF-1, ATF-2, CREB, and C/EBP family members, which in turn can produce specific changes in gene expression (reviewed by Ono and Han, 2000). In NIH3T3 fibroblasts, expression of a constitutive active form of the small GTP-binding protein Cdc42Hs, results in p38 MAP kinase activation that correlates with inhibition of cell cycle progression at the G1/S transition (Molnar et al., 1997). However, the opposite result was obtained when using primary non-adherent fibroblasts (MEFs), i.e. inhibition of p38 MAP kinase activity stimulates proliferation (Philips et al., 2000). The inhibitory role of p38 MAP kinases at the G1/S transition has been correlated with the downregulation of cyclin D1 levels (Lavoie et al., 1996), whereas the faster G1/S transition in primary MEFs may be due to the p38 MAP kinase-dependent increase in cyclin A expression (Philips et al., 2000). The activation of p38 MAP kinases also has a negative role in primary fetal hepatocyte proliferation upon treatment with the protein synthesis inhibitor anisomycin or expression of a constitutive activated form of MKK6 (Awad et al., 2000). Moreover, injection of specific p38 MAP kinase inhibitors in utero of mouse embryos, strongly reduces the transient physiological growth arrest of hepatocytes and the corresponding increase in the mitotic index correlates with cyclin D1 accumulation (Awad and Gruppuso, 2000). Finally, in Kaposi Sarcoma cells, the same mitogenic stimulus can trigger the activation of both ERK and p38 MAP kinases, which have positive and negative roles in cell proliferation respectively (Murakami-Mori et al., 1999). The molecular mechanisms by which p38 MAP kinases regulate G1 progression during the cell cycle are not completely understood. The expression of cyclin A and cyclin D1 can be regulated at both the transcriptional and post-transcriptional levels. For example, p38 MAP kinases can regulate cyclin D1 protein stability by direct phosphorylation on Thr-286, which triggers its ubiquitination and degradation (Casanovas et al., 2000). Interestingly, p38 MAP kinases can also regulate the phosphorylation of the retinoblastoma protein Cell cycle regulation by p38 MAP kinases
Biology of the Cell 93 (2001) 47–51
(pRb), which is an important regulator of the restriction point transition in G1. Thus, p38 MAP kinases can revert pRb-mediated growth suppression by inducing the phosphorylation of pRb in a Cdk/cyclinindependent way. It has also been shown that pRb can be phosphorylated by p38 MAP kinases, perhaps directly, upon Fas receptor stimulation (Wang et al., 1999). Another important regulator of the G1/S transition is p53. This protein can modulate the expression of several genes involved in cell cycle regulation, such as the Cdk inhibitor p21Cip1/Waf1. There is evidence that p53 is an in vitro and in vivo substrate of p38 MAP kinases (Bulavin et al., 1999; Keller et al., 1999). Moreover, different types of genotoxic stresses can induce the phosphorylation of p53 on several serine residues in a p38 MAP kinase-dependent way. This phosphorylation results in the stabilization of the p53 protein by interfering with its interaction with the ubiquitin ligase Mdm2 (She et al., 2000). In summary, p38 MAP kinases can regulate cell proliferation and must be apparently inhibited at the G0/G1 transition. However, activation of p38 MAP kinases after the restriction point (G1/S transition) can either increase or decrease the number of cells that pass through S-phase, depending on the cell type and the stimulus (figure 1). At the molecular level, the control of S phase entry may be due to the regulation of cyclin A and cyclin D1 levels, as well as the regulation of pRb and p53 phosphorylation.
3. G2/M TRANSITION The role of p38 MAP kinases in G2/M control was first proposed in Schizosaccharomyces pombe. The fission yeast mutant spc1- characterized by a G2 delay, elongated phenotype and 2N DNA content, was shown to be mutated in a p38 MAP kinase homologue. The spc1phenotype resembled an S. pombe mitotic arrest and was exacerbated by environmental stresses such as nutrient depletion or high osmolarity (Shiozaki and Russell, 1995). Moreover, the Spc1 (also called Sty1) protein was phosphorylated under the culture conditions that induce mitotic arrest. Genetic analysis indicate that the Spc1 MAP kinase might function independently of two well-known regulators of the mitotic Cdc2 activity, the kinase Wee1 and the phosphatase Cdc25 (Shiozaki and Russell, 1995). These results suggest that the Spc1-induced mitotic arrest might not be mediated by the inhibitory tyrosine phosphorylation of Cdc2. Later reports have confirmed that the Spc1/Sty1 MAP kinase pathway is required for mitosis entry in fission yeast (Shieh et al., 1998). It was also recently reported that Spc1 can regulate the accumulation of Cdc25 protein (apparently due to a reduction in Cdc25 protein degradation) during the normal cell cycle as well as in response to several stresses. The Ambrosino and Nebreda
Biology of the Cell 93 (2001) 47–51
Figure 1. Regulation of the cell cycle by p38 MAP kinases. Inhibition of p38 MAP kinases is required for cells to enter the cell cycle (G0/G1) and be committed to it [1]. After the restriction point (R), p38 MAP kinases need to be inactivated for the G1/S transition in NIH3T3 fibroblasts and primary hepatocytes [2a], whereas p38 MAP kinase activity is required at this stage in T and B lymphocytes [2b]. In several systems from yeast to mammalian cell lines, p38 MAP kinases have to be inactivated for the G2/M transition and M-phase progression [3].
same report confirmed the existence of an additional Cdc25-independent pathway that is controlled by Spc1 and regulates mitosis (Kishimoto and Yamashita, 2000). A pivotal role for p38 MAP kinases has also been proposed in the coordination of cell division with environmental stresses in budding yeast. Osmotic stress in S. cerevisiae results in the activation of the p38 MAP kinase Hog1, which is involved in the subsequent accumulation of intracellular glycerol and G2 delay. The yeast hog1-mutants have a complex phenotype with morphological changes suggesting deregulated coordination between cell growth and the cell cycle (Brewster and Gustin, 1994). The G2 delay triggered by osmotic stress in budding yeast involves changes in Cdc28 (homologue of Cdc2) phosphorylation and activity that are regulated by the tyrosine kinase Swe1 and the MAP kinase Hog1, respectively (Alexander et al., 2001). In seastar, a p38 MAP kinase homologue has been recently identified as a protein whose phosphorylation status changes during oocyte meiotic maturation. This protein, named Mipk, is dephosphorylated on tyrosine during the maturation and rephosphorylated again after fertilization. This result suggests that Mipk might be involved in the maintenance of the G2 arrest and that its inactivation might be required for the progression into M phase (Morrison et al., 2000). However, addition of activated p38 MAP kinases to Xenopus cell-free extracts or co-injection of p38 MAP kinases Cell cycle regulation by p38 MAP kinases
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with the MKK6 activator into cleaving Xenopus embryos induces arrest in M-phase (Takenaka et al., 1998). Mitotic arrest in several mammalian cell types, for example upon nocodazole treatment, also correlates with the activation of p38 MAP kinases. Moreover, p38 MAP kinases might have to be inactivated to allow the increase in MPF activity levels required for entry into mitosis (Takenaka et al., 1998). It has also been recently shown that oxidative stress or genotoxic agents can induce a mitotic arrest or a G2 delay that is dependent on the selective activation of p38 MAP kinases (Kurata, 2000). The molecular mechanisms are not well understood, but again they may vary depending on the stress and the cell type. In the case of γ-irradiation, the G2 arrest correlates with p38 MAP kinase and MKK6 activation and the inhibition of this pathway abrogates the DNA damage checkpoint in SV40-transformed human fibroblasts. At the molecular level, it has been proposed that the activation of the p38γ isoform is specifically required for the mitotic arrest and that p38γ MAP kinase can indirectly regulate the activity of the Chk2 kinase, which in turn phosphorylates Cdc25C (Wang et al., 2000). DNA damage due to UV irradiation also triggers a mitotic arrest. In several cell lines, p38 MAP kinases play an important role in the regulation of the G2/M phase transition following UV irradiation. Inhibition of p38 MAP kinases, either by treatment with the SB202190 inhibitor or by protein depletion using antisense oligonucleotides, abolishes the G2 delay induced by UV irradiation (Bulavin et al., 2001). The molecular mechanisms responsible for the mitotic arrest/G2 delay following p38 MAP kinase activation start to be elucidated. In vitro, p38 MAP kinases can phosphorylate Cdc25B and Cdc25C at the residues required for binding to 14-3-3 proteins. Moreover, treatment of dermal fibroblasts and HeLa cells with SB202190 suggests that p38 MAP kinases are responsible for the UV-induced phosphorylation of Cdc25B at serine 309. This phosphorylation increases the affinity for 14-3-3, which results in the inactivation of Cdc25B. In contrast, the UV-induced binding of Cdc25C to 14-3-3 does not appear to be regulated by SB202190-sensitive p38 MAP kinases in vivo (Bulavin et al., 2001). The activation of p38 MAP kinases is also tightly regulated during thymocyte differentiation. Experiments with transgenic mice expressing a constitutively active MKK6 mutant in immature thymocytes have demonstrated that continued activation of the p38 MAP kinase pathway arrests cell cycle progression and the differentiation process. Immature thymocytes are probably arrested early in mitosis and contain higher levels of cyclin A. Conversely, treatment with the SB203580 inhibitor induces mitotic progression. This demonstrates that p38 MAP kinase inhibition is re Ambrosino and Nebreda
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quired for progression trough mitosis and ultimately differentiation of immature thymocytes (Diehl et al., 2000). In summary, the onset of the mitotic arrest and the G2 delay induced by different DNA damaging agents requires p38 MAP kinase activation. Moreover, p38 MAP kinases appear to be able to regulate the activity of Cdc25 phosphatases in both mammalian cells and yeast. However, it is plausible that other mechanisms are involved. For example, p53 and p21Cip1/Waf1 play an important role in the maintenance of the G2 arrest and p53 is phosphorylated by p38 MAP kinases in UV irradiated cells.
4. CONCLUSIONS New functions for p38 MAP kinases have been elucidated in recent years. One of these functions is the regulation of the cell cycle and checkpoint controls. The p38 MAP kinase pathway has to be inactivated to exit from the quiescent state as well as to elude mitotic arrest (figure 1). Moreover, activation of p38 MAP kinases during the G1/S transition can either inhibit or promote cell cycle progression depending on the stimulus and the cell type (figure 1). The molecular mechanisms mediating these effects are mostly poorly understood and they are likely to differ depending on the experimental systems used. Although it is possible that p38 MAP kinases do not normally regulate the cell cycle, it seems clear that they are involved in coupling cellular growth and proliferation with different types of environmental signals that can damage the cells. Acknowledgments. We thank our colleagues Gustavo Gutierrez, Eusebio Perdiguero and Emma Stavropoulos for critically reading the manuscript.
REFERENCES Alexander, M.R., Tyers, M., Perret, M., Craig, B.M., Fang, K.S., Gustin, M.C., 2001. Regulation of cell cycle progression by swe1p and hog1p following hypertonic stress. Mol. Biol. Cell 12, 53–62. Alonso, G., Ambrosino, C., Jones, M., Nebreda, A.R., 2000. Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength. J. Biol. Chem. 275, 40641–40648. Awad, M.M., Enslen, H., Boylan, J.M., Davis, R.J., Gruppuso, P.A., 2000. Growth regulation via p38 mitogen-activated protein kinase in developing liver. J. Biol. Chem. 275, 38716–38721. Awad, M.M., Gruppuso, P.A., 2000. Cell cycle control during liver development in the rat: evidence indicating a role for cyclin D1 posttranscriptional regulation. Cell Growth Differ. 11, 325–334. Brewster, J.L., Gustin, M.C., 1994. Positioning of cell growth and division after osmotic stress requires a MAP kinase pathway. Yeast 10, 425–439. Bulavin, D.V., Saito, S., Hollander, M.C., Sakaguchi, K., Anderson, C.W., Appella, E., Fornace, A.J. Jr., 1999. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J 18, 6845–6854.
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Biology of the Cell 93 (2001) 47–51 Bulavin, D.V., Higashimoto, Y., Popoff, I.J., Gaarde, W.A., Basrur, V., Potapova, O., Appella, E., Fornace, A.J. Jr., 2001. Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase. Nature 411, 102–107. Casanovas, O., Miro, F., Estanyol, J.M., Itarte, E., Agell, N., Bachs, O., 2000. Osmotic stress regulates the stability of cyclin D1 in a p38SAPK2-dependent manner. J. Biol. Chem. 275, 35091–35097. Cohen, P., 1997. The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol. 7, 353–361. Crawley, J.B., Rawlinson, L., Lali, F.V., Page, T.H., Saklatvala, J., Foxwell, B.M., 1997. T cell proliferation in response to interleukins 2 and 7 requires p38MAP kinase activation. J. Biol. Chem. 272, 15023–15027. Craxton, A., Shu, G., Graves, J.D., Saklatvala, J., Krebs, E.G., Clark, E.A., 1998. p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes. J. Immunol. 161, 3225–3236. Diehl, N.L., Enslen, H., Fortner, K.A., Merritt, C., Stetson, N., Charland, C., Flavell, R.A., Davis, R.J., Rincon, M., 2000. Activation of the p38 mitogen-activated protein kinase pathway arrests cell cycle progression and differentiation of immature thymocytes in vivo. J. Exp. Med. 191, 321–334. Enslen, H., Raingeaud, J., Davis, R.J., 1998. Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J. Biol. Chem. 273, 1741–1748. Goedert, M., Cuenda, A., Craxton, M., Jakes, R., Cohen, P., 1997. Activation of the novel stress-activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6); comparison of its substrate specificity with that of other SAP kinases. EMBO J. 16, 3563–3571. Han, J., Lee, J.D., Bibbs, L., Ulevitch, R.J., 1994. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265, 808–811. Jiang, Y., Chen, C., Li, Z., Guo, W., Gegner, J.A., Lin, S., Han, J., 1996. Characterization of the structure and function of a new mitogenactivated protein kinase (p38beta). J. Biol. Chem. 271, 17920–17926. Jiang, Y., Gram, H., Zhao, M., New, L., Gu, J., Feng, L., Di Padova, F., Ulevitch, R.J., Han, J., 1997. Characterization of the structure and function of the fourth member of the p38 group mitogen-activated protein kinases, p38δ. J. Biol. Chem. 272, 30122–30128. Keller, D., Zeng, X., Li, X., Kapoor, M., Iordanov, M.S., Taya, Y., Lozano, G., Magun, B., Lu, H., 1999. The p38MAPK inhibitor SB203580 alleviates ultraviolet-induced phosphorylation at serine 389 but not serine 15 and activation of p53. Biochem. Biophys. Res. Commun. 261, 464–471. Kishimoto, N., Yamashita, I., 2000. Multiple pathways regulating fission yeast mitosis upon environmental stresses. Yeast. 16, 597–609. Kurata, S., 2000. Selective activation of p38 MAPK cascade and mitotic arrest caused by low level oxidative stress. J. Biol. Chem. 275, 23413–23416. Lavoie, J.N., L’Allemain, G., Brunet, A., Muller, R., Pouyssegur, J., 1996. Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by thep38/HOGMAPK pathway. J. Biol. Chem. 271, 20608–20616. Lechner, C., Zahalka, M.A., Giot, J.F., Moller, N.P., Ullrich, A., 1996. ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation. Proc. Natl. Acad. Sci. USA 93, 4355–4359. Lee, J.C., Laydon, J.T., McDonnell, P.C., Gallagher, T.F., Kumar, S., Green, D., McNulty, D., Blumenthal, M.J., Heys, J.R., Landvatter, S.W., Strickler, J.E., McLaughlin, M.M., Siemens, I.R., Fisher, S.M., Livi, G.P., White, J.R., Adams, J.L., Young, P.R., 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372, 739–746. Mertens, S., Craxton, M., Goedert, M., 1996. SAP kinase-3, a new member of the family of mammalian stress-activated protein kinases. FEBS Lett. 383, 273–276. Molnar, A., Theodoras, A.M., Zon, L.I., Kyriakis, J.M., 1997. Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK. J. Biol. Chem. 272, 13229–13235.
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Biology of the Cell 93 (2001) 47–51 Morrison, D.L., Yee, A., Paddon, H.B., Vilimek, D., Aebersold, R., Pelech, S.L., 2000. Regulation of the meiosis-inhibited protein kinase, a p38(MAPK) isoform, during meiosis and following fertilization of seastar oocytes. J. Biol. Chem. 275, 34236–34244. Murakami-Mori, K., Mori, S., Nakamura, S., 1999. p38MAP kinase is a negative regulator for ERK1/2-mediated growth of AIDS-associated Kaposi’s sarcoma cells. Biochem. Biophys. Res. Commun. 264, 676–682. Nebreda, A.R., Porras, A., 2000. p38 MAP kinases: beyond the stress response. Trends Biochem. Sci. 25, 257–260. Ono, K., Han, J., 2000. The p38 signal transduction pathway: activation and function. Cell. Signal. 12, 1–13. Philips, A., Roux, P., Coulon, V., Bellanger, J.M., Vie, A., Vignais, M.L., Blanchard, J.M., 2000. Differential effect of Rac and Cdc42 on p38 kinase activity and cell cycle progression of nonadherent primary mouse fibroblasts. J. Biol. Chem. 275, 5911–5917. Rausch, O., Marshall, C.J., 1999. Cooperation of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways during granulocyte colony-stimulating factor-induced hemopoietic cell proliferation. J. Biol. Chem. 274, 4096–4105. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., Zamanillo, D., Hunt, T., Nebreda, A.R., 1994. A novel kinase cascade
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51 triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78, 1027–1037. She, Q.B., Chen, N., Dong, Z., 2000. ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation. J. Biol. Chem. 275, 20444–20449. Shieh, J.C., Wilkinson, M.G., Millar, J.B., 1998. The Win1 mitotic regulator is a component of the fission yeast stress-activated Sty1 MAPK pathway. Mol. Biol. Cell 9, 311–322. Shiozaki, K., Russell, P., 1995. Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast. Nature 378, 739–743. Takenaka, K., Moriguchi, T., Nishida, E., 1998. Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest. Science 280, 599–602. Wang, S., Nath, N., Minden, A., Chellappan, S., 1999. Regulation of Rb and E2F by signal transduction cascades: divergent effects of JNK1 and p38 kinases. EMBO J. 18, 1559–1570. Wang, X., McGowan, C.H., Zhao, M., He, L., Downey, J.S., Fearns, C., Wang, Y., Huang, S., Han, J., 2000. Involvement of the MKK6p38gamma cascade in gamma-radiation-induced cell cycle arrest. Mol. Cell. Biol. 20, 4543–4552.
Ambrosino and Nebreda
Review
Cell cycle regulation by p38 MAP kinases Concetta Ambrosino*, Angel R. Nebreda European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Received 30 June 2001; revised 1 August 2001; accepted 15 August 2001
1. INTRODUCTION MAP kinases are signaling intermediaries that allow eukaryotic cells to interpret and respond to many different stimuli. Three major MAP kinase pathways have been identified. These include the extracellular signal-regulated kinases (ERK1 and ERK2), which are mainly activated by mitogens and serum stimulation. In contrast, the c-Jun N-terminal kinases (JNK) and the p38 MAP kinases are strongly activated by environmental and genotoxic stresses. The p38 MAP kinases are also implicated in several aspects of the immune response. The first member of this family to be isolated was a protein (p38), which was rapidly phosphorylated on tyrosine residues upon lipopolysaccharide stimulation (Han et al., 1994). It was also found as an stress-activated protein kinase (Reactivating Kinase or RK) that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock protein Hsp27 (Rouse et al., 1994). Finally, the same protein was identified as a target of pyridinylimidazole drugs (Cytokine-Suppressive antiinflammatory drug Binding Protein or CSBP) that inhibit the production of the pro-inflammatory cytokines interleukin-1 and tumor necrosis factor (Lee et al., 1994). Analysis of the cDNA sequence revealed that this protein (p38/RK/CSBP) was the vertebrate homologue of the Saccharomyces cerevisiae protein kinase Hog1. Other p38 isoforms were subsequently cloned and named p38β, p38γ and p38δ or SAPK2b, SAPK3 and SAPK4, respectively (Jiang et al., 1996, Lechner et al., 1996; Mertens et al., 1996; Goedert et al., 1997; Jiang et al., 1997; Enslen et al., 1998). These four p38 MAP * Correspondence and reprints: fax: [49] 6221 387166. E-mail addresses: [email protected] (C. Ambrosino), [email protected] (A.R. Nebreda).
Cell cycle regulation by p38 MAP kinases
kinase isoforms are 57-73% identical in their amino acid sequences but differ in their expression patterns and sensitivities to chemical inhibitors such as SB203580 and SB202190, which only interacts with p38α and p38β (reviewed by Cohen, 1997). In addition, the p38 MAP kinase isoforms are activated with differential specificity by the MAP kinase kinases MKK3, MKK4 and MKK6 (Enslen et al., 1998; Alonso et al., 2000) and they also have differential substrate specificities, albeit with some overlap (reviewed by Cohen, 1997). The p38 MAP kinase pathway has been traditionally associated with the stress and immune response and more recently with the regulation of apoptosis and some differentiation processes (reviewed by Nebreda and Porras, 2000). Here we will focus on the emergent role of p38 MAP kinases (p38α and p38β, unless otherwise indicated) in the regulation of cell proliferation and cell cycle checkpoints (figure 1).
2. CELL CYCLE ENTRY AND THE G1/S TRANSITION In normally growing mammalian cells, the mitogenic regulation of cell proliferation occurs at the transition from the quiescent state G0 to the G1 phase and during G1 progression until the restriction point (R), where the cells become growth factor independent and committed to enter the S phase. In recent years, it has been shown that p38 MAP kinases can regulate cellular growth in different ways depending on the cell type and the stimulus. For example, the activation of p38 MAP kinases plays an important role in the proliferation of T lymphocytes in response to stimulation with the mitogenic cytokines interleukin-2 and interleukin-7 (Crawley et al., 1997). In addition, mitogenic stimuli for B lymphocytic cell lines and human tonsillar B cells, such as the antibody Ambrosino and Nebreda
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engagement of either CD40 (a receptor that mediates the interaction with CD4+ lymphocytes) or IgM, specifically induce p38 MAP kinase activation (Craxton et al., 1998). However, blocking p38 MAP kinase activity by treatment with SB203580, resulted in the inhibition of the CD40 induced proliferation but not of proliferation induced by stimulation with an anti-IgM antibody. These results illustrate that mitogenic activation of p38 MAP kinases does not necessarily correlate with cell proliferation. JNK and p38 MAP kinase activation also has an important role in the regulation of hematopoietic cell proliferation induced by granulocyte colony stimulating factor (Rausch and Marshall, 1999). In these studies, p38 MAP kinases have been shown to regulate the activity of different transcriptional factors, such as NF-kB, ATF-1, ATF-2, CREB, and C/EBP family members, which in turn can produce specific changes in gene expression (reviewed by Ono and Han, 2000). In NIH3T3 fibroblasts, expression of a constitutive active form of the small GTP-binding protein Cdc42Hs, results in p38 MAP kinase activation that correlates with inhibition of cell cycle progression at the G1/S transition (Molnar et al., 1997). However, the opposite result was obtained when using primary non-adherent fibroblasts (MEFs), i.e. inhibition of p38 MAP kinase activity stimulates proliferation (Philips et al., 2000). The inhibitory role of p38 MAP kinases at the G1/S transition has been correlated with the downregulation of cyclin D1 levels (Lavoie et al., 1996), whereas the faster G1/S transition in primary MEFs may be due to the p38 MAP kinase-dependent increase in cyclin A expression (Philips et al., 2000). The activation of p38 MAP kinases also has a negative role in primary fetal hepatocyte proliferation upon treatment with the protein synthesis inhibitor anisomycin or expression of a constitutive activated form of MKK6 (Awad et al., 2000). Moreover, injection of specific p38 MAP kinase inhibitors in utero of mouse embryos, strongly reduces the transient physiological growth arrest of hepatocytes and the corresponding increase in the mitotic index correlates with cyclin D1 accumulation (Awad and Gruppuso, 2000). Finally, in Kaposi Sarcoma cells, the same mitogenic stimulus can trigger the activation of both ERK and p38 MAP kinases, which have positive and negative roles in cell proliferation respectively (Murakami-Mori et al., 1999). The molecular mechanisms by which p38 MAP kinases regulate G1 progression during the cell cycle are not completely understood. The expression of cyclin A and cyclin D1 can be regulated at both the transcriptional and post-transcriptional levels. For example, p38 MAP kinases can regulate cyclin D1 protein stability by direct phosphorylation on Thr-286, which triggers its ubiquitination and degradation (Casanovas et al., 2000). Interestingly, p38 MAP kinases can also regulate the phosphorylation of the retinoblastoma protein Cell cycle regulation by p38 MAP kinases
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(pRb), which is an important regulator of the restriction point transition in G1. Thus, p38 MAP kinases can revert pRb-mediated growth suppression by inducing the phosphorylation of pRb in a Cdk/cyclinindependent way. It has also been shown that pRb can be phosphorylated by p38 MAP kinases, perhaps directly, upon Fas receptor stimulation (Wang et al., 1999). Another important regulator of the G1/S transition is p53. This protein can modulate the expression of several genes involved in cell cycle regulation, such as the Cdk inhibitor p21Cip1/Waf1. There is evidence that p53 is an in vitro and in vivo substrate of p38 MAP kinases (Bulavin et al., 1999; Keller et al., 1999). Moreover, different types of genotoxic stresses can induce the phosphorylation of p53 on several serine residues in a p38 MAP kinase-dependent way. This phosphorylation results in the stabilization of the p53 protein by interfering with its interaction with the ubiquitin ligase Mdm2 (She et al., 2000). In summary, p38 MAP kinases can regulate cell proliferation and must be apparently inhibited at the G0/G1 transition. However, activation of p38 MAP kinases after the restriction point (G1/S transition) can either increase or decrease the number of cells that pass through S-phase, depending on the cell type and the stimulus (figure 1). At the molecular level, the control of S phase entry may be due to the regulation of cyclin A and cyclin D1 levels, as well as the regulation of pRb and p53 phosphorylation.
3. G2/M TRANSITION The role of p38 MAP kinases in G2/M control was first proposed in Schizosaccharomyces pombe. The fission yeast mutant spc1- characterized by a G2 delay, elongated phenotype and 2N DNA content, was shown to be mutated in a p38 MAP kinase homologue. The spc1phenotype resembled an S. pombe mitotic arrest and was exacerbated by environmental stresses such as nutrient depletion or high osmolarity (Shiozaki and Russell, 1995). Moreover, the Spc1 (also called Sty1) protein was phosphorylated under the culture conditions that induce mitotic arrest. Genetic analysis indicate that the Spc1 MAP kinase might function independently of two well-known regulators of the mitotic Cdc2 activity, the kinase Wee1 and the phosphatase Cdc25 (Shiozaki and Russell, 1995). These results suggest that the Spc1-induced mitotic arrest might not be mediated by the inhibitory tyrosine phosphorylation of Cdc2. Later reports have confirmed that the Spc1/Sty1 MAP kinase pathway is required for mitosis entry in fission yeast (Shieh et al., 1998). It was also recently reported that Spc1 can regulate the accumulation of Cdc25 protein (apparently due to a reduction in Cdc25 protein degradation) during the normal cell cycle as well as in response to several stresses. The Ambrosino and Nebreda
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Figure 1. Regulation of the cell cycle by p38 MAP kinases. Inhibition of p38 MAP kinases is required for cells to enter the cell cycle (G0/G1) and be committed to it [1]. After the restriction point (R), p38 MAP kinases need to be inactivated for the G1/S transition in NIH3T3 fibroblasts and primary hepatocytes [2a], whereas p38 MAP kinase activity is required at this stage in T and B lymphocytes [2b]. In several systems from yeast to mammalian cell lines, p38 MAP kinases have to be inactivated for the G2/M transition and M-phase progression [3].
same report confirmed the existence of an additional Cdc25-independent pathway that is controlled by Spc1 and regulates mitosis (Kishimoto and Yamashita, 2000). A pivotal role for p38 MAP kinases has also been proposed in the coordination of cell division with environmental stresses in budding yeast. Osmotic stress in S. cerevisiae results in the activation of the p38 MAP kinase Hog1, which is involved in the subsequent accumulation of intracellular glycerol and G2 delay. The yeast hog1-mutants have a complex phenotype with morphological changes suggesting deregulated coordination between cell growth and the cell cycle (Brewster and Gustin, 1994). The G2 delay triggered by osmotic stress in budding yeast involves changes in Cdc28 (homologue of Cdc2) phosphorylation and activity that are regulated by the tyrosine kinase Swe1 and the MAP kinase Hog1, respectively (Alexander et al., 2001). In seastar, a p38 MAP kinase homologue has been recently identified as a protein whose phosphorylation status changes during oocyte meiotic maturation. This protein, named Mipk, is dephosphorylated on tyrosine during the maturation and rephosphorylated again after fertilization. This result suggests that Mipk might be involved in the maintenance of the G2 arrest and that its inactivation might be required for the progression into M phase (Morrison et al., 2000). However, addition of activated p38 MAP kinases to Xenopus cell-free extracts or co-injection of p38 MAP kinases Cell cycle regulation by p38 MAP kinases
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with the MKK6 activator into cleaving Xenopus embryos induces arrest in M-phase (Takenaka et al., 1998). Mitotic arrest in several mammalian cell types, for example upon nocodazole treatment, also correlates with the activation of p38 MAP kinases. Moreover, p38 MAP kinases might have to be inactivated to allow the increase in MPF activity levels required for entry into mitosis (Takenaka et al., 1998). It has also been recently shown that oxidative stress or genotoxic agents can induce a mitotic arrest or a G2 delay that is dependent on the selective activation of p38 MAP kinases (Kurata, 2000). The molecular mechanisms are not well understood, but again they may vary depending on the stress and the cell type. In the case of γ-irradiation, the G2 arrest correlates with p38 MAP kinase and MKK6 activation and the inhibition of this pathway abrogates the DNA damage checkpoint in SV40-transformed human fibroblasts. At the molecular level, it has been proposed that the activation of the p38γ isoform is specifically required for the mitotic arrest and that p38γ MAP kinase can indirectly regulate the activity of the Chk2 kinase, which in turn phosphorylates Cdc25C (Wang et al., 2000). DNA damage due to UV irradiation also triggers a mitotic arrest. In several cell lines, p38 MAP kinases play an important role in the regulation of the G2/M phase transition following UV irradiation. Inhibition of p38 MAP kinases, either by treatment with the SB202190 inhibitor or by protein depletion using antisense oligonucleotides, abolishes the G2 delay induced by UV irradiation (Bulavin et al., 2001). The molecular mechanisms responsible for the mitotic arrest/G2 delay following p38 MAP kinase activation start to be elucidated. In vitro, p38 MAP kinases can phosphorylate Cdc25B and Cdc25C at the residues required for binding to 14-3-3 proteins. Moreover, treatment of dermal fibroblasts and HeLa cells with SB202190 suggests that p38 MAP kinases are responsible for the UV-induced phosphorylation of Cdc25B at serine 309. This phosphorylation increases the affinity for 14-3-3, which results in the inactivation of Cdc25B. In contrast, the UV-induced binding of Cdc25C to 14-3-3 does not appear to be regulated by SB202190-sensitive p38 MAP kinases in vivo (Bulavin et al., 2001). The activation of p38 MAP kinases is also tightly regulated during thymocyte differentiation. Experiments with transgenic mice expressing a constitutively active MKK6 mutant in immature thymocytes have demonstrated that continued activation of the p38 MAP kinase pathway arrests cell cycle progression and the differentiation process. Immature thymocytes are probably arrested early in mitosis and contain higher levels of cyclin A. Conversely, treatment with the SB203580 inhibitor induces mitotic progression. This demonstrates that p38 MAP kinase inhibition is re Ambrosino and Nebreda
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quired for progression trough mitosis and ultimately differentiation of immature thymocytes (Diehl et al., 2000). In summary, the onset of the mitotic arrest and the G2 delay induced by different DNA damaging agents requires p38 MAP kinase activation. Moreover, p38 MAP kinases appear to be able to regulate the activity of Cdc25 phosphatases in both mammalian cells and yeast. However, it is plausible that other mechanisms are involved. For example, p53 and p21Cip1/Waf1 play an important role in the maintenance of the G2 arrest and p53 is phosphorylated by p38 MAP kinases in UV irradiated cells.
4. CONCLUSIONS New functions for p38 MAP kinases have been elucidated in recent years. One of these functions is the regulation of the cell cycle and checkpoint controls. The p38 MAP kinase pathway has to be inactivated to exit from the quiescent state as well as to elude mitotic arrest (figure 1). Moreover, activation of p38 MAP kinases during the G1/S transition can either inhibit or promote cell cycle progression depending on the stimulus and the cell type (figure 1). The molecular mechanisms mediating these effects are mostly poorly understood and they are likely to differ depending on the experimental systems used. Although it is possible that p38 MAP kinases do not normally regulate the cell cycle, it seems clear that they are involved in coupling cellular growth and proliferation with different types of environmental signals that can damage the cells. Acknowledgments. We thank our colleagues Gustavo Gutierrez, Eusebio Perdiguero and Emma Stavropoulos for critically reading the manuscript.
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