The Kinetic Mechanism of the Dual Phosphorylation of the ATF2 Transcription Factor by p38 Mitogen-activated Protein (MAP) Kinase α
2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês
10.1074/jbc.m008787200
ISSN1083-351X
AutoresWilliam F. Waas, Herng‐Hsiang Lo, Kevin N. Dalby,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe mitogen-activated protein kinases (MAPKs) are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are the terminal component of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed. Although multistep protein phosphorylation within mitogen-activated protein kinase (MAPK) cascades can dramatically influence the sensitivity of signal propagation, an investigation of the mechanism of multisite phosphorylation by a MAPK has not been reported. Here we report a kinetic examination of the phosphorylation of Thr-69 and Thr-71 of the glutathioneS-transferase fusion protein of thetrans-activation domain of activating transcription factor-2 (GST-ATF2-(1–115)) by p38 MAPKα (p38α) as a model system for the phosphorylation of ATF2 by p38α. Our experiments demonstrated that GST-ATF2-(1–115) is phosphorylated in a two-step distributive mechanism, where p38α dissociates from GST-ATF2-(1–115) after the initial phosphorylation of either Thr-69 or Thr-71. Whereas p38α showed similar specificity for Thr-71 and Thr-69 in the unphosphorylated protein, it displayed a marked difference in specificity toward the mono-phosphoisomers. Phosphorylation of Thr-71 had no significant effect on the rate of Thr-69 phosphorylation, but Thr-69 phosphorylation reduced the specificity,k cat/K M, of p38α for Thr-71 by approximately 40-fold. Computer simulation of the mechanism suggests that the activation of ATF2 by p38α in vivo is essentially Michaelian and provides insight into how the kinetics of a two-step distributive mechanism can be adapted to modulate effectively the sensitivity of a signal transduction pathway. This work also suggests that whereas MAPKs utilize docking interactions to bind substrates, they can be weak and transient in nature, providing just enough binding energy to promote the phosphorylation of a specific substrate. The mitogen-activated protein kinases (MAPKs) are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are the terminal component of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed. Although multistep protein phosphorylation within mitogen-activated protein kinase (MAPK) cascades can dramatically influence the sensitivity of signal propagation, an investigation of the mechanism of multisite phosphorylation by a MAPK has not been reported. Here we report a kinetic examination of the phosphorylation of Thr-69 and Thr-71 of the glutathioneS-transferase fusion protein of thetrans-activation domain of activating transcription factor-2 (GST-ATF2-(1–115)) by p38 MAPKα (p38α) as a model system for the phosphorylation of ATF2 by p38α. Our experiments demonstrated that GST-ATF2-(1–115) is phosphorylated in a two-step distributive mechanism, where p38α dissociates from GST-ATF2-(1–115) after the initial phosphorylation of either Thr-69 or Thr-71. Whereas p38α showed similar specificity for Thr-71 and Thr-69 in the unphosphorylated protein, it displayed a marked difference in specificity toward the mono-phosphoisomers. Phosphorylation of Thr-71 had no significant effect on the rate of Thr-69 phosphorylation, but Thr-69 phosphorylation reduced the specificity,k cat/K M, of p38α for Thr-71 by approximately 40-fold. Computer simulation of the mechanism suggests that the activation of ATF2 by p38α in vivo is essentially Michaelian and provides insight into how the kinetics of a two-step distributive mechanism can be adapted to modulate effectively the sensitivity of a signal transduction pathway. This work also suggests that whereas MAPKs utilize docking interactions to bind substrates, they can be weak and transient in nature, providing just enough binding energy to promote the phosphorylation of a specific substrate. mitogen-activated protein kinase mitogen-activated protein MAP kinase kinase MAP kinase kinase kinase mitogen-activated protein kinase kinase 6 mitogen-activated protein kinase kinase 3 p38 MAP kinase α activating transcription factor 2 extra signal regulated protein kinase MAP or ERK kinase kinase 4 high pressure liquid chromatography c-Jun N-terminal protein kinase bombarded mass fragmentation pattern of a molecule bombarded mass fragmentation pattern of a single m/z ion derived from a MS glutathione S-transferase ATF271P, and ATF269P/71P correspond to GST-ATF2-(1–115) phosphorylated on Thr-69, Thr-71, or both Thr-69 and Thr-71 The mitogen-activated protein kinases (MAPKs)1 are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are implicated in processes ranging from pheromone responses and cell wall formation in yeast to mitogenesis, apoptosis, and stress responses in mammalian cells (1Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (757) Google Scholar, 2Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2248) Google Scholar). They are the terminal components of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed.A MAP kinase cascade is generally composed of three protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK) arranged in a linear hierarchical fashion as shown in Scheme FS1. Although there are a large number of MAPKKKs, which become activated through a variety of mechanisms, MAPKKKs are highly specific for their conjugate MAPKK, which they activate by dual phosphorylation. In turn MAPKKs activate their conjugate MAPKs, also by dual phosphorylation, whereas MAPKs, which are the least specific kinases in these modules, phosphorylate many proteins, typically at more than one site (3Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (434) Google Scholar). Although this simple picture is somewhat obscured by the existence of an abundance of different MAPKKKs and by the existence of MAPKK and MAPK isoforms, the three-tiered hierarchy appears to be well conserved. The kinases do not function in isolation, because protein phosphatases represent the force against which they labor to drive the steady-state concentrations of phosphorylated proteins beyond critical thresholds. There are several protein phosphatases, some such as the MAPK phosphatases are specific for certain MAPKs (4Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (702) Google Scholar, 5Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (710) Google Scholar, 6Keyse S.M. Free Radic. Res. 1999; 31: 341-349Crossref PubMed Scopus (30) Google Scholar) whereas others such as protein phosphatases 1 (7Aggen J.B. Nairn A.C. Chamberlin R. Chem. Biol. 2000; 7: R13-R23Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and 2 (8Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (291) Google Scholar, 9Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 10Goldberg Y. Biochem. Pharmacol. 1999; 57: 321-328Crossref PubMed Scopus (129) Google Scholar) dephosphorylate many cellular proteins.p38α (19Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2401) Google Scholar, 20Lee 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. et al.Nature. 1994; 372: 739-746Crossref PubMed Scopus (3120) Google Scholar) is involved in relaying stress-related signals in mammalian cells. It is regulated by two MAPKKs termed MAP kinase kinase 3 (MKK3) and MAP kinase kinase 6 (MKK6) (21Han J. Lee J.D. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 22Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (314) Google Scholar), which phosphorylate the activation loop of p38α twice, once on a tyrosine and once on a threonine. Several activators of one or both of these enzymes have been identified that include MEKK4 (MAP or ERK kinase kinase) (23Porter A.C. Fanger G.R. Vaillancourt R.R. Oncogene. 1999; 18: 7794-7802Crossref PubMed Scopus (73) Google Scholar, 24Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 25Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), apoptosis-stimulated kinase (26Tobiume K. Inage T. Takeda K. Enomoto S. Miyazono K. Ichijo H. Biochem. Biophys. Res. Commun. 1997; 239: 905-910Crossref PubMed Scopus (67) Google Scholar, 27Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1999) Google Scholar), and transforming growth factor-β-activated kinase (28Hirose T. Fujimoto W. Tamaai T. Kim K.H. Matsuura H. Jetten A.M. Mol. Endocrinol. 1994; 8: 1667-1680PubMed Google Scholar, 29Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 30Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1169) Google Scholar). The basic region-leucine zipper protein activating transcription factor 2 (ATF2) is a substrate of p38α and other stress-activated MAPKs. It is a DNA-binding protein that forms a homodimer or heterodimer with c-Jun, binds to cyclic AMP-response elements (CREs), and stimulates CRE-dependent transcription of genes. Recently, it was also shown to be a histone acyltransferase, specific for histones H2B and H4 (31Kawasaki H. Schiltz L. Chiu R. Itakura K. Taira K. Nakatani Y. Yokoyama K.K. Nature. 2000; 405: 195-200Crossref PubMed Scopus (222) Google Scholar). Increases in its transcriptional activity (32van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (569) Google Scholar), its acyltransferase activity (31Kawasaki H. Schiltz L. Chiu R. Itakura K. Taira K. Nakatani Y. Yokoyama K.K. Nature. 2000; 405: 195-200Crossref PubMed Scopus (222) Google Scholar), and its cellular stability (33Fuchs S.Y. Tappin I. Ronai Z. J. Biol. Chem. 2000; 275: 12560-12564Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) are associated with the dual phosphorylation of ATF2 on Thr-69 and Thr-71.We found that the kinetic mechanism for the dual phosphorylation of the two residues within GST-ATF2-(1–115), Thr-69 and Thr-71, occurs by a two-step mechanism where p38α dissociates from the protein after each phosphorylation event. The kinetics of this process is predicted to have important implications for the activation of ATF2 by p38αin vivo and highlights the potential that two-step distributive mechanisms have for controlling the amplitude sensitivity of signal transduction pathways.DISCUSSIONIn this study we show that the stress-activated MAPK, p38α, dually phosphorylates the transcription factor ATF2 by a two-step (double collision) distributive mechanism and not by a one-step (single collision) processive mechanism. Our observation was surprising, because the manner that substrates are recognized by MAPKs appears to be ideal to promote a processive mechanism of substrate phosphorylation. There are two possible mechanisms of dual phosphorylation that can be distinguished by the relative order of substrate binding (Scheme FS3). The upper pathway in Scheme FS3 represents a processive mechanism of phosphorylation, where the protein substratedoes not dissociate from the enzyme until after the second phosphoryl transfer, k′p, has occurred. The requirements for this mechanism are fairly stringent because nucleotide exchange, substrate repositioning, and phosphoryl transfer mustall occur faster than the dissociation of the mono-phosphorylated intermediate (S*) from the respective ternary or binary complexes. The lower pathway corresponds to the less stringent, two-step, distributive pathway, where S* dissociates from the enzyme after the first phosphorylation, k p, and then recombines before the second, k′p.MAPKs are believed to utilize substrate-docking interactions that appear to be well situated to provide a common platform for the phosphorylation of multiple residues within a substrate through a processive mechanism (3Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (434) Google Scholar, 43Gavin A.C. Nebreda A.R. Curr. Biol. 1999; 9: 281-284Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 44Smith J.A. Poteet-Smith C.E. Malarkey K. Sturgill T.W. J. Biol. Chem. 1999; 274: 2893-2898Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). By using a docking site to maintain contact with a substrate and then simply "sliding" the active site from one phosphorylation site to the next, an MAPK could phosphorylate several residues without dissociating fully from the substrate. The catalytic advantage of such a process, over a nonprocessive mechanism, while not expected to be large for the dual phosphorylation of a protein, could be significant within the context of a signal transduction cascade, where the efficient propagation of a signal through the cascade may be critical. 7Typically a one-step processive enzymatic mechanism will give 50% product ∼2.5-fold faster than a two-step distributive mechanism, assuming comparable rate constants for the binding and chemical steps. Interestingly, it has been proposed and also examined theoretically, although not proven experimentally, that scaffold proteins facilitate the processive phosphorylation of proteins in signal transduction cascades (45Levchenko A. Bruck J. Sternberg P.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5818-5823Crossref PubMed Scopus (382) Google Scholar).ATF2 contains a putative MAPK docking site that is similar to the DEJL docking sequence, (K/R)(K/R)(K/R)X (1–5)(L/I)X(L/I), found in many other MAPK substrates (3Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (434) Google Scholar). This DEJL-like sequence,46KHKHEMTL53, found N-terminal to the phosphorylation sites, Thr-69 and Thr-71, within the trans-activation domain of ATF2, probably interacts with Asp-313 and Asp-316 of p38α and is predicted to be a primary specificity determinant (41Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (667) Google Scholar). Whereas the DEJL-like domain appears to be ideally situated to promote a processive mechanism, it is clear from our data that this does not happen and that the mono-phosphorylated GST-ATF2-(1–115) dissociates from p38α with a rate constant greater than, or equal to 0.6 s−1, the magnitude of GST-ATF2-(1–115) turnover. Previous studies with p38α uncovered a dependence ofk cat, for the phosphorylation of a peptide, on viscosity and also a small thiol effect (46Chen G. Porter M.D. Bristol J.R. Fitzgibbon M.J. Pazhanisamy S. Biochemistry. 2000; 39: 2079-2087Crossref PubMed Scopus (60) Google Scholar). This suggests that phosphoryl transfer is not rate-limiting for the phosphorylation of a peptide substrate and that a viscosity-sensitive conformational change or a product dissociation step is rate-limiting instead. We are currently investigating the kinetic mechanism of p38α in more detail to determine the rate-limiting step for the phosphorylation of GST-ATF2-(1–115). There are several possible reasons why the mechanism of ATF2 phosphorylation by p38α is not processive. One possibility is that the putative docking interactions are not the only interactions required to form a stable p38α-ATF2 complex and that other interactions within or near the active site of p38α are also necessary. This is supported by mutagenesis experiments on p38 isoforms that showed that docking site interactions are not the only interactions that determine the specificity of the interactions between p38 isoforms and protein substrates (47Gum R.J. Young P.R. Biochem. Biophys. Res. Commun. 1999; 266: 284-289Crossref PubMed Scopus (25) Google Scholar). If other interactions are critical for complex formation and become disrupted after the first phosphoryl transfer, ATF2 dissociation might quickly ensue, leading to a distributive mechanism.It will be interesting to determine whether other MAPKs, which exhibit potentially tighter binding interactions, phosphorylate their substrates with a processive mechanism. There have been several reports that suggest that in some cases MAPKs can bind tightly to docking domains. For example, the binding of the MAPK ERK2 to a glutathioneS-transferase fusion protein of the Ets domain transcription factor Elk-1 (GST-Elk1) immobilized on glutathione-agarose beads was shown to be strong enough to survive multiple washes by buffer (48Yang S.H. Yates P.R. Whitmarsh A.J. Davis R.J. Sharrocks A.D. Mol. Cell. Biol. 1998; 18: 710-720Crossref PubMed Scopus (232) Google Scholar). It was shown that the docking site interactions were both necessary and sufficient to maintain the ERK2-Elk-1 complex on the beads. Similar results were observed for several other MAPK/substrate interactions using a similar approach (49Kallunki T. Su B. Tsigelny I. Sluss H.K. Derijard B. Moore G. Davis R. Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (592) Google Scholar). However, it is possible that the tight binding of MAPK-substrate complexes seen on glutathione-agarose beads are due to nonspecific binding associated with the matrix that increases the affinity of the interactions.Although p38α showed similar specificity for Thr-71 and Thr-69 in GST-ATF2-(1–115) (Fig. 2 A), it displayed a marked difference in the specificity toward the mono-phosphoisomers. This demonstrates that although local sequence is not the primary determinant of p38α specificity; it can be very important nevertheless. Cantley and co-workers (42Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar) recently showed that ERK2 has a preference for a proline at the P-2 position, and therefore it is possible that phosphorylation of a threonine at this position can hinder binding of a substrate to p38α.Interestingly, many MAP kinase substrates appear to be phosphorylated, at least twice by a MAP kinase, before they become activated. Numerous examples of protein conformations regulated by single phosphorylations exist in the literature, so dual phosphorylation is not requiredper se. Why then is dual phosphorylation so common in MAPK cascades? It has been suggested that dual phosphorylation provides an added check against the inappropriate low rate activation by another protein kinase present in the cell (15Ferrell Jr., J.E. Bhatt R.R. J. Biol. Chem. 1997; 272: 19008-19016Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). This may be true for the activation of MAPK substrates, because different MAPKs, although similar in both structure and specificity, activate different proteins with different functions in vivo. Another possibility, however, is that multiple phosphorylation mechanisms (by the same enzyme) provide a simple and effective way of influencing the amplitude sensitivity of a pathway. The recent argument by Huang and Ferrell (17Huang C.Y. Ferrell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10078-10083Crossref PubMed Scopus (926) Google Scholar) that a two-step distributive mechanism can lead to anincrease in ultrasensitivity of a signaling pathway is compelling and could help explain the existence of the exquisite switch-like behavior of a MAPK cascade in Xenopus oocytes. Their laboratory investigated how the variable concentration of the maturation-inducing hormone progesterone, which activates the MAPKKK, Mos, is converted into a switch-like activation of MAPK. They concluded that the observed behavior is consistent with a model where at low concentrations of progesterone the MAPK remains inactive, but upon passing through a narrow concentration threshold of progesterone the MAPK becomes fully activated (50Ferrell Jr., J.E. Machleder E.M. Science. 1998; 280: 895-898Crossref PubMed Scopus (848) Google Scholar). Sigmoidal stimulus-response curves are important because they provide a way of switching from off to on over a narrow range of input and provide a threshold that can filter out base-line noise.Computer simulation of our experimentally observed kinetic results (Fig. 4) shows, however, that a distributive mechanism does not necessarily result in an increase in ultrasensitivity in vivo. In fact it appears to offer the potential for considerable variation in the degree of sensitivity. We observed a 40-fold reduction in the specificity of p38α toward ATF2−71P in vitro, and we used this as the basis for our calculations. Rather than model the reaction of p38α specifically, we examined the effect of decreasing the specificity of a MAPK for one of two intermediates formed as part of a two-step distributive mechanism (Scheme FS2 A). We assumed that the intermediates were initially formed in equal amounts from the unphosphorylated substrate but that the MAPK displays a 10-fold decrease in specificity toward one of them. The decrease in specificity was predicted to lead to a 3-fold increase in the range of MAPK activation required to drive the formation of the product from 10% to 90% maximum, assuming that the phosphatase did not discriminate between the phosphorylated products (Fig. 4). This conclusion is qualitatively independent of the level of the contribution from zero-order ultrasensitivity and illustrates how the degree of activation of a system in response to a signal could be adapted by mutations that alter the kinetic parameters of activation.The kinetics of ATF2 activation by p38α suggests that a further increase in ultrasensitivity at this level of the pathway is unnecessary. It will be interesting to determine whether the activation of p38α displays "switch-like" ultrasensitivity in response to cell stresses or whether its response is more hyperbolic, where control is exerted over a wider range of stimulus. It will also be interesting to compare the activation of other p38α substrates such as the transcription factors MEF2 (51Wang X.Z. Ron D. Science. 1996; 272: 1347-1349Crossref PubMed Scopus (738) Google Scholar) and CHOP (52Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. di Padova F. Olson E.N. Ulevitch R.J. Han J. Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (375) Google Scholar), because in each case, activation is brought about by the phosphorylation of two sites that lie close together in primary sequence. It will also be fascinating to determine how the activation of ATF2 by the other subfamily of stress-activated MAPKs, the c-Jun N-terminal protein kinases (JNKs), compares to p38α. Differences in the specificity could reflect differences in the dependence of ATF2 activation on the activity of the two subfamilies.In summary, the dual phosphorylation of protein substrates by a two-step distributive mechanism appears to offer considerable flexibility in the control of the amplitude-sensitivity of a pathway and is likely to contribute significantly to the signal/response profiles of numerous signal transduction pathways. At one extreme a distributive mechanism of multiple phosphorylation can increase the ultrasensitivity of a pathway, as is probably the case for the activation of MAPK by MAPKK in Xenopus oocytes (17Huang C.Y. Ferrell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10078-10083Crossref PubMed Scopus (926) Google Scholar). At the other extreme the same mechanism could help maintain a broader threshold, allowing the system to respond to a wider range of stimulus. These factors are likely to be important for the activation of a large number of enzymes and proteins regulated by MAPK cascades in eukaryotic organisms. The mitogen-activated protein kinases (MAPKs)1 are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are implicated in processes ranging from pheromone responses and cell wall formation in yeast to mitogenesis, apoptosis, and stress responses in mammalian cells (1Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (757) Google Scholar, 2Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2248) Google Scholar). They are the terminal components of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed. A MAP kinase cascade is generally composed of three protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK) arranged in a linear hierarchical fashion as shown in Scheme FS1. Although there are a large number of MAPKKKs, which become activated through a variety of mechanisms, MAPKKKs are highly specific for their conjugate MAPKK, which they activate by dual phosphorylation. In turn MAPKKs activate their conjugate MAPKs, also by dual phosphorylation, whereas MAPKs, which are the least specific kinases in these modules, phosphorylate many proteins, typically at more than one site (3Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (434) Google Scholar). Although this simple picture is somewhat obscured by the existence of an abundance of different MAPKKKs and by the existence of MAPKK and MAPK isoforms, the three-tiered hierarchy appears to be well conserved. The kinases do not function in isolation, because protein phosphatases represent the force against which they labor to drive the steady-state concentrations of phosphorylated proteins beyond critical thresholds. There are several protein phosphatases, some such as the MAPK phosphatases are specific for certain MAPKs (4Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (702) Google Scholar, 5Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (710) Google Scholar, 6Keyse S.M. Free Radic. Res. 1999; 31: 341-349Crossref PubMed Scopus (30) Google Scholar) whereas others such as protein phosphatases 1 (7Aggen J.B. Nairn A.C. Chamberlin R. Chem. Biol. 2000; 7: R13-R23Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and 2 (8Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (291) Google Scholar, 9Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 10Goldberg Y. Biochem. Pharmacol. 1999; 57: 321-328Crossref PubMed Scopus (129) Google Scholar) dephosphorylate many cellular proteins. p38α (19Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2401) Google Scholar, 20Lee 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. et al.Nature. 1994; 372: 739-746Crossref PubMed Scopus (3120) Google Scholar) is involved in relaying stress-related signals in mammalian cells. It is regulated by two MAPKKs termed MAP kinase kinase 3 (MKK3) and MAP kinase kinase 6 (MKK6) (21Han J. Lee J.D. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 22Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (314) Google Scholar), which phosphorylate the activation loop of p38α twice, once on a tyrosine and once on a threonine. Several activators of one or both of these enzymes have been identified that include MEKK4 (MAP or ERK kinase kinase) (23Porter A.C. Fanger G.R. Vaillancourt R.R. Oncogene. 1999; 18: 7794-7802Crossref PubMed Scopus (73) Google Scholar, 24Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 25Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), apoptosis-stimulated kinase (26Tobiume K. Inage T. Takeda K. Enomoto S. Miyazono K. Ichijo H. Biochem. Biophys. Res. Commun. 1997; 239: 905-910Crossref PubMed Scopus (67) Google Scholar, 27Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1999) Google Scholar), and transforming growth factor-β-activated kinase (28Hirose T. Fujimoto W. Tamaai T. Kim K.H. Matsuura H. Jetten A.M. Mol. Endocrinol. 1994; 8: 1667-1680PubMed Google Scholar, 29Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 30Yamaguchi K. Shirakabe K. S
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