Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Mapkap Kinase 2/3 (MK2/3)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_531

Synonyms

Historical Background

Formally, all protein kinases downstream to MAP kinases should be regarded as MAPK-activated protein kinases (MKs). This would include ribosomal S6-kinases (RSKs); mitogen- and stress-activated protein kinases (MSKs); MAP kinase-interacting kinases (MNKs); as well as MK2, MK3, and MK5/PRAK (Roux and Blenis 2004). However, due to the specificity of activation, this group of protein kinases is further subdivided (Fig. 1): RSKs are activated by classical ERKs (Erk1/Erk2) and MSKs and MNKs are phosphorylated by both ERKs and p38 MAPK. MK2 and MK3 are the only MKs which are exclusively activated by p38 MAPK. MK5/PRAK has been described as a “p38-regulated and -activated kinase (PRAK),” but its activation by p38 MAPK is challenged, and alternative activation by atypical MAPKs (Erk3/Erk4) is discussed (Gaestel 2006). For historical reasons RSK was designated MAPKAPK 1 (MK1), and the name MK4 was given to MK2 of sea urchin.
Mapkap Kinase 2/3 (MK2/3), Fig. 1

Classification of protein kinases downstream to MAPKs. For details, see text. Adapted from Gaestel (2006) with modifications

A stress-induced small heat shock protein-kinase activity was described as early as in 1983 (Kim et al. 1983), but identification of this activity as the protein kinases MK2 (Stokoe et al. 1992) and MK3 (McLaughlin et al. 1996) took more than 10 years. Characterization of the physiological roles of these protein kinases is still ongoing.

Structure, Activation, and Expression

According to their primary structure, MK2/3 belong to the family of calcium/calmodulin-dependent protein kinases (Camk). However, their activity does not depend on calcium, but on the phosphorylation of regulatory sites within the catalytic domain and in a hinge region between the catalytic domain and the C-terminus (Ben-Levy et al. 1995, Engel et al. 1995) (see Fig. 2). In human MK2, two phosphorylation sites are located within the catalytic domain, T222 and S272, and a further phosphorylation site is T334 in the hinge region. Similar sites were also identified in MK3. These are proline-directed sites which are phosphorylated by p38 MAPKα,β (Freshney et al. 1994, Rouse et al. 1994). The N-terminus of MK2/3 contains a proline-rich region which is able to bind to SH3 domain-containing proteins in vitro (Plath et al. 1994). The C-terminus of MK2/3 is a very interesting part, since it contains different signals for regulation of subcellular localization (nuclear export signal – NES; nuclear import signal – NLS). Furthermore, the C-terminus may have inhibitory effects on the catalytic domain (Engel et al. 1995) and tightly binds to the activator kinase p38 MAPKα (White et al. 2007). The binding specificity between MK2 and p38 MAPKα depends on two regions of p38, the common docking (CD) domain and the ED motif, and on the NLS of MK2 (Gum and Young 1999, Tanoue et al. 2001).
Mapkap Kinase 2/3 (MK2/3), Fig. 2

Primary structure of MK2 and MK3. MK2 and MK3 display 78% identity in their amino acid sequence and contain N-terminal proline-rich domains (P-rich), central protein-kinase domains (catalytic), and C-terminal regulatory tails which contain a nuclear export signal (NES) and a bi-partite nuclear localization motif (NLS). The NLS overlaps with the p38 MAPK-docking site. At least two regulatory phosphorylation sites are present in both enzymes: one threonine within the catalytic domain at the activation loop (T222) and the other threonine in the hinge region between kinase domain and C-terminal tail

A variant of the cDNA of human MK2 that codes for an alternative C-terminus without NES and NLS has been described (Zu et al. 1994). MK2 also migrates as two (mouse, 46 and 54 kDa; human, 53 and 60 kDa) distinct bands in SDS–PAGE (Stokoe et al. 1992, Cano et al. 1996), which are both absent from MK2-knockout fibroblasts (Kotlyarov et al. 1999), and has two biochemically distinct forms (p43 and p49) in cardiac myocytes (Chevalier and Allen 2000). So far, it is not completely clear whether both bands detected for MK2 correspond to different proteins based on an alternatively spliced transcript or to posttranslational modification/processing of MK2.

MK2 and MK3 are highly expressed in heart and skeletal muscle, but their activity can also be detected in most other tissues and cell lines. Compared to expression of MK2, expression of MK3 is much lower, making it a “minor isoenzyme” of MK2. This explains the clear phenotype of the MK2 deletion (Kotlyarov et al. 1999), which cannot be compensated by MK3 because of its low expression (see below).

The regulation of MK2/3’s subcellular localization is an interesting issue. GFP-tagged, overexpressed MK2/3 is mainly localized in the nucleus and translocated to the cytoplasm after activation (Engel et al. 1998). Whether this mechanism of coupled activation and translocation is of importance to the endogenous enzymes and whether this mechanism is modulated by complex formation of MK2/3 with p38 MAPK is so far not clear.

Substrates

The human kinome contains more than 500 protein kinases, and the human proteome is thought to comprise more than 10,000 different phosphorylation sites. Hence, an average protein kinase should phosphorylate more than 20 amino acid residues of various substrate proteins. Knowing the complete substrate spectrum of MK2/3 could be very helpful for understanding its downstream signaling in different cell types and physiological situations. The optimal phosphorylation site motif for MK2/3 has been defined as (L,F,I)-X-R-(Q,S,T)-L-(pS,pT)-hydrophobic. So far, no difference in substrate-specificity between MK2 and MK3 has been detected (Clifton et al. 1996), although some functional differences between MK2 and MK3 emerged recently (Ehlting et al. 2011; Guess et al. 2013). Several substrates of MK2/3 have been described. Besides the major substrate Hspb1, the following substrates have been identified: myosin II regulatory light chain, lymphocyte-specific protein 1, tyrosine hydroxylase, αB-crystallin, vimentin, serum response factor, transcription factors E47 and ER81, 5-lipoxygenase, poly(A)-binding protein 1, tuberin, hnRNP A0, p16-Arc, LIM-kinase 1 (LIMK), NOGO-B, 14-3-3ζ, tristetraproline (TTP), p66-Shc, Bcl-2-associated athanogene 2, polycomb-group protein Bmi 1, DNA-damage response protein phosphatase Cdc25B/C, and the p53 E3 ubiquitin ligase HDM2 (for references see supplementary table of (Gaestel 2006)). In the last years, further enzymes have been characterized as substrates of MK2/3, such as the ribosomal S6-kinase Rsk (Zaru et al. 2007), the phosphodiesterase-4A5 (Mackenzie et al. 2011), and the ubiquitin-conjugating enzyme Ube2j1 (Menon et al. 2013). Also, further mRNA-binding proteins, such as BRF1 and RBM7 (Maitra et al. 2008; Tiedje et al. 2015), and the autophagy protein beclin1 (Wei et al. 2015) have been identified as MK2 substrates. However, at the moment, not all in vivo substrates of MK2/3 have been verified in vivo. The physiological function of phosphorylation of most of the MK2/3 substrates is far from being completely understood.

Physiological Roles

The physiological role of MK2 became mainly evident from the MK2-knockout mouse, which is viable and fertile but displays resistance against endotoxic shock (Kotlyarov et al. 1999) and collagen-induced arthritis (Hegen et al. 2006) as well as increased susceptibility in a Listeria infection model (Lehner et al. 2002). These phenotypes can be explained by MK2/3-dependent regulation of expression of the inflammatory master cytokine TNF at the posttranscriptional level of mRNA stability and translation (Kotlyarov et al. 1999, Neininger et al. 2002). Mechanistically, this regulation proceeds for AU-rich element (ARE)-containing mRNAs, such as TNF mRNA, via phosphorylation of ARE-binding proteins, such as hnRNP A0 (Rousseau et al. 2002) and TTP (Stoecklin et al. 2004). Recently, a comprehensive model of the regulation of TNF mRNA by MK2 and p38 has been proposed (Fig. 3). In this model, the competition between the two ARE-binding proteins TTP, which destabilizes the TNF mRNA and blocks its translation, and HuR, which stabilizes TNF mRNA and facilitates its translation, is regulated by phosphorylation of TTP. Phosphorylation of TTP decreases its affinity for the ARE and initiates its replacement by HuR (Tiedje et al. 2012, 2014).
Mapkap Kinase 2/3 (MK2/3), Fig. 3

A comprehensive model of the regulation of TNF mRNA by MK2 and p38 in macrophages. Nonphosphorylated TTP binds to the ARE (AUUUA) of TNF mRNA and interferes with translational initiation by competition with eIF4G binding to PABP1 or by inhibition of binding of the 43S preinitiation complex (left). Bacterial lipopolysaccharide (LPS) induces increased expression of TNF mRNA and TTP protein and activates p38 and MK2/3. Phosphorylation of TTP by MK2/3 impairs its capacity to compete with cytoplasmic HuR at the ARE of TNF mRNAs, and phosphorylation of HuR by p38 leads to its translocation from the nucleus (n) to the cytoplasm (c). As a result, HuR shows increased binding to the ARE and stimulates initiation of translation of TNF mRNA facilitating TNF biosynthesis (right). Subsequently, dephosphorylation of TTP by protein phosphatase 2A (PP2A) increases its affinity to ARE and leads to the replacement of HuR by nonphospho-TTP and downregulation of TNF production (from Tiedje et al. 2012)

In a MK2-free genetic background deletion of MK3 leads to a further slight, but significant reduction of TNF production, indicating cooperative action of both enzymes (Ronkina et al. 2007). Apart from displaying catalytic activity, MK2/3 bind to p38 MAPK and mutually stabilize each other by protein complex formation. In MK2 knockout and MK2/3-double knockout mice, a significantly reduced p38 MAPK level is detected (Ronkina et al. 2007). Further physiological roles for MK2/3 include cell cycle checkpoint control, cell migration, and general stress response.

Because of its contribution to the production of inflammatory cytokines and because of the toxicity of p38 MAPK-inhibitors, MK2 becomes increasingly of interest as a target for anti-inflammatory therapy (Gaestel et al. 2009, 2013). First small molecule inhibitors of MK2 and/or MK3 have been reported. Of these, the orally available small molecule MK2 inhibitor of the benzothiophene type, PF-3644022, was demonstrated to be effective in a chronic streptococcal cell-wall-induced arthritis model in rats (Mourey et al. 2010).

Summary

MK2/3 are p38 MAPK-activated protein kinases that are stimulated by different stresses such as heat shock, hypo- and hyperosmolarity, and treatment with anisomycin or arsenite as well as by bacterial lipopolysaccharide (LPS) and chemotaxis-inducing formyl peptides. MK2 and MK3 show different levels of expression and activity, making MK2 the major and MK3 the minor “isoform.” MK2/3 are involved in stress and immune response by the modulation of production of cytokines such as TNF, mainly at the posttranscriptional level, regulating cytokine messenger RNA stability and translation. mRNA-binding substrates such as tristetraproline (TTP), hnRNP A0, and possibly also poly(A)-binding protein 1 are involved in this regulation. The existence of a wide variety of further substrates identified for MK2/3 indicates various other physiological functions for these protein kinases in vivo. One of the major substrates of MK2/3, the small heat shock protein Hspb1, contributes to stabilization of the actin cytoskeleton and acts as a molecular chaperone.

References

  1. Ben-Levy R, Leighton IA, Doza YN, Attwood P, Morrice N, Marshall CJ, et al. Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. EMBO J. 1995;14:5920–30.PubMedPubMedCentralGoogle Scholar
  2. Cano E, Doza YN, Ben-Levy R, Cohen P, Mahadevan LC. Identification of anisomycin-activated kinases p45 and p55 in murine cells as MAPKAP kinase-2. Oncogene. 1996;12:805–12.PubMedGoogle Scholar
  3. Chevalier D, Allen BG. Two distinct forms of MAPKAP kinase-2 in adult cardiac ventricular myocytes. Biochemistry. 2000;39:6145–56.PubMedCrossRefGoogle Scholar
  4. Clifton AD, Young PR, Cohen P. A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress. FEBS Lett. 1996;392:209–14.PubMedCrossRefGoogle Scholar
  5. Ehlting C, Ronkina N, Böhmer O, Albrecht U, Bode KA, Lang KS, et al. Distinct functions of the mitogen-activated protein kinase-activated protein (MAPKAP) kinases MK2 and MK3: MK2 mediates lipopolysaccharide-induced signal transducers and activators of transcription 3 (STAT3) activation by preventing negative regulatory effects of MK3. J Biol Chem. 2011;286(27):24113–24.PubMedPubMedCentralCrossRefGoogle Scholar
  6. Engel K, Schultz H, Martin F, Kotlyarov A, Plath K, Hahn M, et al. Constitutive activation of mitogen-activated protein kinase-activated protein kinase 2 by mutation of phosphorylation sites and an A-helix motif. J Biol Chem. 1995;270:27213–21.PubMedCrossRefGoogle Scholar
  7. Engel K, Kotlyarov A, Gaestel M. Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. EMBO J. 1998;17:3363–71.PubMedPubMedCentralCrossRefGoogle Scholar
  8. Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J, et al. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell. 1994;78:1039–49.PubMedCrossRefGoogle Scholar
  9. Gaestel M. MAPKAP kinases – MKs – two’s company, three’s a crowd. Nat Rev Mol Cell Biol. 2006;7:120–30.PubMedCrossRefGoogle Scholar
  10. Gaestel M. What goes up must come down: molecular basis of MAPKAP kinase 2/3-dependent regulation of the inflammatory response and its inhibition. Biol Chem. 2013;394(10):1301–15.PubMedCrossRefGoogle Scholar
  11. Gaestel M, Kotlyarov A, Kracht M. Targeting innate immunity protein kinase signalling in inflammation. Nat Rev Drug Discov. 2009;8(6):480–99.PubMedCrossRefGoogle Scholar
  12. Guess AJ, Ayoob R, Chanley M, Manley J, Cajaiba MM, Agrawal S, et al. Crucial roles of the protein kinases MK2 and MK3 in a mouse model of glomerulonephritis. PLoS ONE. 2013;8(1):e54239.PubMedPubMedCentralCrossRefGoogle Scholar
  13. Gum RJ, Young PR. Identification of two distinct regions of p38 MAPK required for substrate binding and phosphorylation. Biochem Biophys Res Commun. 1999;266:284–9.PubMedCrossRefGoogle Scholar
  14. Hegen M, Gaestel M, Nickerson-Nutter CL, Lin LL, Telliez JB. MAPKAP kinase 2-deficient mice are resistant to collagen-induced arthritis. J Immunol. 2006;177:1913–7.PubMedCrossRefGoogle Scholar
  15. Kim YJ, Shuman J, Sette M, Przybyla A. Phosphorylation pattern of a 25 Kdalton stress protein from rat myoblasts. Biochem Biophys Res Commun. 1983;117:682–7.PubMedCrossRefGoogle Scholar
  16. Kotlyarov A, Neininger A, Schubert C, Eckert R, Birchmeier C, Volk HD, et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol. 1999;1:94–7.PubMedCrossRefGoogle Scholar
  17. Lehner MD, Schwoebel F, Kotlyarov A, Leist M, Gaestel M, Hartung T. Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection. J Immunol. 2002;168:4667–73.PubMedCrossRefGoogle Scholar
  18. Mackenzie KF, Wallace DA, Hill EV, Anthony DF, Henderson DJP, Houslay DM, et al. Phosphorylation of cAMP-specific PDE4A5 (phosphodiesterase-4A5) by MK2 (MAPKAPK2) attenuates its activation through protein kinase A phosphorylation. Biochem J. 2011;435(3):755–69.PubMedCrossRefGoogle Scholar
  19. Maitra S, Chou C-F, Luber CA, Lee K-Y, Mann M, Chen C-Y. The AU-rich element mRNA decay-promoting activity of BRF1 is regulated by mitogen-activated protein kinase-activated protein kinase 2. RNA. 2008;14(5):950–9.PubMedPubMedCentralCrossRefGoogle Scholar
  20. McLaughlin MM, Kumar S, McDonnell PC, Van Horn S, Lee JC, Livi GP, et al. Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. J Biol Chem. 1996;271:8488–92.PubMedCrossRefGoogle Scholar
  21. Menon MB, Tiedje C, Lafera J, Ronkina N, Konen T, Kotlyarov A, et al. Endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 is a novel substrate of MK2 (MAPKAP kinase-2) involved in MK2-mediated TNFα production. Biochem J. 2013;456(2):163–72.PubMedCrossRefGoogle Scholar
  22. Mourey RJ, Burnette BL, Brustkern SJ, Daniels JS, Hirsch JL, Hood WF, et al. A benzothiophene inhibitor of mitogen-activated protein kinase-activated protein kinase 2 inhibits tumor necrosis factor alpha production and has oral anti-inflammatory efficacy in acute and chronic models of inflammation. J Pharmacol Exp Ther. 2010;333:797–807.PubMedCrossRefGoogle Scholar
  23. Neininger A, Kontoyiannis D, Kotlyarov A, Winzen R, Eckert R, Volk HD, et al. MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels. J Biol Chem. 2002;277:3065–8.PubMedCrossRefGoogle Scholar
  24. Plath K, Engel K, Schwedersky G, Gaestel M. Characterization of the proline-rich region of mouse MAPKAP kinase 2: influence on catalytic properties and binding to the c-abl SH3 domain in vitro. Biochem Biophys Res Commun. 1994;203:1188–94.PubMedCrossRefGoogle Scholar
  25. Ronkina N, Kotlyarov A, Dittrich-Breiholz O, Kracht M, Hitti E, Milarski K, et al. The mitogen-activated protein kinase (MAPK)-activated protein kinases MK2 and MK3 cooperate in stimulation of tumor necrosis factor biosynthesis and stabilization of p38 MAPK. Mol Cell Biol. 2007;27:170–81.PubMedCrossRefGoogle Scholar
  26. Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell. 1994;78:1027–37.PubMedCrossRefGoogle Scholar
  27. Rousseau S, Morrice N, Peggie M, Campbell DG, Gaestel M, Cohen P. Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs. EMBO J. 2002;21:6505–14.PubMedPubMedCentralCrossRefGoogle Scholar
  28. Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev. 2004;68:320–44.PubMedPubMedCentralCrossRefGoogle Scholar
  29. Stoecklin G, Stubbs T, Kedersha N, Wax S, Rigby WF, Blackwell TK, et al. MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay. EMBO J. 2004;23:1313–24.PubMedPubMedCentralCrossRefGoogle Scholar
  30. Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 1992;313:307–13.Google Scholar
  31. Tanoue T, Maeda R, Adachi M, Nishida E. Identification of a docking groove on ERK and p38 MAP kinases that regulates the specificity of docking interactions. EMBO J. 2001;20:466–79.PubMedPubMedCentralCrossRefGoogle Scholar
  32. Tiedje C, Ronkina N, Tehrani M, Dhamija S, Laass K, Holtmann H, et al. The p38/MK2-driven exchange between tristetraprolin and HuR regulates AU-rich element-dependent translation. PLoS Genet. 2012;8(9):e1002977.PubMedPubMedCentralCrossRefGoogle Scholar
  33. Tiedje C, Holtmann H, Gaestel M. The role of mammalian MAPK signaling in regulation of cytokine mRNA stability and translation. J Interf Cytokine Res. 2014;34(4):220–32.CrossRefGoogle Scholar
  34. Tiedje C, Lubas M, Tehrani M, Menon MB, Ronkina N, Rousseau S, et al. p38 MAPK/MK2-mediated phosphorylation of RBM7 regulates the human nuclear exosome targeting complex. RNA. 2015;21(2):262–78.PubMedPubMedCentralCrossRefGoogle Scholar
  35. Wei Y, An Z, Zou Z, Sumpter R, Su M, Zang X, et al. The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation. eLife. 2015;4:e05289.Google Scholar
  36. White A, Pargellis CA, Studts JM, Werneburg BG, Farmer 2nd BT. Molecular basis of MAPK-activated protein kinase 2: p38 assembly. Proc Natl Acad Sci USA. 2007;104:6353–8.PubMedPubMedCentralCrossRefGoogle Scholar
  37. Zaru R, Ronkina N, Gaestel M, Arthur JSC, Watts C. The MAPK-activated kinase Rsk controls an acute Toll-like receptor signaling response in dendritic cells and is activated through two distinct pathways. Nat Immunol. 2007;8(11):1227–35.PubMedCrossRefGoogle Scholar
  38. Zu YL, Wu F, Gilchrist A, Ai Y, Labadia ME, Huang CK. The primary structure of a human MAP kinase activated protein kinase 2. Biochem Biophys Res Commun. 1994;200:1118–24.PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Hannover Medical SchoolInstitute of Cell BiochemistryHannoverGermany