Mapkap Kinase 2/3 (MK2/3)
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
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.
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.
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).
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.
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