MKK6 is a dual specificity mitogen-activated protein kinase that was first cloned by PCR using degenerate primers for the conserved kinase domain of MKKs (Han et al. 1996). MKK6 amino-acid sequence shares about 80% of similarity with MKK3 and 40% with MKK4 (Stein et al. 1996). Two MKK6 isoforms have been described in mouse and human (Cuenda et al. 1996; Han et al. 1996). The bigger isoform contains 334 amino acids and is highly expressed in heart, skeletal muscle, pancreas, and liver, while the smaller isoform of 278 amino acids has been detected only in skeletal muscle (Han et al. 1996).
Regulation of MKK6 Activity
Osmotic stress, ultraviolet light, and anisomycin activate MAP three kinase-1 (MTK1), which in turn phosphorylates MKK6 in a dose-dependent manner in COS and HeLa cells (Takekawa et al. 1997).
Transforming growth factor beta–activated protein kinase 1 (TAK1) has been shown to phosphorylate and activate MKK6 in vitro and in vivo (Moriguchi et al. 1996) and to be required to activate MKK6-p38 pathway during myogenic differentiation (Bhatnagar et al. 2010). Thousand and one amino acid kinase-2 (TAO2) coimmunoprecipitates and phosphorylates MKK6 during myogenic differentiation in the presence of stress stimuli such as sorbitol, sodium chloride, taxol, and nocodazole (Chen and Cobb, 2001). Apoptosis signal-regulating kinase 1 (ASK1) activates MKK6-p38 in response to proinflammatory cytokines and apoptotic stress (Ichijo et al. 1997). Noteworthy, phosphorylation mediated by ASK1 and TAO2 is characterized by a sequential order; first they phosphorylate MKK6 on Thr211 residue and subsequently on Ser207 (Humphreys et al. 2013). PKNα (a fatty-acid-activated and rho-activated serine/threonine protein kinase) has been shown to play an activating role in the MKK6-p38 pathway as an upstream activator of MTK1 and as a scaffold protein, associating with each member of the p38γ MAPK signaling pathway (p38γ, MKK6, and MTK1) (Takahashi et al. 2003). MKK6 activation has also been shown to be mediated by microtubules and Dctn1, a component of the dynein-dynactin complex involved in organelle transportation along the microtubules (Cheung et al. 2004).
Regarding MKK6 inactivation, the serine/threonine protein phosphatase type 2C alpha (PP2Cα) has been shown to dephosphorylate MKK6 in vitro and in vivo (Takekawa et al. 1998). Acetylation of serine 207 and threonine 211 by acetyltransferase YopJ (Yersinia outer protein J) inhibits MKK6 activity, indicating that phosphorylation and acetylation states of the activation loop domain are crucial to regulate MKK6 kinase activity. Importantly, the crystal structure of nonphosphorylated human MKK6 helped to reveal that MKK6 has a unique auto-inhibitory mechanism (different from the ones present in MKK4 or MKK1) which involves three short α-helices configured in the activation loop region (Matsumoto et al. 2012; Min et al. 2009).
In addition to modulation of MKK6 activity by direct enzymatic reactions, regulation of its protein and mRNA abundance have been shown to impact on MKK6 levels and thus in the magnitude of MKK6-p38 signaling. For instance, a negative feedback has been described in vitro and in vivo where one of MKK6 substrates, p38alpha, destabilizes MKK6 mRNA by promoting the dissociation of HuR from MKK6 3’UTR (Ambrosino et al. 2003). In accordance with this data, MKK3 deficient mice, which show lower p38 activation than WT mice, present higher amounts of MKK6 protein (Ma et al. 2007); however, whether p38α absence was indeed responsible for the observed increased MKK6 levels was not studied.
Upon activation, MKK6 phosphorylates p38α, p38β, p38γ, and p38δ on their regulatory threonine and tyrosine residues within a tripeptide motif (Thr-Gly-Tyr) located in the activation loop of p38 (Raingeaud et al. 1995). The specific recognition of MKK6-p38 binding is mediated by a consensus docking domain (Lys/Arg–Xaa3–Leu/Ile–Xaa–Leu/Ile) present in the N-terminal region of MKK6 (Enslen et al. 2000).
MKK3 and MKK4, who share highly similar catalytic domains to MKK6, can also phosphorylate and activate p38 mitogen-activated protein kinase (MAPK) family members (Derijard et al. 1995; Raingeaud et al. 1996). Genetic and biochemical evidence demonstrates nonredundant and selective functions for these MAPKKs in regulating the activity of p38 kinases in response to specific stimuli. For instance, p38α is activated by MKK6 and MKK3 in response to TNFα stimuli, while in response to UV light p38α is phosphorylated and activated by MKK6, MKK3, and also MKK4. Environmental stress activates MKK6 and MKK3, which in turn activate p38β and p38γ. However, MKK6 activation by TNFα results in activation of p38γ, while MKK3 activation by TNFα, UV light, osmotic shock, and anisomycin results in activation of p38δ (Remy et al. 2010). The complexity of MAPKs’ activation may reflect the ability to respond to multiple stimuli.
While phosphorylation of MKK6 in serine 207 and threonine 211 is an activatory event, MKK6 acetylation of these same residues by YopJ (Yersinia Outer protein J) has been shown to inhibit MKK6 activity. Therefore, MKK6 acetylation may be the mechanism by which YopJ blocks the innate immune response (Mukherjee et al. 2006).
Functional Roles of MKK6 Activity
Role of MKK6 in Cell Fate
MKK6-p38 pathway has been shown to promote differentiation of several cell types; the most studied being the myogenic lineage. MKK6 knockout mice are viable, fertile, and do not show developmental or tissue abnormalities. This data indicates that other kinases, most likely MKK3 and MKK4 who share highly homologous kinase domains, could compensate MKK6 loss in most important biological processes. Nevertheless, absence of MKK6 (or its activation impairment) as well as MKK6-forced activation have revealed important specific roles for MKK6 during cell fate decisions. For instance, MKK6−/− mice show impairment in double positive thymocyte apoptosis, indicating that MKK6 plays a role in thymocyte maturation (Tanaka et al. 2002).
MKK6 plays a crucial role in promoting myogenesis through the activation of p38 pathway (Zetser et al. 1999). Myogenic differentiation stimuli such as cell-to-cell contact activate the CDO-JLP immunoglobulin-scaffold protein complex, which in turn activates the MKK6-p38-α/β pathway (Takaesu et al. 2006). Other ligands, such as TNFα and amphoterin/RAGE, have been shown to induce myogenic differentiation through MKK6-p38 activation (Sorci et al. 2004). MKK6 exerts its promyogenic functions mainly through the p38 alpha kinase isoform (Perdiguero et al. 2007), which mediates the assembly of an active myogenic transcriptional complex by direct phosphorylation of its components: MEF2 myogenic transcriptional factors (Zhao et al. 1999; Rampalli et al. 2007), SWI/SNF (SWItch/Sucrose NonFermentable) chromatin remodeling subunits (Simone et al. 2004, Forcales et al. 2012), and E47 (Lluís et al. 2005). p38 beta and gamma have been reported to promote and inhibit muscle gene expression by different groups (Wang et al. 2008; Gillespie et al. 2009).
Importantly, the MKK6-p38 pathway has been shown to play a role in the survival of differentiated neurons. In response to calcium influx, the activation of MKK6-p38 pathway induces MEF2C phosphorylation in serine 387 and this event may activate the expression of survival genes and repress the expression of apoptotic genes (Mao et al. 1999).
Intriguingly, MKK6 activation can reprogram neutrophils towards the monocytic lineage in mice and human cells (Köfel et al. 2014). Upon MKK6-p38 activation by transducing mouse and human peripheral blood neutrophils with a dominant activated MKK6, the granulopoietic C/EBPa transcriptional factor is degraded, which facilitates monocytic differentiation. GM-CSF/TNFα/IL-1β inflammatory cytokines promoted MKK6 phosphorylation with concomitant c-Jun induction and C/EBPa degradation; this event was accompanied with the neutrophil to monocyte transdifferentiation. Surprisingly, MKK6-p38 inhibitors were not used in order to monitor whether this pathway is not only sufficient but also required for neutrophil to monocyte reprogramming.
MKK6 and Inflammation
MKK6 activation occurs in inflammatory diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA) (Chabaud-Riou and Firestein 2004). Blocking MKK6-p38 activity has been shown to attenuate these inflammatory diseases. For instance, a dominant negative MKK6 impairs the production of inflammatory cytokines (IL-6, IL-8) and the protease MMP-3 in IL-1 stimulated fibroblasts-like synoviocytes (Inoue et al. 2005). Furthermore, in a passive K/B × N mouse serum transfer model of arthritis, MKK6−/− mice show attenuation of arthritis, cartilage destruction, and bone erosion (Yoshizawa et al. 2009). In this study, MKK6 would be contributing to the colocalization of p38 with its substrate MK2 (MAPKAPK2).
In macrophages, LPS induces MKK6 phosphorylation and its binding with IRAK2. This event is required for the posttranscriptional control of cytokine and chemokine expression involved in the innate immune response (Wan et al. 2009).
Chronic inflammatory reactions are present in Alzheimer disease where glial cells release cytokines that are toxic to neurons and induce their degeneration (Akiyama et al. 2000). Importantly, activated MKK6 has been shown to localize with pathological structures such as neurofibrillary tangles, senile plaques, neuropil threads, and granular structures, only in the hippocampal and cortical regions of individuals with Alzheimer disease (Zhu et al. 2001). The authors suggest that activated MKK6-p38 pathway could promote cell death of damaged neurons and contribute like this to neurodegeneration, a hallmark of Alzheimer disease.
MKK6-p38 pathway plays an important cardioprotective role in the heart. In an in vivo model of infarction, transgenic mice hearts overexpressing MKK6 show a better functional recovery and less injury than nontransgenic mice (Martindale et al. 2005). One possible mechanism could be that MKK6-p38 activation in these MKK6-transgenic mice induced the expression of the heat shock protein αB-crystallin and the antiapoptotic Bcl-2 protein, which have been shown to play important cardioprotective roles.
All these works suggest that in an inflammatory environment, whether MKK6 is activated in cytokine producing cells or in cells receiving those inflammatory signals could result in opposite biological outcomes that include apoptosis versus cell survival.
MKK6 Role in Cancer
A tumor suppressor role has been described by different groups for the MKK6-p38 pathway, in part due to the observation that MKK6-p38 activation induces apoptosis in several cell lines, and this effect can be blocked by specifically inhibiting p38 with the SB203580 drug (Olson and Hallahan 2004). In addition, MKK6-p38 pathway is a critical regulator in G2-checkpoint, inducing cell cycle arrest in response to DNA damage (Wang et al. 2000). Fibroblasts isolated from MKK6−/− mice show an increased proliferation rate in serum-free medium compared to WT fibroblasts, correlating with the maintained expression of D-cyclins and the presence of phosphorylated Rb. Importantly, subcutaneous injection of these MKK6−/− fibroblasts into nude mice induces larger tumors compared to the WT fibroblasts (Brancho et al. 2003). Furthermore, a deficient MKK6-p38 signaling has been shown in certain types of rhabdomyosarcomas, where restoring a persistent activation of p38 MAPK pathway by the constitutively active mutant MKK6EE leads to tumor growth arrest and terminal differentiation (Puri et al. 2000). A similar approach was used in nude mice that were injected with HeLa (human cervical carcinoma) cells expressing a constitutively active MKK6 and showed reduced tumor formation (Timofeev et al. 2005). MKK6 levels were shown to be lower in 20 cases of liver cancer (Iyoda et al. 2003), suggesting that reduced MKK6-p38 signaling could represent an antiapoptotic mechanism giving growth advantages to these cells.
Thus, all these experiments indicate that absence of MKK6 signaling results in tumor progression, whereas activation of the MKK6-p38 pathway is sufficient to suppress in vivo tumorigenesis. In addition, an anti-metastatic effect has been attributed to the MKK6-p38 pathway; ectopic expression of MKK6 in an ovarian carcinoma cell line prevented the metastatic process when these cells were injected intraperitoneally (Hickson et al. 2006).
All these works reveal that cancer disease is highly heterogenic; a multistep process involving many players in a complex combinatorial system. Thus, it is not surprising to find MKK6-p38 kinase pathway involved in what may seem at first contradictory outcomes. Regarding this issue, some light has been shed by a recent work that demonstrated a dual role for p38a kinase in colorectal cancer, by displaying tumor suppression activity at early stages of the disease and promoting cell survival and thus fueling the oncogenic process at later phases (Gupta et al. 2014); however, whether MKK6 was the responsible kinase for p38 activation in colorectal cancer was not addressed.
Mitogen-activated protein kinase kinase-6 (MKK6) belongs to the MAPK kinase (MAPKK) family of enzymes, which specifically activates p38 MAPK. MKK6 has a similar sequence to MKK3 (80%) and to MKK4 (40%). Cellular stresses such as osmotic shock, UV irradiation, hypoxia, cytokine stimulation, and cell-to-cell contact activate the MKK6-p38 pathway by activating upstream kinases (MAPKKKs) including Ask1 (apoptosis signal-regulating kinase 1), Tak1 (transforming growth factor beta–activated kinase 1), Tao2 (1001 amino acids kinases 2), and Mtk1 (Mekk4/MAP three kinase 1). All of these protein kinases phosphorylate MKK6 on serine 207 and threonine 211 resulting in MKK6 activation. Active MKK6 phosphorylates p38α, β, δ, and γ MAPKs, on their regulatory threonine and tyrosine residues, activating p38 MAPK signaling. MKK6 is widely expressed in mammalian tissues with higher accumulation in skeletal muscle, where it plays a crucial role during myogenic differentiation. In cancer, several loss of function and gain of function assays support a tumor suppressor role for MKK6 activity; however, some reports are also showing protumorigenic features of MKK6. MKK6 activation in an inflammatory environment also seems to lead to antagonistic results.
In summary, MKK6 has major distinct roles in several biological processes. The specificity of its action depends on multiple factors such as the diversity of its activators, the selectivity of the available substrates, the concomitant signaling pathways being activated or repressed in a particular situation, the duration and magnitude of the activation, and the assembly with scaffold proteins among others. Because of its prominent signaling role in differentiation, inflammatory diseases, and cancer, the exploration of drugs targeting MKK6 activity is a challenging field to develop new therapeutic approaches.
- Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR, Eikelenboom P, Emmerling M, Fiebich BL, Finch CE, Frautschy S, Griffin WS, Hampel H, Hull M, Landreth G, Lue L, Mrak R, Mackenzie IR, McGeer PL, O’Banion MK, Pachter J, Pasinetti G, Plata-Salaman C, Rogers J, Rydel R, Shen Y, Streit W, Strohmeyer R, Tooyoma I, Van Muiswinkel FL, Veerhuis R, Walker D, Webster S, Wegrzyniak B, Wenk G, Wyss-Coray T. Inflammation and Alzheimer’s disease. Neurobiol Aging. 2000;21(3):383–421.PubMedPubMedCentralCrossRefGoogle Scholar
- Ambrosino C, Mace G, Galban S, Fritsch C, Vintersten K, Black E, Gorospe M, Nebreda AR. Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase. Mol Cell Biol. 2003;23(1):370–81. PubMed PMID: 12482988; PubMed Central PMCID: PMC140674.Google Scholar
- Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacobsen A, Byrne CJ, Heuer ML, Larsson E, Antipin Y, Reva B, Goldberg AP, Sander C, Schultz N. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2(5):401–4.Google Scholar
- Forcales SV, Albini S, Giordani L, Malecova B, Cignolo L, Chernov A, Coutinho P, Saccone V, Consalvi S, Williams R, Wang K, Wu Z, Baranovskaya S, Miller A, Dilworth FJ, Puri PL. Signal-dependent incorporation of MyoD-BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex. EMBO J. 2012;31(2):301–16. doi: 10.1038/emboj.2011.391.PubMedCrossRefGoogle Scholar
- Gupta J, del Barco Barrantes I, Igea A, Sakellariou S, Pateras IS, Gorgoulis VG, Nebreda AR. Dual function of p38Œ± MAPK in colon cancer: suppression of colitis-associated tumor initiation but requirement for cancer cell survival. Cancer Cell. 2014;25(4):484–500. doi: 10.1016/j.ccr.2014.02.019. Epub 2014 Mar 27. PubMedGoogle Scholar
- Humphreys JM, Piala AT, Akella R, He H, Goldsmith EJ. Precisely ordered phosphorylation reactions in the p38 mitogen-activated protein (MAP) kinase cascade. J Biol Chem. 2013;288(32):23322–30. doi: 10.1074/jbc.M113.462101. Epub 2013 Jun 6. PubMed PMID: 23744074; PubMed Central PMCID: PMC3743502.
- Huth HW, Albarnaz JD, Torres AA, Bonjardim CA, Ropert C. MEK2 controls the activation of MKK3/MKK6-p38 axis involved in the MDA-MB-231 breast cancer cell survival: correlation with cyclin D1 expression. Cell Signal. 2016;28(9):1283–91. doi: 10.1016/j.cellsig.2016.05.009. Epub 2016 May 13. PubMed PMID: 27181679.
- Igea A, Nebreda AR. The stress kinase p38a as a target for cancer therapy. Cancer Res. 2015;75(19):3997–4002. doi: 10.1158/0008-5472.CAN-15-0173. Epub 2015 Sep 16. Review. PubMed PMID: 26377941.
- Köfel R, Meshcheryakova A, Warszawska J, Hennig A, Wagner K, Jörgl A, Gubi D, Moser D, Hladik A, Hoffmann U, Fischer MB, van den Berg W, Koenders M, Scheinecker C, Gesslbauer B, Knapp S, Strobl H. Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation. Blood. 2014;124(17):2713–24.CrossRefGoogle Scholar
- Ma FY, Tesch GH, Flavell RA, Davis RJ, Nikolic-Paterson DJ. MKK3-p38 signaling promotes apoptosis and the early inflammatory response in the obstructed mouse kidney. Am J Physiol Renal Physiol. 2007;293(5):F1556–63. Epub 2007 Aug 8. PubMed PMID: 17686961.Google Scholar
- Martindale JJ, Wall JA, Martinez-Longoria DM, Aryal P, Rockman HA, Guo Y, Bolli R, Glembotski CC. Overexpression of mitogen-activated protein kinase kinase 6 in the heart improves functional recovery from ischemia in vitro and protects against myocardial infarction in vivo. J Biol Chem. 2005;280(1):669–76.PubMedCrossRefGoogle Scholar
- Matsumoto T, Kinoshita T, Matsuzaka H, Nakai R, Kirii Y, Yokota K, Tada T. Crystal structure of non-phosphorylated MAP2K6 in a putative auto-inhibition state. J Biochem. 2012;151(5):541–9. doi: 10.1093/jb/mvs023. Epub 2012 Mar 1. PubMed PMID: 22383536
- Moriguchi T, Toyoshima F, Gotoh Y, Iwamatsu A, Irie K, Mori E, Kuroyanagi N, Hagiwara M, Matsumoto K, Nishida E. Purification and identification of a major activator for p38 from osmotically shocked cells. Activation of mitogen-activated protein kinase kinase 6 by osmotic shock, tumor necrosis factor-alpha, and H2O2. J Biol Chem. 1996;271(43):26981–8.PubMedCrossRefGoogle Scholar
- Olson JM, Hallahan AR. p38 MAP kinase: a convergence point in cancer therapy. Trends Mol Med. 2004;10(3):125–9. Review. PubMed PMID: 15102355Google Scholar
- Parray AA, Baba RA, Bhat HF, Wani L, Mokhdomi TA, Mushtaq U, Bhat SS, Kirmani D, Kuchay S, Wani MM, Khanday FA. MKK6 is upregulated in human esophageal, stomach, and colon cancers. Cancer Invest. 2014;32(8):416–22. doi: 10.3109/07357907.2014.933236. Epub 2014 Jul 14. PubMed PMID: 25019214
- Raingeaud J, Whitmarsh AJ, Barrett T, Dérijard B, Davis RJ. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol. 1996;16(3):1247–55. PubMed PMID: 8622669; PubMed Central PMCID: PMC231107.Google Scholar
- Takekawa M, Maeda T, Saito H. Protein phosphatase 2Calpha inhibits the human stress-responsive p38 and JNK MAPK pathways. EMBO J. 1998;17(16):4744–52. PubMed PMID: 9707433; PubMed Central PMCID: PMC1170803.Google Scholar
- Wan Y, Xiao H, Affolter J, Kim TW, Bulek K, Chaudhuri S, Carlson D, Hamilton T, Mazumder B, Stark GR, Thomas J, Li X. Interleukin-1 receptor-associated kinase 2 is critical for lipopolysaccharide-mediated post-transcriptional control. J Biol Chem. 2009;284(16):10367–75.PubMedPubMedCentralCrossRefGoogle Scholar