MAP kinase-activated protein kinase 2 (MAPKAP-K2, MK2; Gene ID: 9261) is a 400 AA (46kDa) large enzyme that plays a central role mainly in the inflammatory response and cytokines production. It belongs to the serine/threonine-protein kinase family and is also involved in endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling, DNA damage response and transcriptional regulation.1-4
Following stress, it is phosphorylated (at Thr-222, Ser-272 and Thr-334) and activated by MAP kinase p38-alpha/MAPK14, leading to phosphorylation of substrates.5 Phosphorylation of Thr-334 (located between the kinase domain and the C-terminal regulatory domain) may serve as a switch for MK2 nuclear import and export. Phosphorylated MK2 masks the nuclear localization signal at its C-terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.6, 7
The heterodimer of MK2 with p38-alpha/MAPK14 forms a stable complex: molecules are positioned 'face to face' so that the ATP-binding sites of both kinases are at the heterodimer interface.8, 9 Other important interaction partners of MK2 have been identified, such as AKT110, HSP27/HspB111, 12, HSF113, PHC2 and SHC114.
Besides phosphorylation another important regulatory post-translational modification of MK2 is described: Sumoylation, which inhibits the protein kinase activity.15
Recently the inhibition of MK2/3 has been identified as an emerging strategy to manipulate the inflammatory response as a therapeutic option.16-18 Small-molecule pharmaceutical inhibitors SB-203580 or genistein block the activation of MK2.19, 20
All these data, together with the growing number of publication on the complex network organization (http://gopubmed.org/web/gopubmed/) underline the importance of MK2 in biomedical research.21
To enable researches to answer the many open questions regarding MAPK-activated protein kinase 2 faster and easier we have now developed our highly reliable MK2-Trap. MK2-Trap (just like our widely used GFP-Trap®) is based on our high quality alpaca antibody fragments (VHH, sdAb, nanobody) coupled to agarose beads. It facilitates the biochemical analysis of MK2 and its interacting partners. Specifically pull down human, murine or hamster MK2 from any biological sample (e.g. cell extracts or tissue material) irrespective of the phosphorylation status or other modifications. Efficiently perform subsequent analysis like Western Blot, Mass spectroscopy or enzyme activity measurements.
As our collaboration partner Prof. Dr. Matthias Gaestel (Head of Institute of Physiological Chemistry, Hannover Medical University, Germany) stated: “The MK2-Trap beads are great for pull-down from lysate; nearly full depletion of MK2 from the supernatant and also nice Co-IP of p38! Thus the beads will be well suited to purify sufficient material of endogenous MK2 for biochemical analysis.“
In a next step we will soon also introduce our MK2-Chromobody® for the intracellular live-cell analysis of endogenous MK2. Just write me an email or leave a comment below if you are interested in this exciting new research tool and want to learn more!
1. Clifton, A.D.; Young, P.R.; 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, 209-214.
2. Kobayashi, M.; Nishita, M.; Mishima, T., et al. MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration. The EMBO journal. 2006, 25, 713-726.
3. Manke, I.A.; Nguyen, A.; Lim, D., et al. MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. Molecular cell. 2005, 17, 37-48.
4. Kopper, F.; Bierwirth, C.; Schon, M., et al. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity. Proceedings of the National Academy of Sciences of the United States of America. 2013, 110, 16856-16861.
5. Ben-Levy, R.; Leighton, I.A.; Doza, Y.N., et al. Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. The EMBO journal. 1995, 14, 5920-5930.
6. Meng, W.; Swenson, L.L.; Fitzgibbon, M.J., et al. Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export. J Biol Chem. 2002, 277, 37401-37405.
7. Reinhardt, H.C.; Hasskamp, P.; Schmedding, I., et al. DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization. Molecular cell. 2010, 40, 34-49.
8. ter Haar, E.; Prabhakar, P.; Liu, X., et al. Crystal structure of the p38 alpha-MAPKAP kinase 2 heterodimer. J Biol Chem. 2007, 282, 9733-9739.
9. White, A.; Pargellis, C.A.; Studts, J.M., et al. Molecular basis of MAPK-activated protein kinase 2:p38 assembly. Proceedings of the National Academy of Sciences of the United States of America. 2007, 104, 6353-6358.
10. Rane, M.J.; Coxon, P.Y.; Powell, D.W., et al. p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. J Biol Chem. 2001, 276, 3517-3523.
11. Stokoe, D.; Engel, K.; Campbell, D.G., et al. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 1992, 313, 307-313.
12. Lavoie, J.N.; Lambert, H.; Hickey, E., et al. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. Molecular and cellular biology. 1995, 15, 505-516.
13. Wang, X.; Khaleque, M.A.; Zhao, M.J., et al. Phosphorylation of HSF1 by MAPK-activated protein kinase 2 on serine 121, inhibits transcriptional activity and promotes HSP90 binding. J Biol Chem. 2006, 281, 782-791.
14. Yannoni, Y.M.; Gaestel, M.; Lin, L.L. P66(ShcA) interacts with MAPKAP kinase 2 and regulates its activity. FEBS Lett. 2004, 564, 205-211.
15. Chang, E.; Heo, K.S.; Woo, C.H., et al. MK2 SUMOylation regulates actin filament remodeling and subsequent migration in endothelial cells by inhibiting MK2 kinase and HSP27 phosphorylation. Blood. 2011, 117, 2527-2537.
16. Ronkina, N.; Kotlyarov, A.; Gaestel, M. MK2 and MK3--a pair of isoenzymes? Frontiers in bioscience : a journal and virtual library. 2008, 13, 5511-5521.
17. Gaestel, M. What goes up must come down: molecular basis of MAPKAP kinase 2/3-dependent regulation of the inflammatory response and its inhibition. Biological chemistry. 2013, 394, 1301-1315.
18. Gaestel, M.; Kotlyarov, A.; Kracht, M. Targeting innate immunity protein kinase signalling in inflammation. Nature reviews. Drug discovery. 2009, 8, 480-499.
19. Maulik, N.; Yoshida, T.; Zu, Y.L., et al. Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. The American journal of physiology. 1998, 275, H1857-1864.
20. Liao, Q.C.; Xiao, Z.S.; Qin, Y.F., et al. Genistein stimulates osteoblastic differentiation via p38 MAPK-Cbfa1 pathway in bone marrow culture. Acta pharmacologica Sinica. 2007, 28, 1597-1602.
21. Gaestel, M. MAPKAP kinases - MKs - two's company, three's a crowd. Nature reviews. Molecular cell biology. 2006, 7, 120-130.
Posted by Kourosh on Tue, May 13, 2014
Yes! Our latest paper reporting on screening for protein-protein interaction inhibitors with F2H is now online: http://www.ncbi.nlm.nih.gov/pubmed/24476585.
This is also my first first-author paper from my postdoctoral studies at ChromoTek! Together with Big Pharma (Janssen Pharmaceutica NV) we worked on this project, blind-testing compounds on p53/Mdm2 and p53/Mdm4 interactions, and this was a very rewarding experience. Even more exciting was that our independent results aligned extremely well! All right, coming from basic science, one is just used to things not always working that smoothly. The greater was my surprise how easy and fast it was to screen protein interactions with microscopy here, just using GFP- and RFP-fusion proteins.
Fluorescence images show bait and prey interaction in the cellular F2H assay.
Upper row: GFP-bait forms a bright green spot in the nucleus of F2H cells. RFP-prey interacts with the GFP-bait and forms a co-localizing red spot.
Lower row: Upon incubation with an inhibitor red spot disappears, red signal is disperse => interaction is disrupted
In my former lab, cell-based analysis of protein-protein interactions by microscopy would mean trying your luck with FRET. And FRET always came in a package with 4-hour late night slots at the confocal, freezing in that spaghetti-top (own fault) and hoping that the software does not crash and your settings will not go to Nirvana, because you still need 18 more cells to get that statistics. And pray that no one comes up with an idea to try a “couple of inhibitors” of this interaction! In different concentrations!
Anyways, screening for inhibitors of protein-protein interactions went much more efficient with F2H. It takes only half a second to look at a cell to say if it is an interaction or not. You should still check about 50 cells per condition – which all in all takes a few minutes. Thus, for our paper I screened 20 compounds in 8 different concentrations on p53/Mdm2 and p53/Mdm4 interactions. This would have taken me ages with cell-based FRET. Disclosure: to speed things up even more, we let an automated 96-well microscope image the interactions and run high-content analysis.
F2H-Assay proved useful when testing not only small-molecule inhibitors, but also stapled peptides, and for real-time monitoring of protein-protein interaction dynamics in living cells as well. Our collaboration with Sir D. Lane’s group on these topics resulted in a few more papers, all published in 2013: http://www.ncbi.nlm.nih.gov/pubmed/23214419, http://www.ncbi.nlm.nih.gov/pubmed/23653682, http://www.ncbi.nlm.nih.gov/pubmed/24278380.
Being so happy with our F2H success, I really wonder if there are easier ways to see proteins interacting?
Posted by Larisa Yurlova on Thu, Mar 20, 2014
From October 14th on we will be travelling all across the US to meet with customers and interested scientists to talk about their projects and how an Alpaca might be able to launch their next discovery...
Are you interested in the Alpaca antibody advantage, free samples and a nice chat about your projects? If you are close to one of the following locations, get in touch with us and meet us!
October 15th: MIT, Cambridge
October 16th: John Hopkins University, College Park
October 17th: University of Maryland, College Park
October 18th: NCI, Bethesda
October 21st: Medical University of South Carolina, Charleston
October 22nd: McArdle Laboratory, Madison
October 24th: Northwestern University, Evanston
October 25th: Purdue University, West Lafayette
October 28th: Indiana University, Bloomington
November 1st: MSKCC, New York City
November 4th: Rockefeller University, NYC
November 7th: Parkinson's Institute, Sunnyvale
November 10th to 13th: Society for Neuroscience Annual Meeting, San Diego
November 14th: Salk Institute, La Jolla
November 15th: University of California, Riverside