Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis


Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Long MLCK1 is specifically recruited to the PAMR in response to inflammatory stimuli.
Fig. 2: Identification and preliminary characterization of small molecules that bind to IgCAM3.
Fig. 3: Divertin reverses acute TNF-induced MLC phosphorylation and barrier dysfunction in vitro.
Fig. 4: Divertin prevents acute TNF-induced barrier dysfunction in mouse jejunum in vivo and human jejunal mucosae ex vivo.
Fig. 5: Divertin corrects increased intestinal permeability in IL-10 knockout mice without significant systemic, mucosal, or epithelial toxicities.
Fig. 6: Divertin corrects intestinal permeability defects and limits the progression of experimental inflammatory bowel disease.

Data availability

All requests for raw and analyzed data and materials are promptly reviewed to verify if the request is subject to any intellectual property or confidentiality obligations. Human participants were de-identified and no further data are available. Any data and materials that can be shared will be released via a Material Transfer Agreement. The crystal structure data are available as Protein Data Bank code: 6C6M (


  1. 1.

    Odenwald, M. A. & Turner, J. R. The intestinal epithelial barrier: a therapeutic target? Nat. Rev. Gastroenterol. Hepatol. 14, 9–21 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    Clayburgh, D. R. et al. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest. 115, 2702–2715 (2005).

    CAS  Article  Google Scholar 

  3. 3.

    Turner, J. R. Intestinal mucosal barrier function in health and disease. Nat. Rev. Immunol. 9, 799–809 (2009).

    CAS  Article  Google Scholar 

  4. 4.

    Lazar, V. & Garcia, J. G. A single human myosin light chain kinase gene (MLCK; MYLK) transcribes multiple nonmuscle isoforms. Genomics 57, 256–267 (1999).

    CAS  Article  Google Scholar 

  5. 5.

    Blue, E. K. et al. 220- and 130-kDa MLCKs have distinct tissue distributions and intracellular localization patterns. Am. J. Physiol., Cell Physiol. 282, C451–C460 (2002).

    CAS  Article  Google Scholar 

  6. 6.

    Kamm, K. E. & Stull, J. T. Dedicated myosin light chain kinases with diverse cellular functions. J. Biol. Chem. 276, 4527–4530 (2001).

    CAS  Article  Google Scholar 

  7. 7.

    Clayburgh, D. R. et al. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability. J. Biol. Chem. 279, 55506–55513 (2004).

    CAS  Article  Google Scholar 

  8. 8.

    Birukov, K. G. et al. Differential regulation of alternatively spliced endothelial cell myosin light chain kinase isoforms by p60(Src). J. Biol. Chem. 276, 8567–8573 (2001).

    CAS  Article  Google Scholar 

  9. 9.

    Wainwright, M. S. et al. Protein kinase involved in lung injury susceptibility: evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment. Proc. Natl Acad. Sci. USA 100, 6233–6238 (2003).

    CAS  Article  Google Scholar 

  10. 10.

    Su, L. et al. TNFR2 activates MLCK-dependent tight junction dysregulation to cause apoptosis-mediated barrier loss and experimental colitis. Gastroenterology 145, 407–415 (2013).

    CAS  Article  Google Scholar 

  11. 11.

    Somlyo, A. V. et al. Myosin light chain kinase knockout. J. Muscle Res. Cell Motil. 25, 241–242 (2004).

    CAS  Article  Google Scholar 

  12. 12.

    Zolotarevsky, Y. et al. A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease. Gastroenterology 123, 163–172 (2002).

    CAS  Article  Google Scholar 

  13. 13.

    Wang, F. et al. IFN-γ-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction. Gastroenterology 131, 1153–1163 (2006).

    CAS  Article  Google Scholar 

  14. 14.

    Powrie, F. et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1, 553–562 (1994).

    CAS  Article  Google Scholar 

  15. 15.

    Williams, A. F. & Barclay, A. N. The immunoglobulin superfamily: domains for cell surface recognition. Annu. Rev. Immunol. 6, 381–405 (1988).

    CAS  Article  Google Scholar 

  16. 16.

    Holden, H. M., Ito, M., Hartshorne, D. J. & Rayment, I. X-ray structure determination of telokin, the C-terminal domain of myosin light chain kinase, at 2.8 A resolution. J. Mol. Biol. 227, 840–851 (1992).

    CAS  Article  Google Scholar 

  17. 17.

    Turner, J. R. et al. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am. J. Physiol. 273, C1378–C1385 (1997).

    CAS  Article  Google Scholar 

  18. 18.

    Owens, S. E., Graham, W. V., Siccardi, D., Turner, J. R. & Mrsny, R. J. A strategy to identify stable membrane-permeant peptide inhibitors of myosin light chain kinase. Pharm. Res. 22, 703–709 (2005).

    CAS  Article  Google Scholar 

  19. 19.

    He, W. Q. et al. Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice. Gastroenterology 135, 610–620 (2008).

    CAS  Article  Google Scholar 

  20. 20.

    Marchiando, A. M. et al. Caveolin-1-dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo. J. Cell Biol. 189, 111–126 (2010).

    CAS  Article  Google Scholar 

  21. 21.

    Graham, W. V. et al. Tumor necrosis factor-induced long myosin light chain kinase transcription is regulated by differentiation-dependent signaling events. Characterization of the human long myosin light chain kinase promoter. J. Biol. Chem. 281, 26205–26215 (2006).

    CAS  Article  Google Scholar 

  22. 22.

    NCI Developmental Therapeutics Program. Datawarehouse index results; (2004).

  23. 23.

    Su, L. et al. Targeted epithelial tight junction dysfunction causes immune activation and contributes to development of experimental colitis. Gastroenterology 136, 551–563 (2009).

    CAS  Article  Google Scholar 

  24. 24.

    Berg, D. J. et al. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses. J. Clin. Invest. 98, 1010–1020 (1996).

    CAS  Article  Google Scholar 

  25. 25.

    Kühn, R., Löhler, J., Rennick, D., Rajewsky, K. & Müller, W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263–274 (1993).

    Article  Google Scholar 

  26. 26.

    Odenwald, M. A. & Turner, J. R. Intestinal permeability defects: is it time to treat? Clin. Gastroenterol. Hepatol. 11, 1075–1083 (2013).

    Article  Google Scholar 

  27. 27.

    Buhner, S. et al. Genetic basis for increased intestinal permeability in families with Crohn’s disease: role of CARD15 3020insC mutation? Gut 55, 342–347 (2006).

    CAS  Article  Google Scholar 

  28. 28.

    Prager, M. et al. Myosin IXb variants and their pivotal role in maintaining the intestinal barrier: a study in Crohn’s disease. Scand. J. Gastroenterol. 49, 1191–1200 (2014).

    CAS  Article  Google Scholar 

  29. 29.

    Wapenaar, M. C. et al. Associations with tight junction genes PARD3 and MAGI2 in Dutch patients point to a common barrier defect for coeliac disease and ulcerative colitis. Gut 57, 463–467 (2008).

    CAS  Article  Google Scholar 

  30. 30.

    Wyatt, J., Vogelsang, H., Hübl, W., Waldhöerr, T. & Lochs, H. Intestinal permeability and the prediction of relapse in Crohn’s disease. Lancet 341, 1437–1439 (1993).

    CAS  Article  Google Scholar 

  31. 31.

    Arrieta, M. C., Madsen, K., Doyle, J. & Meddings, J. Reducing small intestinal permeability attenuates colitis in the IL10 gene-deficient mouse. Gut 58, 41–48 (2009).

    CAS  Article  Google Scholar 

  32. 32.

    Vetrano, S. et al. Unique role of junctional adhesion molecule-A in maintaining mucosal homeostasis in inflammatory bowel disease. Gastroenterology 135, 173–184 (2008).

    CAS  Article  Google Scholar 

  33. 33.

    Lin, H. B., Cadete, V. J., Sawicka, J., Wozniak, M. & Sawicki, G. Effect of the myosin light chain kinase inhibitor ML-7 on the proteome of hearts subjected to ischemia-reperfusion injury. J. Proteomics 75, 5386–5395 (2012).

    CAS  Article  Google Scholar 

  34. 34.

    Clayburgh, D. R., Musch, M. W., Leitges, M., Fu, Y. X. & Turner, J. R. Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo. J. Clin. Invest. 116, 2682–2694 (2006).

    CAS  Article  Google Scholar 

  35. 35.

    Pearson, A. D., Eastham, E. J., Laker, M. F., Craft, A. W. & Nelson, R. Intestinal permeability in children with Crohn’s disease and coeliac disease. Br. Med. J. (Clin. Res. Ed.) 285, 20–21 (1982).

    CAS  Article  Google Scholar 

  36. 36.

    Gruber, R. et al. Diverse regulation of claudin-1 and claudin-4 in atopic dermatitis. Am. J. Pathol. 185, 2777–2789 (2015).

    CAS  Article  Google Scholar 

  37. 37.

    Mirzapoiazova, T. et al. Non-muscle myosin light chain kinase isoform is a viable molecular target in acute inflammatory lung injury. Am. J. Respir. Cell Mol. Biol. 44, 40–52 (2011).

    CAS  Article  Google Scholar 

  38. 38.

    Xu, J. et al. Nonmuscle myosin light-chain kinase mediates neutrophil transmigration in sepsis-induced lung inflammation by activating β2 integrins. Nat. Immunol. 9, 880–886 (2008).

    CAS  Article  Google Scholar 

  39. 39.

    Brown, G. R. et al. Tumor necrosis factor inhibitor ameliorates murine intestinal graft-versus-host disease. Gastroenterology 116, 593–601 (1999).

    CAS  Article  Google Scholar 

  40. 40.

    Bennett, J. et al. Blood–brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE. J. Neuroimmunol. 229, 180–191 (2010).

    CAS  Article  Google Scholar 

  41. 41.

    Kebir, H. et al. Human TH17 lymphocytes promote blood–brain barrier disruption and central nervous system inflammation. Nat. Med. 13, 1173–1175 (2007).

    CAS  Article  Google Scholar 

  42. 42.

    Moravcevic, K., Oxley, C. L. & Lemmon, M. A. Conditional peripheral membrane proteins: facing up to limited specificity. Structure 20, 15–27 (2012).

    CAS  Article  Google Scholar 

  43. 43.

    Sagona, A. P. et al. PtdIns(3)P controls cytokinesis through KIF13A-mediated recruitment of FYVE-CENT to the midbody. Nat. Cell Biol. 12, 362–371 (2010).

    CAS  Article  Google Scholar 

  44. 44.

    Webb, B. A. et al. A histidine cluster in the cytoplasmic domain of the Na-H exchanger NHE1 confers pH-sensitive phospholipid binding and regulates transporter activity. J. Biol. Chem. 291, 24096–24104 (2016).

    CAS  Article  Google Scholar 

  45. 45.

    Buschmann, M. M. et al. Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux. Mol. Biol. Cell 24, 3056–3068 (2013).

    CAS  Article  Google Scholar 

  46. 46.

    Keegan, R. M. & Winn, M. D. Automated search-model discovery and preparation for structure solution by molecular replacement. Acta Crystallogr. D 63, 447–457 (2007).

    CAS  Article  Google Scholar 

  47. 47.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  Article  Google Scholar 

  48. 48.

    Bricogne, G. et al. BUSTER version 2.11.7. (Global Phasing Ltd., 2017).

  49. 49.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  50. 50.

    Irwin, J. J. & Shoichet, B. K. ZINC: a free database of commercially available compounds for virtual screening. J. Chem. Inf. Model. 45, 177–182 (2005).

    CAS  Article  Google Scholar 

  51. 51.

    Berglund, J. J., Riegler, M., Zolotarevsky, Y., Wenzl, E. & Turner, J. R. Regulation of human jejunal transmucosal resistance and MLC phosphorylation by Na+-glucose cotransport. Am. J. Physiol. Gastrointest. Liver Physiol 281, G1487–G1493 (2001).

    CAS  Article  Google Scholar 

  52. 52.

    Turner, J. R. & Black, E. D. NHE3-dependent cytoplasmic alkalinization is triggered by Na+-glucose cotransport in intestinal epithelia. Am. J. Physiol., Cell Physiol 281, C1533–C1541 (2001).

    CAS  Article  Google Scholar 

  53. 53.

    Wang, F. et al. Interferon-γ and tumor necrosis factor-α synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am. J. Pathol. 166, 409–419 (2005).

    CAS  Article  Google Scholar 

  54. 54.

    Salic, A. & Mitchison, T. J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl Acad. Sci. USA 105, 2415–2420 (2008).

    CAS  Article  Google Scholar 

  55. 55.

    Badri, K. R. et al. Blood pressure homeostasis is maintained by a P311-TGF-β axis. J. Clin. Invest. 123, 4502–4512 (2013).

    CAS  Article  Google Scholar 

  56. 56.

    Oishi, K. et al. Agonist-induced isometric contraction of smooth muscle cell-populated collagen gel fiber. Am. J. Physiol., Cell Physiol. 279, C1432–C1442 (2000).

    CAS  Article  Google Scholar 

  57. 57.

    Russo, J. M. et al. Distinct temporal-spatial roles for rho kinase and myosin light chain kinase in epithelial purse-string wound closure. Gastroenterology 128, 987–1001 (2005).

    CAS  Article  Google Scholar 

Download references


This study was supported by National Institutes of Health (NIH) grant nos. R01DK61931 (J.R.T.), R01DK068271 (J.R.T.), R24DK099803 (J.R.T.), R01GM081030 (L.W.M.), R01AG048793 (S.C.M.), P30CA014599 (University of Chicago Comprehensive Cancer Center), P30DK034854 (the Harvard Digestive Disease Center), and T32HL007237 (W.V.G., A.M.M.). Work was also supported by the Broad Medical Research Program (IBD-022, to J.R.T.), the Department of Defense (W81XWH-09-1-0341, to J.R.T.), a Catalyst Award from the Chicago Biomedical Consortium (J.R.T., L.W.M.), the National Natural Science Foundation of China (81470804 and 31401229 to W.H.). The Berkeley Center for Structural Biology is supported in part by the NIH, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under contract no. DE-AC02-05CH11231.

Author information




J.R.T. conceptualized the study. W.V.G., W.H., A.M.M., J.Z., G.S., H.-S.L., A.B., M.L.D.M.O., Z.-H.J., W.C., H.Z., Yitang Wang, J.G., J.W., H.J.R., Yingmin Wang, and J.R.T. carried out the investigations. S.B.S., D.O., S.C.M., L.W.M., and J.R.T. managed the resources. H.Z., J.G., and D.O. managed the software. W.V.G., W.H., W.C., and J.R.T. carried out the visualization. W.V.G. and, J.R.T. wrote the original manuscript draft. All authors reviewed and edited the draft. J.R.T. and W.H. acquired the funding. These pairs of authors contributed equally: W.V.G. and W.H., and A.M.M. and J.Z.

Corresponding author

Correspondence to Jerrold R. Turner.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 IgCAM3 drug-binding pocket and in silico candidate identification.

a, Crystal structure of human IgCAM3. The colors indicate hydrophobic (blue) or hydrophilic (orange) residues. b, Predicted ΔG scored for the 139,735 molecules docked into the region of small molecule binding. Two groups of molecules with either very low or intermediate ΔG are indicated by symbols that coincide with those in Fig. 2c. The inset shows the IgCAM3 binding pocket (red), with predicted docking of selected small molecules.

Extended Data Fig. 2 Divertin binds to recombinant IgCAM3.

a, IgCAM3 shown as a ribbon diagram with the location of tryptophan 447 (W447) and three residues in the putative drug-binding pocket, leucine 449 (L449), glutamine 457 (Q457), and aspartic acid 481 (D481). b, Changes in peak fluorescence of wild-type IgCAM3, mutant IgCAM3, and NATA in the presence of increasing concentrations of NSC55937 reveal a shift in the maximum wavelength of wild-type IgCAM3 only. c, Fluorescence emission spectrum of wild-type IgCAM3 in the presence of increasing NSC55937 concentrations demonstrates dose-dependent tryptophan fluorescence quenching and a red shift in the maximum emission wavelength, indicative of NSC55937 binding to IgCAM3. NSC55937 was used at 0 μM (red), 10 μM (orange), 33 μM (yellow), 100 μM (green), 333 μM (blue), and 1000 μM (violet). d, Mutant IgCAM3 (Leu449Arg, Gln457Lys, and Asp481Val) abolishes the ability of NSC55937 to quench IgCAM3 tryptophan fluorescence across all concentrations of divertin. e, Fluorescence emission spectra of the tryptophan analog (1 mM) in the presence of increasing concentrations of NSC55937. No fluorescence quenching occurred. Data are representative of three or more independent experiments with similar results.

Extended Data Fig. 3 Divertin delays the development of experimental inflammatory bowel disease.

a, Fourteen days after T cell transfer, mice were treated with daily intraperitoneal injections of saline (green) or divertin (red). n = 10 independent animals per condition. The mean ± s.e.m. is shown. **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. b, Divertin-treated mice were protected from the weight loss experienced by saline-treated (green) mice. n = 10 independent animals per condition. the mean ± s.e.m. is shown. **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. c, Divertin significantly increased survival during adoptive transfer colitis. n = 10 independent animals per condition. *P < 0.05 by Gehan–Breslow–Wilcoxon test. d, Intestinal permeability on day 56. Data are normalized to recovery from a healthy wild-type mouse. n = 7 (saline), n = 10 (divertin). **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. e, Mucosal TNF on day 56 was significantly reduced by divertin treatment (red). n = 7 (saline), n = 10 (divertin). *P < 0.05 by unpaired, two-sided t-test with Welch’s correction. f, Colon lengths on day 56. Images of representative colons are shown, with their lengths corresponding to the labels on the y axis. n = 7 (saline) or n = 10 (divertin) independent animals per condition. **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. g, Colonic histopathology scores on day 56. n = 7 (saline) or n = 10 (divertin) independent animals per condition. **P < 0.01 by unpaired, two-sided t-test with Welch’s correction. h, Histopathology shows crypt loss (asterisk) and crypt abscesses (arrow) in the mucosa from a saline-treated mouse and partial goblet cell preservation in the mucosa from a divertin-treated mouse (arrowhead). Bar, 50 μm. The insets show complete cross sections of colon, with the boxes indicating the areas shown at higher magnification. Bar, 250 μm. Images are representative of three independent experiments with similar results. The experiment shown in this figure used female mice as T cell donors and immunodeficient recipients. Results were similar in two independent studies that, in combination, included nine saline-treated and nine divertin-treated male mice.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Graham, W.V., He, W., Marchiando, A.M. et al. Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis. Nat Med 25, 690–700 (2019).

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing