TNF Activates FK506 Binding Protein 8 (FKBP8) Interactions That Direct Myosin Light Chain Kinase 1 (MLCK1) Recruitment to the Perijunctional Actomyosin Ring and Drive Epithelial Barrier Loss

Background Intestinal barrier dysfunction is thought to contribute to progression of inflammatory bowel disease and systemic disorders. We recently reported that a specific myosin light chain kinase splice variant, MLCK1, is recruited to the apical perijunctional actomyosin ring of intestinal epithelial in both acute and chronic diarrheal disease. Disruption of MLCK1 recruitment restores tight junction barrier function and blocks progression of immune‐mediated colitis without toxicities associated with enzymatic inhibition, e.g., defective wound repair or smooth muscle function. We sought to further define the mechanisms responsible for MLCK1 recruitment to the perijunctional actomyosin ring. Objective To define MLCK1‐binding partners and the means by which they regulate intracellular targeting of MLCK1. Methods Yeast‐2‐hybrid (Y2H) screening and microscale thermophoresis (MST) were used to discover and define MLCK1‐interacting proteins. Caco‐2 BBe cells, some stably expressing MLCK1‐EGFP, were cultured on Transwells. Immunofluorescence microscopy and proximity ligation assay (PLA) were used to detect intracellular protein distributions and interactions. Tight junction barrier function was measured as transepithelial electrical resistance (TER). Results Y2H using MLCK1‐specific sequences captured the FK506‐binding/peptidylprolyl isomerase domain of FK506‐binding protein 8 (FKBP8, aka FKBP38). MST analysis showed that the recombinant MLCK1 N‐terminus bound directly to full‐length FKBP8 (K d =~5μM). This interaction was mediated by the FK506‐binding/peptidylprolyl isomerase domain and could be blocked by FK506 (tacrolimus). Consistent with a role in barrier regulation, FK506 induced dose‐dependent TER increases in cultured epithelial monolayers. Immunofluorescent microscopy demonstrated overlapping distributions of MLCK1‐EGFP and FKBP8 at the perijunctional actomyosin ring, lateral membranes, and within the cytoplasm of intestinal epithelial cells, but PLA detected only limited direct interactions between MLCK1 and FKBP8. Tumor necrosis factor (TNF) triggered MLCK1 recruitment to the perijunctional actomyosin ring, markedly increased MLCK1‐FKBP8 interactions, as assessed by PLA, and induced barrier loss. FK506 addition to TNF‐treated monolayers displaced MLCK1 from the perijunctional ring and restored barrier function. Conclusions TNF activates FKBP8 binding to MLCK1. This interaction is critical for TNF‐induced MLCK1 recruitment to the perijunctional actomyosin ring and subsequent barrier loss. Disruption of the MLCK1‐FKBP8 interaction could, in part, explain the efficacy of FK506 in severe Crohn's disease. We speculate that specific targeting of the MLCK1‐FKBP8 interaction may have therapeutic benefit in many diseases associated with intestinal barrier dysfunction. Support or Funding Information Supported by the NIH (DK R01 DK068271) and the NNSF of China (81800464). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .