IgCAM domain 3 is necessary for basal and TNF‐induced MLCK1 trafficking in intestinal epithelial cells

Abstract

Tumor necrosis factor (TNF), a cytokine central to inflammatory bowel disease (IBD), causes tight junction barrier loss via myosin light chain kinase (MLCK) activation. Two splice variants, MLCK1 and MLCK2, which differ by a single immunoglobulin‐cell adhesion molecule (IgCAM) domain, IgCAM3, are expressed in human intestinal epithelia. Based on our previous data showing that MLCK1, which includes IgCAM3, is concentrated at the perijunctional actomyosin ring (PAMR) in vivo, we hypothesized that IgCAM3 is essential for MLCK1 trafficking. Analysis of EGFP‐tagged MLCK1 and MLCK2 stably expressed in Caco‐2 intestinal epithelial monolayers showed that MLCK1 concentrates at the PAMR while MLCK2 distributes more broadly and associates with basal stress fibers. TNF increased MLCK1 trafficking to the PAMR, but had little effect on MLCK2 localization. To assess the potential contributions roles of other MLCK domains to MLCK1 trafficking, EGFP‐tagged MLCK constructs composed of IgCAM domains 1–2‐3, 1–2‐3–4, and 1–2‐4, which lack DXR motifs and enzymatic domains. Both MLCK1‐IgCAM1–3 and MLCK1‐IgCAM1–4 concentrated at the PAMR, while MLCK1‐IgCAM3 and MLCK2‐IgCAM1–4 did not. These data show that IgCAM3 is necessary, but only MLCK1‐IgCAM1–3 is sufficient for MLCK1 trafficking to the PAMR. We speculate that IgCAM3‐mediated trafficking may be required for TNF‐induced barrier loss.