Distinct steady-state properties and TNF responses of epithelial long myosin light chain kinase (MLCK) splice variants

Abstract

Intestinal epithelia express two long MLCK splice variants, MLCK1 and MLCK2. We have previously shown that disruption of inflammation-induced MLCK1 recruitment to the perijunctional actomyosin ring prevents barrier loss and attenuates disease progression. Here we sought to define the domains responsible for distinct MLCK1 and MLCK2 behaviors. Quantitative analysis of human biopsies demonstrated specific increases in MLCK1 expression and perijunctional localization in Crohn's disease. When expressed in cultured intestinal epithelial cells, we found, as expected, that MLCK1 is most concentrated at the perijunctional actomyosin ring. In contrast, MLCK2 is predominantly associated with basal F-actin stress fibers. Immunoglobulin-cell adhesion molecule domain 3 (IgCAM3) must be critical for MLCK1 recruitment, as that domain is incomplete in MLCK2. Consistent with this, truncation mutants consisting of N-terminal IgCAM domains 1-4, without C-terminal catalytic domains, localized similarly to full-length MLCK1 and MLCK2, respectively. Further mutagenesis allowed identification of IgCAM2 and IgCAM3 domains as the minimal region required for MLCK1 recruitment. Although IgCAM3 does not concentrate perijunctionally, it can act as a dominant negative effector that limits steady-state and TNF-induced MLCK1 recruitment and barrier loss. Together, the demonstration of selective MLCK1 upregulation and perijunctional recruitment in Crohn's disease and identification of domains required for perijunctional MLCK1 recruitment provide a conceptual understanding and structural data needed for development of therapeutic means of blocking MLCK1-mediated barrier loss without the toxicity of enzymatic MLCK inhibition.

Paracellular permeability and tight junction regulation in gut health and disease

“…Although the underlying mechanisms have not been defined, splicing seems to be precisely regulated during differentiation, as MLCK2 is expressed throughout the crypt-villus axis but MLCK1 expression is limited to the upper villus 157 . Moreover, the increased MLC phosphorylation in active Crohn's disease is specifically associated with perijunctional MLCK1 recruitment 159,160 (Fig. 4).…”