Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function

Abstract

Although the C-terminal cytoplasmic tail of the tight junction protein occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13-induced, claudin-2-dependent barrier loss. Thus, occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.

Figure 1.

CK2 decreases epithelial barrier function by amplifying paracellular ion flux. (A) CK2 inhibitors elevated Caco-2 TER in a dose-dependent manner after 5 h of treatment. (B) CK2 inhibition caused maximal TER increases within 3 h. (C) Chemical inhibition or CK2 knockdown resulted in comparable TER increases after 6 h of DMAT (50 µM) treatment. (D) Biionic potentials of sodium, methylamine, ethylamine, tetramethylammonium, tetraethylammonium, and N-methyl-d-glucamine were measured 4 h after TBCA (50 µM) addition.

Figure 2.

CK2-mediated barrier regulation is occludin dependent. (A) Effect of CK2 inhibition (50 µM DMAT) on distribution of transmembrane TJ proteins (green) and ZO-1 (red). Bar, 10 µm. (B) Quantitative analysis of occludin enrichment at the TJ. (C) Quantitative analysis of number of occludin-containing vesicles. (D) CK2 inhibition (50 µM DMAT) failed to elevate TER of occludin knockdown monolayers.

Figure 3.

CK2 inhibition stabilizes occludin at the TJ in vitro and in vivo. (A) Kymographs of EGFP-TAMP FRAP, with or with out DMAT treatment (50 µM) at bicellular TJ regions. Bar, 3 µm. (B) Effect of CK2 inhibition (50 µM DMAT) on EGFP-TAMP mobile fractions. (C) EGFP-occludin mobile fraction after CK2 knockdown or chemical CK2 inhibition (50 µM DMAT). (D) Effect of CK2 inhibition (50 µM DMAT) on EGFP-occludin FRAP in mouse jejunum in vivo.

Figure 4.

S408 phosphorylation is required for CK2-mediated regulation of occludin dynamics and barrier function. (A and B) FRAP analysis of EGFP-occludin mutants expressed in occludin knockdown Caco-2 cells. Bar, 3 µm. (C) Expression of EGFP or EGFP-occludin mutants in occludin knockdown (KD) Caco-2.

Figure 5.

Occludin S408 dephosphorylation enhances interactions with claudin-1, claudin-2, and ZO-1, and their exchange at the TJ. (A) GST-occludin C-terminal tails (383–522) immobilized on glutathione-agarose were used to probe Caco-2 lysates, and recovered proteins assessed by SDS-PAGE immunoblot. (B) GST-occludin C-terminal tails were used to capture VSV G-ZO-1-U5-GuK. (C) GST-occludin C-terminal tails were used to probe control and ZO-1 knockdown Caco-2 lysates, as above. (D) FRAP of mRFP1-ZO-1 was assessed in control and occludin knockdown monolayers as well as occludin knockdown monolayers expressing wild-type, S408D, or S408A EGFP-occludin. (E) FRAP of mRFP1-tagged claudin-1, claudin-2, and claudin-4 were assessed in occludin knockdown monolayers either alone or with wild-type, S408D, or S408A EGFP-occludin. (F) FRAP analysis of mRFP1-claudin-2 expressed in wild-type, occludin knockdown, or ZO-1 knockdown monolayers as well as ZO-1 knockdown monolayers expressing wild-type, ΔPDZ1, or ΔU5-GuK EGFP-ZO-1. (G) FRAP of wild-type, S408D, and S408A EGFP-occludin in ZO-1 knockdown monolayers. (H) EGFP-ZO-1 fluorescent recovery at the center (circles) and edges (squares) of elongated bleach regions. Bar, 6 µm. Chemical CK2 inhibition throughout this figure was with 50 µM TBCA

Figure 6.

CK2-mediated barrier regulation requires ZO-1 and claudin-2. (A and B) TER and PNa⁺/PCl⁻ responses of control and occludin knockdown monolayers to CK2 inhibition. (C and D) TER and PNa⁺/PCl⁻ responses of control and ZO-1 knockdown monolayers to CK2 inhibition. (E) TER and PNa⁺/PCl⁻ after claudin-1 and claudin-2 knockdown. (F and G) TER and PNa⁺/PCl⁻ responses of control and claudin-2 knockdown monolayers to CK2 inhibition. (H and I) TER and PNa⁺/PCl⁻ responses of control, claudin-1, claudin-2, and double-knockdown monolayers to CK2 inhibition. (J) Tight junction protein expression in Caco-2 and T84 monolayers, before or after 12 h of IL-13 treatment (1 ng/ml). (K and L) Effect of CK2 inhibition (50 µM TBCA) on TER and PNa⁺/PCl⁻ of control and IL-13–treated T84 monolayers.

Figure 7.

Model of CK2-mediated regulation of TJ structure, dynamic behavior, and barrier function through occludin S408 phosphorylation. Proposed organization, interactions, and dynamic behavior of proteins involved in TJ regulation by CK2. (A) CK2-mediated phosphorylation of S408 enhances occludin self-association, increases the occludin mobile fraction, and reduces occludin association with ZO-1, claudin-1, and claudin-2. This promotes flux across claudin-2, and, to a lesser extent, claudin-1 pores, thereby increasing paracellular cation flux. (B) When dephosphorylated at S408, occludin is stabilized at the TJ through enhanced association with ZO-1 via the U5-GuK domain. ZO-1 also facilitates indirect interactions between occludin and claudin-2, which associates with ZO-1 via the PDZ1 domain, thereby acting as a scaffold to organize a complex. As a result, dynamic behaviors of occludin, ZO-1, claudin-1, and claudin-2 converge, and function of claudin-1 and claudin-2 paracellular pores is reduced.