Phosphorylation of claudin-2 on serine 208 promotes membrane retention and reduces trafficking to lysosomes

Abstract

SummaryClaudins are critical components of epithelial and endothelial tight junction seals, but their post-transcriptional regulation remains poorly understood. Several studies have implicated phosphorylation in control of claudin localisation and/or function, but these have focused on single sites or pathways with differing results, so that it has been difficult to draw general functional conclusions. In this study, we used mass spectrometry (MS) analysis of purified claudin-2 from MDCK II cells and found that the cytoplasmic tail is multiply phosphorylated on serines, a threonine and tyrosines. Phos-tag SDS PAGE revealed that one site, S208, is heavily constitutively phosphorylated in MDCK II cells and in mouse kidney; this site was targeted for further study. Mutational analysis revealed that the phosphomimetic mutant of claudin-2, S208E, was preferentially localised to the plasma membrane while claudin-2 S208A, which could not be phosphorylated at this site, both immunolocalized and co-fractionated with lysosomal markers. Mutations at sites that were previously reported to interfere with plasma membrane targeting of claudin-2 reduced phosphorylation at S208, suggesting that membrane localisation is required for phosphorylation; however phosphorylation at S208 did not affect binding to ZO-1 or ZO-2 Administration of forskolin or PGE2 resulted in dephosphorylation at S208 and transient small increases in transepithelial electrical resistance (TER). Together these data are consistent with phosphorylation at S208 playing a major role in the retention of claudin-2 at the plasma membrane.

12/15-Lipoxygenase-dependent ROS production is required for diet-induced endothelial barrier dysfunction

“…A large body of evidence shows that interference in the interactions between TJ proteins perturbs the TJs affecting the barrier permeability of the endothelium ( 38 ). It was further observed that the TJ formation or disintegration depends on the phosphorylation state of TJ proteins (39)(40)(41). However, most of these studies were focused on epithelial TJs, and therefore, the role of phosphorylation of TJ proteins in the preservation or perturbation of endothelial TJ integrity and its barrier function has not been well-studied.…”

Phosphorylation at Ser-181 of Oncogenic KRAS Is Required for Tumor Growth

“…To formally prove this hypothesis, detection of KRAS phosphorylation was necessary. Since no suitable antibodies are available, we used the Phos-Tag-based approach (31,32) to determine the oncogenic KRAS phosphorylation status in the generated tumors. This method is based on the fact that a complex formation between the phosphate group of a phosphorylated protein and a divalent metal ion in Phos-Tag reduces the mobility of the phospho-protein during the electrophoresis separation, thus allowing resolution of phosphorylated and non-phosphorylated proteins into different bands.…”

Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins

“…Although most of the 23 human claudins end in a similar Tyr-Val canonical PDZ binding motif (see Fig. 1), their more upstream residues are both shared and distinct (6). The mechanisms that regulate claudin function remain poorly defined but of considerable interest.…”

“…For example, the cytoplasmic tails of many claudins are documented to be phosphorylated at multiple positions including serines, threonines, and tyrosines (15). Changes in phosphorylation have been correlated with changes in barrier properties, trafficking, and turnover, cited in Van Itallie et al (6), although no direct evidence has yet been provided that phosphorylation alters affinity of the PDZ domaintarget interaction. Similar correlative findings have been reported after introducing phosphomimicking mutations (i.e.…”

“…mutating the Ser/Thr/Tyr residue to Asp or Glu) although the mechanisms underlying these changes are not defined (16,17). We were particularly interested in testing a role for the tyrosine at the P Ϫ6 , which we had previously shown could be phosphorylated in vivo (6) and because this position has a tyrosine residue in 9 of the 23 human claudins (Fig. 1).…”