Marjan Gucek

National Institutes of Health, 베서스다, Maryland, United States

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Publications (3)9.93 Total impact

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    ABSTRACT: The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier forming proteins like occludin and the claudin family, scaffolding proteins like ZO-1, and some cytoskeletal, signaling and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins which are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N- or C-terminus, expressing these fusion proteins in MDCK epithelial cells and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Out of a predicted proteome of ≈ 9000 we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C- terminal ends. Those proximal to the N-terminus were enriched in transmembrane tight junction proteins and those proximal to the C-terminus were enriched in cytoskeletal proteins. We also identified many unexpected, but easily rationalized proteins. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution which could be generally applied to defining subcellular protein compartmentalization.
    Journal of Biological Chemistry 04/2013; DOI:10.1074/jbc.M113.466193 · 4.60 Impact Factor
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    ABSTRACT: Claudins 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 localization 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 MS analysis of purified claudin-2 from MDCK II cells and found that the cytoplasmic tail is multiply phosphorylated on serines, 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 localized 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 which were previously reported to interfere with plasma membrane targeting of claudin-2 reduced phosphorylation at S208, suggesting that membrane localization 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 TER. Together these data are consistent with phosphorylation at S208 playing a major role in the retention of claudin-2 at the plasma membrane.
    Journal of Cell Science 07/2012; DOI:10.1242/jcs.111237 · 5.33 Impact Factor