Cyclic AMP induces phosphorylation of claudin-5 immunoprecipitates and expression of claudin-5 gene in blood–brain-barrier endothelial cells via protein kinase A-dependent and -independent pathways

Ishizaki, Tsutomu; Chiba, Hideki; Kojima, Takashi; Fujibe, Masato; Soma, Tamotsu; Miyajima, Hideaki; Nagasawa, Kunihiko; Wada, Ikuo; Sawada, Norimasa

doi:10.1016/s0014-4827(03)00354-9

Cited by 181 publications

(133 citation statements)

References 52 publications

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“…Our results suggest that PKCε's role in metastasis may be related to its ability to impair the barrier function (and cell adhesion) through phosphorylation of TJ proteins such as claudin-4. These findings are consistent with a number of studies that have previously shown that phosphorylation of various claudin proteins can decrease TJ integrity [32,35,[51][52][53][54], although phosphorylation of claudin-1 has also been shown to promote barrier function [29,31]. However, it is important to note that TJ disruption likely occurs through multiple mechanisms.…”

Section: Discussionsupporting

confidence: 90%

“…Interestingly, several studies have demonstrated the involvement of various kinases in the phosphorylation and regulation of claudin proteins [29][30][31][32][33][34][35][36][37], and we have recently shown that phosphorylation of claudin-3 by PKA can affect TJ properties in ovarian cancer cells [38]. Protein kinase C (PKC) isoforms are present in ovarian cancer and are known to modulate TJ function by phosphorylation of the proteins in the complex [24,34,[39][40][41][42][43], but it is unclear whether PKC can directly phosphorylate and regulate claudin proteins.…”

Section: Introductionmentioning

confidence: 99%

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Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells

D’Souza

Indig

Morin

2007

Experimental Cell Research

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Claudin proteins belong to a large family of transmembrane proteins essential to the formation and maintenance of tight junctions (TJs). In ovarian cancer, TJ protein claudin-4 is frequently overexpressed and may have roles in survival and invasion, but the molecular mechanisms underlying its regulation are poorly understood. In this report, we show that claudin-4 can be phosphorylated by protein kinase C (PKC) at Thr189 and Ser194 in ovarian cancer cells and overexpression of a claudin-4 mutant protein mimicking the phosphorylated state results in the disruption of the barrier function. Furthermore, upon phorbol ester-mediated PKC activation of OVCA433 cells, TJ strength is decreased and claudin-4 localization is altered. Analyses using PKC inhibitors and siRNA suggest that PKCε, an isoform typically expressed in ovarian cancer cells, may be important in the TPAmediated claudin-4 phosphorylation and weakening of the TJs. Furthermore, immunofluorescence studies showed that claudin-4 and PKCε are co-localized at the TJs in these cells. The modulation of claudin-4 activity by PKCε may not only provide a mechanism for disrupting TJ function in ovarian cancer, but may also be important in the regulation of TJ function in normal epithelial cells.

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells

D’Souza

Indig

Morin

2007

Experimental Cell Research

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“…For example, PKA-mediated phosphorylation of claudin-3 in an ovarian cancer cell line was associated with a decrease in TER (26), but PKA-dependent phosphorylation of claudin-5 in cultured blood-brain barrier endothelial cells treated with cAMP increased TER (52). As is true for all phosphorylation sites identified in claudins to date, claudin-3 phosphorylation by PKA occurred in the COOHterminal cytoplasmic domain, on Thr192 (26).…”

Section: Phosphorylation Of Claudinsmentioning

confidence: 94%

Biology of claudins

Angelow¹,

Ahlstrom²,

Yu³

2008

American Journal of Physiology-Renal Physiology

307

294

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Claudins are a family of tight junction membrane proteins that regulate paracellular permeability of epithelia, likely by forming the lining of the paracellular pore. Claudins are expressed throughout the renal tubule, and mutations in two claudin genes are now known to cause familial hypercalciuric hypomagnesemia with nephrocalcinosis. In this review, we discuss recent advances in our understanding of the physiological role of various claudins in normal kidney function, and in understanding the fundamental biology of claudins, including the molecular basis for selectivity of permeation, claudin interactions in tight junction formation, and regulation of claudins by protein kinases and other intracellular signals.

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“…However, only a few studies have clearly demonstrated phosphorylation of claudins. For example, phosphorylation of claudin-1 by mitogen-activated protein kinases (21) and protein kinase C (22), as well as phosphorylation of claudin-5 by cAMP-dependent protein kinase (PKA) (23,24), has been reported. Also, WNK4 kinase has been shown to phosphorylate claudin-3 and -4 (25).…”

mentioning

confidence: 99%

Phosphorylation of Claudin-3 at Threonine 192 by cAMP-dependent Protein Kinase Regulates Tight Junction Barrier Function in Ovarian Cancer Cells

D’Souza

Agarwal

Morin

2005

Journal of Biological Chemistry

194

147

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Claudins are integral membrane proteins essential in the formation and function of tight junctions (TJs). Disruption of TJs, which have essential roles in cell permeability and polarity, is thought to contribute to epithelial tumorigenesis. Claudin-3 and -4 are frequently overexpressed in ovarian cancer, but the molecular pathways involved in the regulation of these proteins are unclear. Interestingly, several studies have demonstrated a role for phosphorylation in the regulation of TJ complexes, although evidence for claudin phosphorylation is scarce. Here, we showed that claudin-3 and -4 can be phosphorylated in ovarian cancer cells. In vitro phosphorylation assays using glutathione S-transferase fusion constructs demonstrated that the C terminus of claudin-3 is an excellent substrate for cAMP-dependent protein kinase (PKA). Using site-directed mutagenesis, we identified a PKA phosphorylation site at amino acid 192 in the C terminus of claudin-3. Overexpression of the protein containing a T192D mutation, mimicking the phosphorylated state, resulted in a decrease in TJ strength in ovarian cancer cell line OVCA433. Our results suggest that claudin-3 phosphorylation by PKA, a kinase frequently activated in ovarian cancer, may provide a mechanism for the disruption of TJs in this cancer. In addition, our findings may have general implications for the regulation of TJs in normal epithelial cells.

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Cyclic AMP induces phosphorylation of claudin-5 immunoprecipitates and expression of claudin-5 gene in blood–brain-barrier endothelial cells via protein kinase A-dependent and -independent pathways

Cited by 181 publications

References 52 publications

Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells

Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells

Biology of claudins

Phosphorylation of Claudin-3 at Threonine 192 by cAMP-dependent Protein Kinase Regulates Tight Junction Barrier Function in Ovarian Cancer Cells

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