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First published online 11 April 2006
doi: 10.1242/jcs.02901

Journal of Cell Science 119, 1781-1789 (2006)
Published by The Company of Biologists 2006
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Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions

Akira Ikari1,2,*, Satomi Matsumoto1, Hitoshi Harada1, Kuniaki Takagi1, Hisayoshi Hayashi3, Yuichi Suzuki3, Masakuni Degawa4 and Masao Miwa2

1 Department of Environmental Biochemistry and Toxicology, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
2 Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
3 Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
4 Department of Molecular Toxicology and COE Program for the 21st Century, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan


Figure 1
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  		Fig. 1. Phosphorylation of paracellin-1 by cAMP. (A-F) Whole membrane fractions were prepared from MDCK cells expressing FLAG (Mock) or FLAG-tagged PCLN-1 (PCLN-1). The fractions (30 µg) were applied to SDS-polyacrylamide gel and then immunoblotted with antibodies raised against PCLN-1 (A), FLAG (B), occludin (C), claudin-1 (D), claudin-4 (E) or ZO-1 (F). To quantify the expression levels of these proteins, the amounts of occludin, claudin-1, claudin-4 and ZO-1 in the mock cells were normalized as 100%. In the PCLN-1-expressing cells, the relative expression levels of occludin, claudin-1, claudin-4 and ZO-1 were 98.6±8.5% (n=4), 104±4.1% (n=6), 102±9.0% (n=6) and 98.2±2.5% (n=4), respectively. (G-J) MDCK cells expressing FLAG-tagged PCLN-1 were treated with 50 µM H-89, 10 µM PKI or 50 µM DDA for 1 hour. When used, 100 µM 8-Br-cAMP (cAMP) was treated for 1 hour after the addition of DDA. Whole membrane fractions (30 µg) were immunoblotted with anti-FLAG antibody (G,I). The whole membrane fractions (500 µg) were incubated with protein G-Sepharose and anti-FLAG antibody. The immune pellets were immunoblotted with anti-phosphoserine (P-Ser) antibody (H,J).

 

Figure 2
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  		Fig. 2. Effects of PCLN-1 expression on paracellular permeability. (A) Mock (open columns) and FLAG-tagged PCLN-1-expressing MDCK cells (closed columns) were plated on Snapwell polyester filters. TER values were measured at 7 days after plating. (B,C) Mock (open columns) and FLAG-tagged PCLN-1-expressing cells (closed columns) were cultured for 7 days. The absolute permeability for Na+ (PNa) and Cl- (PCl) and relative permeability of Na+ to Cl- (PNa/PCl) were calculated as described in the Materials and Methods. **P<0.01, compared with mock cells. NS, not significant compared with mock cells.

 

Figure 3
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  		Fig. 3. Effects of PKA inhibitors on paracellular permeability. (A,B,D) Mock (open columns) and FLAG-tagged PCLN-1-expressing MDCK cells (closed columns) were treated with 50 µM H-89, 10 µM PKI or 50 µM DDA for 1 hour. When used, 100 µM 8-Br-cAMP (cAMP) was treated for 1 hour after the addition of DDA. TER, FITC-dextran-4k flux and Mg2+ transport from the apical to basal compartments were measured after treatment with each drug. *P<0.05 and **P<0.01, compared with control. ##P<0.01, compared with DDA only. NS, not significant compared with the control. (C) Mg2+ transport from the apical to basal compartments was measured in mock (open circles) and FLAG-tagged PCLN-1-expressing MDCK cells (closed circles). After incubation at 37°C for 0.5, 1 or 2 hours, the basal compartment media were collected and subjected to Mg2+ measurement. **P<0.01, compared with mock cells.

 

Figure 4
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  		Fig. 4. Effects of PKA inhibitors on PCLN-1 distribution. Mock and FLAG-tagged PCLN-1-expressing MDCK cells were treated with 50 µM H-89, 10 µM PKI, 50 µM DDA or 50 µM DDA for 1 hour. When used, 100 µM 8-Br-cAMP (cAMP) was treated for 1 hour after the addition of DDA. (A) The Triton X-100-soluble (60 µg) and -insoluble fractions (30 µg) were immunoblotted with anti-FLAG antibody. (B) The whole membrane fractions (500 µg) were incubated with protein G-Sepharose and anti-FLAG antibody to immunoprecipitate (IP). The immune pellets were immunoblotted (IB) with anti-ZO-1 antibody (upper) or anti-PCLN-1 (lower) antibodies. Whole membrane fractions (30 µg) were immunoblotted with anti-ZO-1 antibody (middle).

 

Figure 5
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  		Fig. 5. Localization of PCLN-1 in the TJ. FLAG-tagged PCLN-1-expressing MDCK cells were treated with 50 µM H-89, 10 µM PKI, 50 µM DDA or 50 µM DDA for 1 hour. When used, 100 µM 8-Br-cAMP (cAMP) was treated for 1 hour after the addition of DDA. Cells were double stained with anti-FLAG antibody (green) and anti-ZO-1 antibody (red). The x-y sections represent the area of the TJ. Right panels (x-z) show the vertical sections indicated by the triangles at the merged images. Bar, 10 µm.

 

Figure 6
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  		Fig. 6. Dysfunction of PCLN-1 by the S217A mutation. (A) Schematic description of the GST-fused cytoplasmic C-terminal domain of PCLN-1. (B) Proteins were fractionated on gels and stained with Coomassie Brilliant Blue (CBB) for the detection of GST proteins. The proteins bound to beads were incubated with the activated PKA subunit, and were then immunoblotted with anti-phosphoserine antibody (P-Ser). (C-E) Whole membrane fractions (30 µg) were immunoblotted with anti-FLAG (C), anti-claudin-1 (D) or anti-claudin-4 antibodies (E). As compared with cells expressing WT PCLN-1, the relative expression levels in cells expressing the S217A mutant PCLN-1 were 102±6.0% (claudin-1) and 97.1±9.0% (claudin-4), respectively. (F,H) Whole membrane fractions (500 µg) were incubated with protein G-Sepharose and anti-FLAG antibody to immunoprecipitate (IP). The immune pellets were immunoblotted (IB) with (F, upper) anti-phosphoserine (P-Ser), (F, lower) anti-PCLN-1, or (H, upper) anti-ZO-1 antibodies. Whole membrane fractions (30 µg) were immunoblotted with anti-FLAG or anti-ZO-1 antibodies (H, lower). (G) The Triton X-100-soluble (60 µg) and -insoluble fractions (30 µg) were immunoblotted with anti-FLAG-antibody. (I-K) TER, FITC-dextran-4k flux and Mg2+ transport were measured in MDCK cells expressing FLAG (Mock), FLAG-tagged WT PCLN-1 (WT), or FLAG-tagged S217A mutant PCLN-1 (S217A). **P<0.01. NS, not significant.

 

Figure 7
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  		Fig. 7. Lysosomal targeting of PCLN-1 by dephosphorylation. (A,B) MDCK cells expressing WT PLCN-1 were grown to a confluent condition on cover glasses. Cells were incubated in the absence (A) and presence (B) of 50 µM H-89 for 1 hour. The cells were then double stained with anti-FLAG antibody (green) and organelle markers (red) of lysosomes (lysotracker), Golgi (furin) or early endosomes (EE, early endosomal antigen 1). (C) MDCK cells expressing the S217A mutant PCLN-1 were double stained with anti-FLAG antibody (green) and ZO-1 (red) or organelle markers (red) of lysosomes, Golgi or EE. The x-y sections represent the area of the TJ. Bar, 10 µm.

 

Figure 8
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  		Fig. 8. Degradation of dephosphorylated PCLN-1 in the lysosome. (A) MDCK cells expressing WT PLCN-1 were treated with 50 µM H-89, 50 µM H-89 plus 10 µM lactacystin, or 50 µM H-89 plus 100 µM chloroquine for 3 hours in the presence of 10 µM cycloheximide. (B) MDCK cells expressing the S217A mutant PLCN-1 were treated with 10 µM lactacystin or 100 µM chloroquine for 3 hours in the presence of 10 µM cycloheximide. Whole membrane fractions (30 µg) were immunoblotted with anti-ZO-1 (A,B, upper) or anti-FLAG antibody antibodies (A,B, middle). The whole membrane fractions (500 µg) were incubated with protein G-Sepharose and anti-FLAG antibody to immunoprecipitate (IP). The immune pellets were immunoblotted (IB) with anti-ubiquitin antibody (Ub; A,B, lower).

 

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© The Company of Biologists Ltd 2006