Phosphorylation of zona occludens-2 by protein kinase C epsilon regulates its nuclear exportation.
Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCepsilon. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.
Chamorro
David
D
Departments of Physiology, Biophysics, and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Mexico, D.F., 07360, Mexico.
Alarcón
Lourdes
L
Ponce
Arturo
A
Tapia
Rocio
R
González-Aguilar
Héctor
H
Robles-Flores
Martha
M
Mejía-Castillo
Teresa
T
Segovia
José
J
Bandala
Yamir
Y
Juaristi
Eusebio
E
González-Mariscal
Lorenza
L
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D062827:Zonula Occludens-2 Protein
D005231:Fatty Acids, Unsaturated; D008565:Membrane Proteins; D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; D062827:Zonula Occludens-2 Protein; D010768:Phosphoserine; D051744:Protein Kinase C-epsilon; C038753:leptomycin B