RESUMEN

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.

Asunto(s)

Núcleo Celular/enzimología , Proteínas de la Membrana/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Perros , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Ácidos Grasos Insaturados/farmacología , Inmunoprecipitación , Proteínas de la Membrana/biosíntesis , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Factores de Tiempo , Transfección , Proteína de la Zonula Occludens-2