Abstract
Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase Cα (PKCα) targeting to the cell-cell contacts. We have previously shown that, upon treatment of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKCα (hPKCα) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKCα, previously identified in a subpopulation of human tumors, induces the loss of this selective targeting. The D294G mutant is instead targeted to the entire plasma membrane, including the cell-cell contacts, and the duration of the first rapid and transient translocation induced by TRH (42) is longer than that of the wild-type enzyme (93.3 versus 22.5 s), coinciding with the duration of the [Ca2+]i increase. We found that in the presence or absence of PMA, RACK1 is never localized at the cell-cell contacts nor was it coimmunoprecipitated with hPKCα wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and β-catenin at the cell-cell contacts and their exclusion from the rest of the plasma membrane. Upon disruption of the F-actin network with phalloidin or cytochalasin D, wild-type hPKCα translocates but did not accumulate at the plasma membrane and β-catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accumulation of the D294G mutant. These results show that the presence of PKCα at the cell-cell contacts is a regulated process which depends upon the integrity of both PKCα and the actin microfilament network.
ACKNOWLEDGMENTS
We thank Danièle Gourdji for providing the GH3B6 cells, Catherine Legraverend and Corinne Prévostel for help in preparation of the manuscript, Tony Ng for providing experimental protocols for coimmunoprecipitation, and Xavier Bonnefont and Teddy Fauquier for help in confocal analyses.
A.V. was supported by the Association pour la Recherche contre le Cancer (ARC) and by the Ligue Nationale contre le Cancer. The confocal microscope was financed by grants from INSERM, Région Languedoc-Roussillon, ARC, and the Fondation pour la Recherche Médicale. This work was supported by grant 5695 from the ARC.