A single point mutation in the V3 region affects protein kinase C alpha targeting and accumulation at cell-cell contacts

Given the importance of intercellular adhesion for many regulatory processe s, we have investigated the control of protein kinase Ca (PKC alpha) target ing to the cell-cell contacts. We have previously shown that, upon treatmen t of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKC alpha (hPKC alpha) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKC alpha, previously identified in a subpopulation of h uman 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 versu s 22.5 s), coinciding with the duration of the [Ca2+](1) increase. We found that in the presence or absence of PMA, RACK1 is never localized at the ce ll-cell contacts nor was it coimmunoprecipitated with hPKC alpha wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulatio n resulted in the presence of F-actin and beta -catenin at the cell-cell co ntacts and their exclusion from the rest of the plasma membrane. Upon disru ption of the F-actin network with phalloidin or cytochalasin D, wild-type h PKC alpha. translocates but did not accumulate at the plasma membrane and b eta -catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accum ulation of the D294G mutant. These results show that the presence of PKC al pha at the cell-cell contacts is a regulated process which depends upon the integrity of both PKC alpha and the actin microfilament network.