A single point mutation in the V3 region affects protein kinase Calpha targeting and accumulation at cell-cell contacts.
Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase Calpha (PKCalpha) 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 PKCalpha (hPKCalpha) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKCalpha, 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 [Ca(2+)](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 hPKCalpha wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and beta-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 hPKCalpha translocates but did not accumulate at the plasma membrane and beta-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 PKCalpha at the cell-cell contacts is a regulated process which depends upon the integrity of both PKCalpha and the actin microfilament network.
Vallentin
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INSERM U469, 34094 Montpellier Cedex 5, France.
Lo
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Joubert
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D000199:Actins; D000818:Animals; D002450:Cell Communication / Q000235:genetics*; D002460:Cell Line; D003599:Cytoskeleton; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D017354:Point Mutation; D011493:Protein Kinase C / Q000235:genetics*; D051571:Protein Kinase C-alpha
D000199:Actins; D007527:Isoenzymes; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha