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Molecular and Cellular Biology, May 2001, p. 3351-3363, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3351-3363.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

A Single Point Mutation in the V3 Region Affects Protein Kinase Calpha Targeting and Accumulation at Cell-Cell Contacts

Alice Vallentin, Thi-Chang Lo, and Dominique Joubert*

INSERM U469, 34094 Montpellier Cedex 5, France

Received 12 September 2000/Returned for modification 21 November 2000/Accepted 16 February 2001

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 [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 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.

* Corresponding author. Mailing address: INSERM U469, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France. Phone: (33) 467-142-918. Fax: (33) 467-542-432. E-mail: joubert{at}u469.montp.inserm.fr.

Molecular and Cellular Biology, May 2001, p. 3351-3363, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3351-3363.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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