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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
C
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
C
(PKC
) 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 PKC
(hPKC
) is selectively
targeted to the cell-cell contacts (42). Here we show that
the D294G mutation of hPKC
, 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 hPKC
wild type or the D294G mutant. In contrast, PMA treatment or long-term
TRH stimulation resulted in the presence of F-actin and
-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 hPKC
translocates but did not accumulate
at the plasma membrane and
-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 PKC
at the cell-cell
contacts is a regulated process which depends upon the integrity of
both PKC
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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