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J. Biol. Chem., Vol. 278, Issue 4, 2348-2355, January 24, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/4/2348    most recent M210093200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hu, T. Articles by Exton, J. H. Search for Related Content PubMed PubMed Citation Articles by Hu, T. Articles by Exton, J. H. Social Bookmarking                      What's this?

Mechanisms of Regulation of Phospholipase D1 by Protein Kinase C^*

Tianhui Hu and John H. Exton

From the Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

It has been suggested that protein-protein interaction is important for protein kinase C (PKC)  to activate phospholipase D1 (PLD1). To determine the one or more sites on PKC that are involved in binding to PLD1, fragments containing the regulatory domain, catalytic domain, and C1-C3 domain of PKC were constructed and shown to be functional, but they all failed to bind and activate PLD1 in vivo and in vitro. A C-terminal 23-amino acid (aa) deletion mutant of PKC was also found to be inactive. To define the binding/activation site(s) in the C terminus of PKC, 1- to 11-aa deletion mutants were made in this terminus. Deletion of up to 9 aa did not alter the ability of PKC to bind and activate PLDl, whereas a 10-aa deletion was inactive. The residue at position 10 was Phe⁶⁶³. Mutations of this residue (F663D and F663A) caused loss of binding, activation, and phosphorylation of PLD1, indicating that Phe⁶⁶³ is essential for these activities. Time course experiments showed that the activation of PLD1 by PMA was much faster than its phosphorylation, and its activity decreased as phosphorylation increased with time. Staurosporine, a PKC inhibitor, completely inhibited PLD1 phosphorylation in response to 4-phorbol 12-myristate 13-acetate PMA and blocked the later decrease in PLD activity. The same results were found with the D481E mutant of PKC, which is unable to phosphorylate PLD1. These results indicate that neither the regulatory nor catalytic domains of PKC alone can bind to or activate PLD1 and that a residue in the C terminus of PKC (Phe⁶⁶³) is required for these effects. The initial activation of PLD1 by PMA is highly correlated with the binding of PKC. Although PKC can phosphorylate PLD1, this is a relatively slow process and is associated with inactivation of the enzyme.

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