Roles of Ionic Residues of the C1 Domain in Protein Kinase C-α Activation and the Origin of Phosphatidylserine Specificity

Lenka Bittova, Robert V. Stahelin, Wonhwa Cho

Research output: Contribution to journalArticle

102 Scopus citations

Abstract

On the basis of extensive structure-function studies of protein kinase C-α (PKC-α), we have proposed an activation mechanism for conventional PKCs in which the C2 domain and the C1 domain interact sequentially with membranes (Medkova, M., and Cho, W. (1999) J. Biol. Chem. 274, 19852-19861). To further elucidate the interactions between the C1 and C2 domains during PKC activation and the origin of phosphatidylserine specificity, we mutated several charged residues in two C1 domains (C1a and C1b) of PKC-α. We then measured the membrane binding affinities, activities, and monolayer penetration of these mutants. Results indicate that cationic residues of the C1a domain, most notably Arg77, interact nonspecifically with anionic phospholipids prior to the membrane penetration of hydrophobic residues. The mutation of a single aspartate (Asp55) in the C1a domain to A1a or Lys resulted in dramatically reduced phosphatidylserine specificity in vesicle binding, activity, and monolayer penetration. In particular, D55A showed much higher vesicle affinity, activity, and monolayer penetration power than wild type under non-activating conditions, i.e. with phosphatidylglycerol and in the absence of Ca2+, indicating that Asp55 is involved in the tethering of the C1a domain to another part of PKC-α, which keeps it in an inactive conformation at the resting state. Based on these results, we propose a refined model for the activation of conventional PKC, in which phosphatidylserine specifically disrupts the C1a domain tethering by competing with Asp55, which then leads to membrane penetration and diacylglycerol binding of the C1a domain and PKC activation.

Original languageEnglish (US)
Pages (from-to)4218-4226
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number6
DOIs
StatePublished - Feb 9 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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