Format

Send to

Choose Destination
J Mol Biol. 2004 Jan 23;335(4):1117-29.

Role of the lysine-rich cluster of the C2 domain in the phosphatidylserine-dependent activation of PKCalpha.

Author information

1
Department de Bioqui;mica y Biologi;a Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30100 Murcia, Spain.

Abstract

The C2 domain of PKCalpha is a Ca(2+)-dependent membrane-targeting module involved in the plasma membrane localization of the enzyme. Recent findings have shown an additional area located in the beta3-beta4 strands, named the lysine-rich cluster, which has been demonstrated to be involved in the PtdIns(4,5)P(2)-dependent activation of the enzyme. Nevertheless, whether other anionic phospholipids can bind to this region and contribute to the regulation of the enzyme's function is not clear. To study other possible roles for this cluster, we generated double and triple mutants that substituted the lysine by alanine residues, and studied their binding and activation properties in a Ca(2+)/phosphatidylserine-dependent manner and compared them with the wild-type protein. It was found that some of the mutants exerted a constitutive activation independently of membrane binding. Furthermore, the constructs were fused to green fluorescent protein and were expressed in fibroblast cells. It was shown that none of the mutants was able to translocate to the plasma membrane, even in saturating conditions of Ca(2+) and diacylglycerol, suggesting that the interactions performed by this lysine-rich cluster are a key event in the subcellular localization of PKCalpha. Taken together, the results obtained showed that these lysine residues might be involved in two functions: one to establish an intramolecular interaction that keeps the enzyme in an inactive conformation; and the second, once the enzyme has been partially activated, to establish further interactions with diacylglycerol and/or acidic phospholipids, leading to the full activation of PKCalpha.

PMID:
14698304
DOI:
10.1016/j.jmb.2003.10.080
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center