Dataset Information

Determination of the calcium-binding sites of the C2 domain of protein kinase Calpha that are critical for its translocation to the plasma membrane.

ABSTRACT: The C2 domain is a conserved protein module present in various signal-transducing proteins. To investigate the function of the C2 domain of protein kinase Calpha (PKCalpha), we have generated a recombinant glutathione S-transferase-fused C2 domain from rat PKCalpha, PKC-C2. We found that PKC-C2 binds with high affinity (half-maximal binding at 0.6 microM) to lipid vesicles containing the negatively charged phospholipid phosphatidylserine. When expressed into COS and HeLa cells, most of the PKC-C2 was found at the plasma membrane, whereas when the cells were depleted of Ca2+ by incubation with EGTA and ionophore, the C2 domain was localized preferentially in the cytosol. Ca2+ titration was performed in vivo and the critical Ca2+ concentration ranged from 0.1 to 0.32 microM. We also identified, by site-directed mutagenesis, three aspartic residues critical for that Ca2+ interaction, namely Asp-187, Asp-246 and Asp-248. Mutation of these residues to asparagine, to abolish their negative charge, resulted in a domain expressed as the same extension as wild-type protein that could interact in vitro with neither Ca2+ nor phosphatidylserine. Overexpression of these mutants into COS and HeLa cells also showed that they cannot localize at the plasma membrane, as demonstrated by immunofluorescence staining and subcellular fractionation. These results suggest that the Ca2+-binding site might be involved in promoting the interaction of the C2 domain of PKCalpha with the plasma membrane in vivo.

SUBMITTER: Corbalan-Garcia S

PROVIDER: S-EPMC1220004 | biostudies-other | 1999 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. PtdIns(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P(2) and/or PtdIns(3,4,5)P(3) from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.

Dataset Information

Determination of the calcium-binding sites of the C2 domain of protein kinase Calpha that are critical for its translocation to the plasma membrane.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets