Determination of the calcium-binding sites of the C2 domain of protein kinase Calpha that are critical for its translocation to the plasma membrane.
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.
Corbalán-García
S
S
Departamento de Bioqu approximately ímica y Biolog approximately ía Molecular (A). Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080-Murcia, Spain.
Rodríguez-Alfaro
J A
JA
Gómez-Fernández
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D020013:Calcium Signaling / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D002135:Calcium-Binding Proteins; D007527:Isoenzymes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha