Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

Abstract

The C2 domain of protein kinase Cα (PKCα) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca²⁺signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca²⁺-activated PKCα and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP₂), dramatically enhances the Ca²⁺-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCα Ca²⁺-binding loops (CBLs) and PIP₂binding site (β-strands 3–4) into a different C2 domain exhibits native Ca²⁺-triggered targeting to plasma membrane and recognizes PIP₂. Conversely, a hybrid containing the CBLs but lacking the PIP₂site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP₂. Similarly, PKCα C2 domains possessing mutations in the PIP₂site target primarily to TGN and fail to recognize PIP₂. Overall, these findings demonstrate that the CBLs are essential for Ca²⁺-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on β-strands 3–4, which bind to plasma membrane PIP₂.