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 PIP2 binding 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 PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCα C2 domains possessing mutations in the PIP2 site 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.

Ceramide-1-phosphate Binds Group IVA Cytosolic Phospholipase a2 via a Novel Site in the C2 Domain

“…Subsequently, a number of other C2 domains have been shown to bind lipids through their ␤-groove in both Ca 2ϩ -dependent and Ca 2ϩ -independent manners (45)(46)(47). Although most C2 domains reported to bind lipids through their ␤-groove interact nonspecifically with phosphatidylinositides, such as PtdIns(4,5)P 2 , the cPLA 2 ␣ C2 domain is one of the first C2 domains demonstrated to harbor such selectivity for anionic lipids, only displaying an affinity increase with C1P.…”

Structural insights into the Ca 2+ and PI(4,5)P 2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

“…This revealed an additional lipidbinding site located in the polybasic region formed by β3-β4 strands that preferentially binds to PI(4,5)P 2 (11)(12)(13)(14)(15). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10,(16)(17)(18)(19).…”

Structural and mechanistic insights into the association of PKCα-C2 domain to PtdIns(4,5)P 2

“…The second region is a polybasic cluster that is located at the concave surface of the C2 domain formed by strands ␤3 and ␤4. Recent studies indicate that this region might bind specifically to PtdIns(4,5)P 2 in a Ca 2ϩ -dependent manner (1,(17)(18)(19)(20)(21).To gain insight into the structural and functional basis for the PtdIns(4,5)P 2 -dependent membrane targeting of the PKC␣-C2 domain, we determined the 3D structures of the ternary and quaternary complexes of the C2 domain of PKC␣, crystallized in presence of Ca 2ϩ and PtdIns(4,5)P 2 or Ca 2ϩ , PtdIns(4,5)P 2 and PtdSer. In addition, the crystallographic results were validated in living cells by site-directed mutagenesis of the residues involved in the PtdIns(4,5)P 2 -C2 domain interaction.…”

“…The second region is a polybasic cluster that is located at the concave surface of the C2 domain formed by strands ␤3 and ␤4. Recent studies indicate that this region might bind specifically to PtdIns(4,5)P 2 in a Ca 2ϩ -dependent manner (1,(17)(18)(19)(20)(21).…”