Abstract
The C2 domains of conventional protein kinase C (PKC) have been implicated in their Ca2+-dependent membrane binding. The C2 domain of PKC-alpha contains several Ca2+ ligands that bind multiple Ca2+ ions and other putative membrane binding residues. To understand the roles of individual Ca2+ ligands and protein-bound Ca2+ ions in the membrane binding and activation of PKC-alpha, we mutated five putative Ca2+ ligands (D187N, D193N, D246N, D248N, and D254N) and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration of PKC-alpha. Altered properties of these mutants indicate that individual Ca2+ ions and their ligands have different roles in the membrane binding and activation of PKC-alpha. The binding of Ca2+ to Asp187, Asp193, and Asp246 of PKC-alpha is important for the initial binding of protein to membrane surfaces. On the other hand, the binding of another Ca2+ to Asp187, Asp246, Asp248, and Asp254 induces the conformational change of PKC-alpha, which in turn triggers its membrane penetration and activation. Among these Ca2+ ligands, Asp246 was shown to be most essential for both membrane binding and activation of PKC-alpha, presumably due to its coordination to multiple Ca2+ ions. Furthermore, to identify the residues in the C2 domain that are involved in membrane binding of PKC-alpha, we mutated four putative membrane binding residues (Trp245, Trp247, Arg249, and Arg252). Membrane binding and enzymatic properties of two double-site mutants (W245A/W247A and R249A/R252A) indicate that Arg249 and Arg252 are involved in electrostatic interactions of PKC-alpha with anionic membranes, whereas Trp245 and Trp247 participate in its penetration into membranes and resulting hydrophobic interactions. Taken together, these studies provide the first experimental evidence for the role of C2 domain of conventional PKC as a membrane docking unit as well as a module that triggers conformational changes to activate the protein.
Highlights
The protein kinase C (PKC)1 family is a set of serine/threonine kinases that transduce the myriad of signals activating cellular functions and proliferation [1,2,3]
Design and Physical Properties of PKC-␣ Mutants—According to the model structure of the C2 domain of PKC-␣ shown in Fig. 1C, five Ca2ϩ-binding aspartyl residues can be classified into three groups; calcium binding region 1 (CBR1) ligands, which primarily coordinate CA1 (Asp187 and Asp193), CBR2 ligands, which mainly coordinate CA2 (Asp248 and Asp254), and Asp246, which coordinates both Ca2ϩ ions
From crystal structures of C2 domains of phospholipase C-␦1 and cytosolic phospholipase A2, it has been proposed that CBR1 and CBR3 are involved in binding to membranes [10, 23]: CBR3 in membrane penetration and CBR1 in interfacial contact with the lipid head group
Summary
The protein kinase C (PKC)1 family is a set of serine/threonine kinases that transduce the myriad of signals activating cellular functions and proliferation [1,2,3]. It has been proposed that the C2 domain of conventional PKCs is involved in this Ca2ϩ-dependent membrane binding activity [4]. We measured the PKC activity of wild type and mutants as a function of free Ca2ϩ concentration and the PS content in vesicles.
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