Synergistic effect of Pb(2+) and phosphatidylinositol 4,5-bisphosphate on C2 domain-membrane interactions.
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abstract
Ca(2+)-responsive C2 domains are peripheral membrane modules that target their host proteins to anionic membranes upon binding Ca(2+) ions. Several C2 domain-containing proteins, such as protein kinase C isoenzymes (PKCs), have been identified as molecular targets of Pb(2+), a known environmental toxin. We demonstrated previously that the C2 domain from PKC (C2) binds Pb(2+) with high affinity and undergoes membrane insertion in the Pb(2+)-complexed form. The objective of this work was to determine the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) on the C2-Pb(2+) interactions. Using nuclear magnetic resonance (NMR) experiments, we show that Pb(2+) and PIP(2) synergistically enhance each other's affinity for C2. Moreover, the affinity of C2 for PIP(2) increases upon progressive saturation of the metal-binding sites. Combining the NMR data with the results of protein-to-membrane Frster resonance energy transfer and vesicle sedimentation experiments, we demonstrate that PIP(2) can influence two aspects of C2-Pb(2+)-membrane interactions: the affinity of C2 for Pb(2+) and the association of Pb(2+) with the anionic sites on the membrane. Both factors may contribute to the toxic effect of Pb(2+) resulting from the aberrant modulation of PKC activity. Finally, we propose a mechanism for Pb(2+) outcompeting Ca(2+) from membrane-bound C2.
