Toxic Metal Ions Promote Self-Association and Replace Structural Zinc Ions in the Regulatory Region of Protein Kinase C

Abstract

Protein Kinase C (PKC) isoenzymes are a family of kinases that control cell proliferation, migration, and apoptosis. The hallmark of PKC activation is their translocation to lipid membranes that occurs in response to Ca2+ and diacylglycerol. Previous studies have shown that PKC activity is modulated by toxic metal ions such as Pb2+ and Cd2+. Our objective was to understand the effect of these metal ions on the structure and function of the membrane-binding regulatory region of PKC. Our efforts focused on the PKCα region that consists of two peripheral membrane-binding domains, C1B and C2. Using a combination of biophysical techniques, we demonstrate that interactions of Pb2+ and Cd2+ with C1B-C2 have profound and distinct consequences for the biophysical and functional properties of this region. While both Cd2+ and Pb2+ can spontaneously replace structural Zn2+ ions in C1B, NMR spectroscopy revealed that only Cd2+ was able to support the proper fold and ligand-binding function of the protein. In addition, Cd2+ promotes the formation of well-defined C1B-C2 oligomers that we were able to detect and characterize using small-angle X-ray scattering experiments and negative-stain electron microscopy. In contrast to Cd2+, Pb2+ cannot support the proper fold of C1B but acts successfully as a functional surrogate of Ca2+ in driving protein-membrane association. Our work demonstrates the potential diversity of responses of signaling proteins to toxic metal ions and suggests that molecular mechanisms of Pb2+ and Cd2+ toxicity are distinct. This work was supported by Welch Foundation grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 GM108998.