Atypical protein kinase C-zeta isoform, a member of phospholipid-dependent serine/threonine kinases, plays a role in modulating cell proliferation, apoptosis and malignant transformation. The physiological function of PKC-zeta can be regulated by its subcellular localization, phosphorylation state, and conformational change caused by allosteric regulators. Perturbation at any of these regulation points disrupts signaling by PKC-zeta and may provide novel therapeutic opportunities for cancer treatment. To study the structure and function of the enzyme and the phosphorylation regulation of its kinase activity, we expressed both the full-length protein and the catalytic domain of PKC-zeta in a Baculovirus/insect cell overexpression system and purified the proteins by column chromatography. Ion exchange chromatography of these two proteins revealed three species in three separate elution peaks. All three species of each protein migrated to a single band in SDS-PAGE. Characterization of the three species of the catalytic domain showed in Western blotting experiments that the level of phosphorylation at threonine 410 in the activation loop was different and one of them had no phosphorylation observed. However, they are all fully active in a kinase activity assay, indicating that the kinase activity of the PKC-zeta catalytic domain does not require activation loop phosphorylation. These different species may represent different phosphorylation states or conformational variations of the enzyme that can be reassembled into an active conformation in a substrate binding-dependent manner. The catalytic domain of PKC-zeta expressed from E. coli lacked post-translational modification or phosphorylation at threonine 410 and showed kinase activity. The kinase activity increased when the catalytic domain was phosphorylated by PDK-1. It is possible that activation loop phosphorylation increases the binding affinity with substrate protein and enhances the kinase activity of the PKC-zeta catalytic domain. However, the phosphorylation is not required for basal kinase activity. In contrast, the threonine 410 phosphorylation of the full-length PKC-zeta plays a role in destabilizing the N-terminal pseudosubstrate binding, which is followed by dramatic conformational changes and structural rearrangements that facilitate the binding of substrate protein and catalysis within the active site.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]