Activation loop phosphorylation-independent kinase activity of human protein kinase C ζ

Abstract

Atypical protein kinase C ζ (PKCζ) plays an important role in cell proliferation and survival. PKCζ and its truncated form containing only the kinase domain, CATζ, have been reported to be activated by the phosphorylation of threonine 410 in the activation loop. We expressed both the full length PKCζ and CATζ in a baculovirus/insect cell overexpression system and purified the proteins for biochemical characterization. Ion exchange chromatography of CATζ revealed three species with different levels of phosphorylation at Thr-410 and allowed the isolation of the CATζ protein devoid of phosphorylation at Thr-410. All three species of CATζ were active and their activity was not correlated with phosphorylation at Thr-410, indicating that the kinase activity of CATζ did not depend solely on activation loop phosphorylation. Tyrosine phosphorylation was detected in all three species of CATζ and the full length PKCζ. Homology structural modeling of PKCζ revealed a conserved, predicted-to-be phosphorylated tyrosine residue, Tyr-428, in the close proximity of the RD motif of the catalytic loop and of Thr-410 in the activation loop. The structural analysis indicated that phospho-Tyr-428 would interact with two key, positively-charged residues to form a triad conformation similar to that formed by phospho-Thr-410. Based on these observations, it is possible that the Thr-410 phosphorylation-independent kinase activity of CATζ is regulated by the phosphorylation of Tyr-428. This alternative mode of PKCζ activation is supported by the observed stimulation of PKCι kinase activity upon phosphorylation at the equivalent site by Abl, and may be involved in resistance to drug-induced apoptosis. © 2007 Wiley-Liss, Inc.