Novel Functions of the Phospholipase D2-Phox Homology Domain in Protein Kinase Cζ Activation

Abstract

It has been established that protein kinase Cζ (PKCζ) participates in diverse signaling pathways and cellular functions in a wide variety of cells, exhibiting properties relevant to cellular survival and proliferation. Currently, however, the regulation mechanism of PKCζ remains elusive. Here, for the first time, we determine that phospholipase D2 (PLD2) enhances PKCζ activity through direct interaction in a lipase activity-independent manner. This interaction of the PLD2-Phox homology (PX) domain with the PKCζ-kinase domain also induces the activation loop phosphorylation of PKCζ and downstream signal stimulation, as measured by p70 S6 kinase phosphorylation. Furthermore, only the PLD2-PX domain directly stimulates PKCζ activity in vitro, and it is necessary for the formation of the ternary complex with phosphoinositide-dependent kinase 1 and PKCζ. The mutant that substitutes the triple lysine residues (Lys¹⁰¹, Lys¹⁰², and Lys¹⁰³) within the PLD2-PX domain with alanine abolishes interaction with the PKCζ-kinase domain and activation of PKCζ. Moreover, breast cancer cell viability is significantly affected by PLD2 silencing. Taken together, these results suggest that the PLD2-mediated PKCζ activation is induced by its PX domain performing both direct activation of PKCζ and assistance of activation loop phosphorylation. Furthermore, we find it is an important factor in the survival of breast cancer cells.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We gratefully acknowledge Alex Toker for kindly providing anti-pPKCζ antibody (pT410) to our laboratory.

This work was supported by the programs of 21st Frontier Proteomics and the National Research Laboratory of the Ministry of Science and Technology, Republic of Korea.