We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury: however, the exact role of DAG kinase (DGK)zeta, an isoform expressed in intestine, during this process is unknown. We Sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGK zeta siRNA transfection decreased H2O2-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGK zeta also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKr, rapidly translocated to the cytoplasm following H2O2 treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGK zeta, suggesting DGK is all upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells. (c) 2008 Elsevier Inc. All rights reserved.