Analysis of Protein Kinase D1’s role during reactive oxygen species-mediated signaling

Abstract

Protein Kinase D1 (PKD1) is a critical component of cellular responses to reactive oxygen species (ROS)-mediated apoptosis. Previous studies demonstrated that upon oxidative stress, PKD1 is activated through a signaling pathway mediated by the Src/Abl non-receptor tyrosine kinases. Subsequently, PKD1’s translocation to different cellular compartments can regulate gene expression and promote cell survival through the NF-kB signaling pathway. Additionally, PKD1 deficiency increases ROS sensitivity in mouse embryonic fibroblasts (MEFs), leading to mitochondrial membrane depolarization and apoptosis when exposed to oxidative stress. Our study examined PKD1’s protective role against oxidative stress-induced apoptosis and its impact on gene expression. Using wild-type (WT) and PKD1-deficient (PKD1-/-) MEFs, we found that the absence of PKD1 significantly increases oxidative stressinduced apoptosis, as evidenced by H₂O₂ stimulation and serum starvation experiments. To better understand the molecular mechanisms underlying PKD1's protective function, RNASeq analysis was performed, comparing gene expression levels between WT and PKD1-/- MEFs. RNA-Seq analysis revealed differentially expressed genes (DEGs) between WT and PKD1-/- genotypes, with 383 DEGs in primary and 1705 DEGs in immortalized MEFs. Notably, BCL2L11 (BIM), a pro-apoptotic protein, was more than two-fold down-regulated in both primary and immortalized PKD1-/- MEFs, suggesting its interaction with PKD1 on the mitochondrial outer membrane through the mitochondrial apoptosis pathway. In addition, KEGG pathway analysis highlighted enriched differentially expressed pathways, including those associated with apoptosis, cell survival, and response to oxidative stress. This study sheds light on how PKD1 defends against oxidative stress and influences gene expression, potentially contributing to the development of treatments for oxidative stress-linked diseases.