Protein kinase C-δ interacts with and phosphorylates ARD1

Protein kinase C-δ (PKCδ) is a diacylglycerol-dependent, calcium-independent novel PKC isoform that is engaged in various cell signaling pathways, such as cell proliferation, apoptosis, inflammation, and oxidative stress. In this study, we searched for proteins that bind PKCδ using a yeast two-hybrid assay and identified murine arrest-defective 1 (mARD1) as a binding partner. The interaction between PKCδ and mARD1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Furthermore, recombinant PKCδ phosphorylated full-length mARD1 protein. The NetPhos online prediction tool suggested PKCδ phosphorylates Ser⁸⁰ , Ser¹⁰⁸ , and Ser¹¹⁴ residues of mARD1 with the highest probability. Based on these results, we synthesized peptides containing these sites and examined their phosphorylations using recombinant PKCδ. Autoradiography confirmed these sites were efficiently phosphorylated. Consequent mass spectrometry and peptide sequencing in combination with MALDI-TOF MS/MS confirmed that Ser⁸⁰ and Ser¹⁰⁸ were major phosphorylation sites. The alanine mutations of Ser⁸⁰ and Ser¹⁰⁸ abolished the phosphorylation of mARD1 by PKCδ in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser⁸⁰ of ARD1 was phosphorylated by PKCβI and PKCζ isotypes with the highest selectivity, while Ser¹⁰⁸ and/or Ser¹¹⁴ were phosphorylated by PKCγ with activities comparable to that of the PKCδ isoform. Overall, these results suggest the possibility that PKCδ transduces signals by regulating phosphorylation of ARD1.