Protein kinase C-δ interacts with and phosphorylates ARD1

Protein kinase C-delta (PKC delta) 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 PKC delta using a yeast two-hybrid assay and identified murine arrest-defective 1 (mARD1) as a binding partner. The interaction between PKC delta and mARD1 was confirmed by glutathioneS-transferase pull-down and co-immunoprecipitation assays. Furthermore, recombinant PKC delta phosphorylated full-length mARD1 protein. The NetPhos online prediction tool suggested PKC delta phosphorylates Ser(80), Ser(108), and Ser(114)residues of mARD1 with the highest probability. Based on these results, we synthesized peptides containing these sites and examined their phosphorylations using recombinant PKC delta. Autoradiography confirmed these sites were efficiently phosphorylated. Consequent mass spectrometry and peptide sequencing in combination with MALDI-TOF MS/MS confirmed that Ser(80)and Ser(108)were major phosphorylation sites. The alanine mutations of Ser(80)and Ser(108)abolished the phosphorylation of mARD1 by PKC delta in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser(80)of ARD1 was phosphorylated by PKC beta I and PKC zeta isotypes with the highest selectivity, while Ser(108)and/or Ser(114)were phosphorylated by PKC gamma with activities comparable to that of the PKC delta isoform. Overall, these results suggest the possibility that PKC delta transduces signals by regulating phosphorylation of ARD1.