Protein kinase C-δ interacts with and phosphorylates ARD1.

Abstract

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.

• Authors: Kwang-Hoon Chun, Seung-Ju Cho, Ji-Won Lee, Ji Hae Seo, Kyu-Won Kim, Seung-Ki Lee
• Journal: Journal of cellular physiology (J Cell Physiol) Vol. 236 Issue 1 Pg. 379-391 (01 2021) ISSN: 1097-4652 [Electronic] United States
• PMID: 32542692 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2020 Wiley Periodicals LLC.

Chemical References

• Peptides
• Protein Isoforms
• Serine
• N-Terminal Acetyltransferase A
• Naa10 protein, mouse
• N-Terminal Acetyltransferase E
• Prkcd protein, mouse
• Protein Kinase C-delta

Topics

• Amino Acid Sequence
• Animals
• Cell Line
• Cell Line, Tumor
• HEK293 Cells
• HeLa Cells
• Humans
• Mice
• N-Terminal Acetyltransferase A (metabolism)
• N-Terminal Acetyltransferase E (metabolism)
• Oxidative Stress (physiology)
• Peptides (metabolism)
• Phosphorylation (physiology)
• Protein Isoforms (metabolism)
• Protein Kinase C-delta (metabolism)
• Serine (metabolism)
• Signal Transduction (physiology)