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 coimmunoprecipitation assays. Furthermore, recombinant PKCδ phosphorylated fulllength mARD1 protein. The NetPhos online prediction tool suggested PKCδ 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 PKCδ. 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 PKCδ in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser 80 of ARD1 was phosphorylated by PKCβI and PKCζ isotypes with the highest selectivity, while Ser 108 and/or Ser 114 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.

Prenylated PALM2 Promotes the Migration of Esophageal Squamous Cell Carcinoma Cells Through Activating Ezrin

“…1 F ). RAP2B and UCHL1 have been studied for their structure, localization, and functions, and their prenylation modifications have also been confirmed and can affect their functions ( 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ). Consequently, PALM2, which had the CAAX motif at the carboxyl terminus but had unclear cellular localization and molecular function, was chosen for further study.…”

“…PALM2 also has many predicted sites of phosphorylation ( https://services.healthtech.dtu.dk/services/NetPhos-3.1/ ) ( supplemental Fig. S4 D ) ( 41 ), ubiquitination ( http://gpsuber.biocuckoo.cn/ ), and acetylation ( http://pail.biocuckoo.org/ ). In our previous proteomics data ( 22 ), we detected seven phosphorylated sites (S260, S262, S371, S376, S378, S382, S383) predicted online.…”

Phosphorylation of the Autographa californica multiple nucleopolyhedrovirus polyhedron envelope protein plays an important role in the formation of the occlusion body envelope

“…Phosphorylation sites were predicted using the NetPhos 3.0 prediction online service (http://www.cbs.dtu.dk/services/NetPhos/). Full-length PEP sequences were used for the predictions, and sites that scored ≥0.5 were defined as candidate phosphorylation sites [34]. Homologues of PEP were searched against the NCBI protein database with the blast algorithm.…”

Identification of Phytoconstituents as Potent Inhibitors of Casein Kinase-1 Alpha Using Virtual Screening and Molecular Dynamics Simulations

“…Casein kinase-1 alpha (CK1α) belongs to the serine/threonine family of kinases and functions primarily as a regulator in various signaling pathways [1]. Human kinases are considered as attractive drug targets for cancer therapy [2][3][4][5][6][7]. Like other kinases, CK1α performs multiple biological processes such as cell division, cell cycle, beta-catenin destruction and cell morphogenesis, signal transduction, WNT signaling pathway, etc.…”