title: Generation and characterization of ATP analog-specific protein kinase Cδ.
abstract: To better study the role of PKCδ in normal function and disease, we developed an ATP analog-specific (AS) PKCδ that is sensitive to specific kinase inhibitors and can be used to identify PKCδ substrates. AS PKCδ showed nearly 200 times higher affinity (Km) and 150 times higher efficiency (kcat/Km) than wild type (WT) PKCδ toward N(6)-(benzyl)-ATP. AS PKCδ was uniquely inhibited by 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1NA-PP1) and 1-(tert-butyl)-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2MB-PP1) but not by other 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) analogs tested, whereas WT PKCδ was insensitive to all PP1 analogs. To understand the mechanisms for specificity and affinity of these analogs, we created in silico WT and AS PKCδ homology models based on the crystal structure of PKCι. N(6)-(Benzyl)-ATP and ATP showed similar positioning within the purine binding pocket of AS PKCδ, whereas N(6)-(benzyl)-ATP was displaced from the pocket of WT PKCδ and was unable to interact with the glycine-rich loop that is required for phosphoryl transfer. The adenine rings of 1NA-PP1 and 2MB-PP1 matched the adenine ring of ATP when docked in AS PKCδ, and this interaction prevented the potential interaction of ATP with Lys-378, Glu-428, Leu-430, and Phe-633 residues. 1NA-PP1 failed to effectively dock within WT PKCδ. Other PP1 analogs failed to interact with either AS PKCδ or WT PKCδ. These results provide a structural basis for the ability of AS PKCδ to efficiently and specifically utilize N(6)-(benzyl)-ATP as a phosphate donor and for its selective inhibition by 1NA-PP1 and 2MB-PP1. Such homology modeling could prove useful in designing molecules to target PKCδ and other kinases to understand their function in cell signaling and to identify unique substrates.
authors.affiliation: From the Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, Ohio 44242.
authors.forename: Varun
authors.initials: V
authors.lastname: Kumar
authors.affiliation: From the Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, Ohio 44242.
authors.forename: Yi-Chinn
authors.initials: YC
authors.lastname: Weng
authors.affiliation: the Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, Ohio 44272.
authors.forename: Werner J
authors.initials: WJ
authors.lastname: Geldenhuys
authors.affiliation: the Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, California 94608, and.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: From the Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, Ohio 44242.
authors.forename: Xiqian
authors.initials: X
authors.lastname: Han
authors.affiliation: the Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, California 94608, and the Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas, Austin, Texas 78712.
authors.forename: Robert O
authors.initials: RO
authors.lastname: Messing
authors.affiliation: From the Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, Ohio 44242, the Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, California 94608, and wchou2@kent.edu.
authors.forename: Wen-Hai
authors.initials: WH
authors.lastname: Chou
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000378:metabolism*; D011687:Purines / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D013481:Superoxides / Q000737:chemistry
chemical_list: D011687:Purines; D005973:Glutamine; D013481:Superoxides; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D051745:Protein Kinase C-delta; D007930:Leucine; D008239:Lysine; C030985:purine
keywords: ATP; Chemical Biology; Protein Kinase C (PKC); Protein Phosphorylation; Stroke