| First Author | Kumar V | Year | 2015 |
| Journal | J Biol Chem | Volume | 290 |
| Issue | 4 | Pages | 1936-51 |
| PubMed ID | 25505183 | Mgi Jnum | J:217880 |
| Mgi Id | MGI:5616014 | Doi | 10.1074/jbc.M114.598698 |
| Citation | Kumar V, et al. (2015) Generation and Characterization of ATP Analog-specific Protein Kinase Cdelta. J Biol Chem 290(4):1936-51 |
| abstractText | To better study the role of PKCdelta in normal function and disease, we developed an ATP analog-specific (AS) PKCdelta that is sensitive to specific kinase inhibitors and can be used to identify PKCdelta substrates. AS PKCdelta showed nearly 200 times higher affinity (Km) and 150 times higher efficiency (kcat/Km) than wild type (WT) PKCdelta toward N(6)-(benzyl)-ATP. AS PKCdelta 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 PKCdelta was insensitive to all PP1 analogs. To understand the mechanisms for specificity and affinity of these analogs, we created in silico WT and AS PKCdelta homology models based on the crystal structure of PKCiota. N(6)-(Benzyl)-ATP and ATP showed similar positioning within the purine binding pocket of AS PKCdelta, whereas N(6)-(benzyl)-ATP was displaced from the pocket of WT PKCdelta 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 PKCdelta, 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 PKCdelta. Other PP1 analogs failed to interact with either AS PKCdelta or WT PKCdelta. These results provide a structural basis for the ability of AS PKCdelta 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 PKCdelta and other kinases to understand their function in cell signaling and to identify unique substrates. |
