Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase Cdelta in the intact cell.
The role of the protein kinase C (PKC) family of serine/threonine kinases in cellular differentiation, proliferation, apoptosis, and other responses makes them attractive therapeutic targets. The activation of PKCs by ligands in vivo varies depending upon cell type; therefore, methods are needed to screen the potency of PKCs in this context. Here we describe a genetically encoded chimera of native PKCdelta fused to yellow- and cyan-shifted green fluorescent protein, which can be expressed in mammalian cells. This chimeric protein kinase, CY-PKCdelta, retains native or near-native activity in the several biological and biochemical parameters that we tested. Binding assays showed that CY-PKCdelta and native human PKCdelta have similar binding affinity for phorbol 12,13-dibutyrate. Analysis of translocation by Western blotting and confocal microscopy showed that CY-PKCdelta translocates from the cytosol to the membrane upon treatment with ligand, that the translocation has similar dose dependence as that of endogenous PKCdelta, and that the pattern of translocation is indistinguishable from that of the green fluorescent protein-PKCdelta fusion well characterized from earlier studies. Treatment with phorbol ester of cells expressing CY-PKCdelta resulted in a dose-dependent increase in FRET that could be visualized in situ by confocal microscopy or measured fluorometrically. By using this construct, we were able to measure the kinetics and potencies of 12 known PKC ligands, with respect to CY-PKCdelta, in the intact cell. The CY-PKCdelta chimera and the in vivo assays described here therefore show potential for high throughput screening of prospective PKCdelta ligands within the context of cell type.
Braun
Derek C
DC
Department of Biology, Gallaudet University, Washington, D. C. 20002, USA.
Garfield
Susan H
SH
Blumberg
Peter M
PM
D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D015240:Phorbol 12,13-Dibutyrate / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology*; D051745:Protein Kinase C-delta; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008024:Ligands; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; C497259:Prkcd protein, mouse; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta