Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases.
Conventional and novel protein kinase C (PKC) isozymes transduce the abundance of signals mediated by phospholipid hydrolysis; however redundancy in regulatory mechanisms confounds dissecting the unique signaling properties of each of the eight isozymes constituting these two subgroups. Previously, we created a genetically encoded reporter (C kinase activity reporter (CKAR)) to visualize the rate, amplitude, and duration of agonist-evoked PKC signaling at specific locations within the cell. Here we designed a reporter, δCKAR, that specifically measures the activation signature of one PKC isozyme, PKC δ, in cells, revealing unique spatial and regulatory properties of this isozyme. Specifically, we show two mechanisms of activation: 1) agonist-stimulated activation at the plasma membrane (the site of most robust PKC δ signaling), Golgi, and mitochondria that is independent of Src and can be triggered by phorbol esters and 2) agonist-stimulated activation in the nucleus that requires Src kinase activation and cannot be triggered by phorbol esters. Translocation studies reveal that the G-protein-coupled receptor agonist UTP induces the translocation of PKC δ into the nucleus by a mechanism that depends on the C2 domain and requires Src kinase activity. However, translocation from the cytosol into the nucleus is not required for the Src-dependent regulation of nuclear activity; a construct of PKC δ prelocalized to the nucleus continues to be activated by UTP by a mechanism dependent on Src kinase activity. These data identify the nucleus as a signaling hub for PKC δ that is driven by receptor-mediated signaling pathways (but not phorbol esters) and differs from signaling at plasma membrane and Golgi in that it is controlled by Src family kinases.
Kajimoto
Taketoshi
T
Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA.
Sawamura
Seishiro
S
Tohyama
Yumi
Y
Mori
Yasuo
Y
Newton
Alexandra C
AC
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004075:Diglycerides / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010716:Phosphatidylinositols / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D051745:Protein Kinase C-delta / Q000737:chemistry*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism*
D004075:Diglycerides; D010716:Phosphatidylinositols; D020033:Protein Isoforms; D019061:src-Family Kinases; D051745:Protein Kinase C-delta