Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases

J Biol Chem. 2010 Dec 31;285(53):41896-910. doi: 10.1074/jbc.M110.184028. Epub 2010 Oct 19.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Cell Membrane / metabolism
  • Chlorocebus aethiops
  • Diglycerides / chemistry*
  • Fluorescence Resonance Energy Transfer
  • Golgi Apparatus / metabolism
  • HeLa Cells
  • Humans
  • Phosphatidylinositols / chemistry*
  • Plasmids / metabolism
  • Protein Isoforms
  • Protein Kinase C-delta / chemistry*
  • Signal Transduction
  • src-Family Kinases / metabolism*

Substances

  • Diglycerides
  • Phosphatidylinositols
  • Protein Isoforms
  • src-Family Kinases
  • Protein Kinase C-delta