Visualization of protein kinase C activity in live cells in response to natural stimulation of endogenous receptors

Abstract

Protein kinase C (PKC) encompasses a family of Ser/Thr kinases whose members transduce the multitude of signals that stimulate phospholipid hydrolysis. PKC isozymes typically reside in the cytosol of unstimulated cells, but quickly translocate to cellular membranes following generation of diacylglycerol and, for conventional PKCs, Ca2+. This membrane translocation has traditionally functioned as the hallmark of protein kinase C activation. We have recently generated a genetically-encoded reporter that allows visualization of protein kinase C activity in real time in live cells (Violin, et al. 2003). This reporter is named CKAR for C Kinase Activity Reporter and is composed of two FRET compatible fluorescent moieties, a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP), which flank a PKC-specific substrate peptide adjacent to an FHA2 phosphopeptide binding module. Here we report on monitoring PKC responses downstream of endogenous receptors using CKAR that has been targeted to different intracellular locations. Specifically, we report on the results of targeting CKAR to plasma membrane, cytosol, mitochondria, Golgi, and nucleus. We first characterize the range of each targeted reporter in COS 7 cells and show that phosphorylation by PKC can be achieved at all of these locations. Next, we demonstrate varying magnitude and duration of phosphorylation by PKC at these regions in response to natural stimuli and begin to investigate underlying causes for the differences. Preliminary data suggest differences in localized second messenger turnover and regional phosphatase activity are key regulators of PKC signaling. Thus, these reporters allow dissection of the spatio-temporal dynamics of PKC signaling in live cells upon stimulation of endogenous receptors by natural ligands. NIH grant GM43154