Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

Abstract

Activation of the two ubiquitous families of protein kinases, protein kinase A (PKA) and protein kinase C (PKC), is thought to be independently coupled to stimulation of G␣ s and G␣ q , respectively. Live-cell confocal imaging of protein kinase C fluorescent protein fusion constructs revealed that simultaneous activation of G␣ s and G␣ q resulted in a differential translocation of the conventional PKC␣ to the plasma membrane while the novel PKC␦ was recruited to the membrane of the endoplasmic reticulum (ER). We demonstrate that the PKC␦ translocation was driven by a novel G␣ s -cyclic AMP-EPAC-RAP-PLC pathway resulting in specific diacylglycerol production at the membrane of the ER. Membrane-specific phosphorylation sensors revealed that directed translocation resulted in phosphorylation activity confined to the target membrane. Specific stimulation of PKC␦ caused phosphorylation of the inositol-1,4,5-trisphosphate receptor and dampening of global Ca 2؉ signaling revealed by graded flash photolysis of caged inositol-1,4,5-trisphosphate. Our data demonstrate a novel signaling pathway enabling differential decoding of incoming stimuli into PKC isoform-specific membrane targeting, significantly enhancing the versatility of cyclic AMP signaling, thus demonstrating the possible interconnection between the PKA and PKC pathways traditionally treated independently. We thus provide novel and elementary understanding and insights into intracellular signaling events.

Does Erythropoietin Regulate TRPC Channels in Red Blood Cells?

“…The latter aspect is likely to be relevant for the rather fragile mouse RBCs. Confocal imaging was performed as previously described [24], in detail we used a multibeam array scanner (Infinity-4, VisiTech Int., Sunderland, UK) with a 491 nm DPSS laser (Calypso, Cobolt, Solna, Sweden). The confocal scanner was attached to an inverted microscope (TE2000-U, Nikon, Tokyo, Japan) utilising a 60x objective.…”

Differential targeting of cPKC and nPKC decodes and regulates Ca2+ and lipid signalling

“…By coexpression of fluorescent protein markers of intracellular membranes, we attempted to understand the process of PKCδ translocation in living cells. Quantitative co-localization analysis of its subcellular targeting revealed that following ATP stimulation the endoplasmic reticulum (ER) membrane was the main target for PKCδ recruitment rather than Golgi or mitochondrial membranes [18]. Other nPKC subfamily member, e.g.…”

“…By employing a combination of multiple pharmacological and molecular biological methods with optical techniques, we dissected the cAMP/Epac/PLCε signalling pathway and demonstrated that the activation of PLCε accelerates the production of DAG at the ER membrane, DAG ER [18]. DAG ER can be specifically recognized by the C1 domain of PKCδ and recruits PKCδ to the ER membrane (X. Hui, G. Reither, L. Kaestner and P. Lipp, unpublished work).…”

“…DAG ER can be specifically recognized by the C1 domain of PKCδ and recruits PKCδ to the ER membrane (X. Hui, G. Reither, L. Kaestner and P. Lipp, unpublished work). As long as intracellular cAMP is increased, DAG at the ER membrane accumulates and eventually recruits nPKCs to the ER [18]. The diffusible second messenger cAMP in fact serves the role of a signal transporter, delivering the signal information from the plasma membrane to inner membranes, where Epac predominantly actives Rap proteins rather than Ras [35].…”

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors

“…6 -9, compartment-dependent translocation of PKC isoforms to the plasma membrane, the nuclear membrane, or the Golgi complex determines specific intracellular responses by placing PKC isoforms in proximity to their interaction partners. By using genetically encoded FRET-based sensors of organelle-specific PKC activity, recent studies provide evidence that location-specific DAG production enables recruitment of PKC isoforms to different intracellular membranes (10,11). Spatially restricted activation of components downstream of G q has been shown to modulate several types of ion channels in a receptor species-dependent fashion, e.g.…”