Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals.

The specificity of many signal transduction pathways relies on the temporal coordination of different second messenger signals. Here we found a molecular mechanism which guarantees that conventional protein kinase C (PKC) isoforms are sequentially activated by calcium and diacylglycerol signals. Receptor stimuli that triggered repetitive calcium spikes induced a parallel repetitive translocation of GFP-tagged PKCgamma to the plasma membrane. While calcium acted rapidly, diacylglycerol binding to PKCgamma was initially prevented by a pseudosubstrate clamp, which kept the diacylglycerol-binding site inaccessible and delayed calcium- and diacylglycerol-mediated kinase activation. After termination of calcium signals, bound diacylglycerol prolonged kinase activity. The properties of this molecular decoding machine make PKCgamma responsive to persistent diacylglycerol increases combined with high- but not low-frequency calcium spikes.

Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

Reversible desensitization of inositol trisphosphate-induced calcium release provides a mechanism for repetitive calcium spikes.

Repetitive transient increases in cytosolic calcium concentration (calcium spikes or calcium oscillations) are a common mode of signal transduction in receptor-mediated cell activation. Repetitive calcium spikes are initiated by phospholipase C-mediated production of inositol 1,4,5-trisphosphate (InsP3) and are thought to be generated by a positive feedback mechanism in which calcium potentiates its own release, a negative feedback mechanism by which calcium release is terminated, and a slow recovery process that defines the time interval between calcium spikes. The molecular mechanisms that terminate each calcium spike and define the spike frequency are not yet known. Here we show, in intact rat basophilic leukemia cells, that calcium responses induced by InsP3 are diminished for a period of 30-60 s following an InsP3-induced calcium spike. The sensitivity of calcium release for InsP3 was probed by UV laser-mediated photorelease of InsP3, and calcium responses were monitored by fluorescence calcium imaging. A maximal loss in sensitivity (desensitization) was observed for InsP3 increases that resulted in a near maximal calcium spike and was expressed as an 80-100% reduction in the calcium response to an equal amount of InsP3, released 10 s after the first UV pulse. When the amount of released InsP3 in the second pulse was increased 2-3-fold, desensitization was overcome and a second calcium response of equal amplitude to the first was produced. A power dependence of 3.2 was measured between the amount of released InsP3 and the amplitude of the triggered calcium response, explaining how a small decrease in InsP3 sensitivity can lead to a nearly complete reduction in the calcium response. Desensitization was abolished by the addition of the calcium buffers BAPTA and EGTA and could be induced by microinjection of calcium, suggesting that it is a calcium-dependent process. Half-maximal desensitization was observed at a free calcium concentration of 290 nM and increased with a power of 3.7 with peak calcium concentration. These studies suggest that reversible desensitization of InsP3-induced calcium release serves as a "saw-tooth" parameter that controls the termination of each spike and the frequency of calcium spikes.

Regulation of nuclear calcium concentration.

Transient increases in nuclear calcium concentration have been shown to activate gene expression and other nuclear processes. It has been suggested that nuclear calcium signals are controlled by a mechanism that is independent of calcium signalling in the cytosol. This would be possible if calcium diffusion is slow and a separate calcium release mechanism is localized to the nuclear region. Alternatively, the nuclear envelope could act as a diffusion barrier for calcium ions released either inside or outside the nucleus. It has also been proposed that inositol 1,4,5-trisphosphate (InsP3) can be generated inside the nucleus and that there are calcium release channels in the inner membrane of the nuclear envelope. Most of the experimental evidence supporting these hypotheses is based on the calibration of nuclear and cytosolic calcium concentrations. However, recent studies suggest that the local calibration of calcium indicators may not be accurate. We propose that nuclear calcium signals can be investigated by a different approach that does not rely on accurate calibration of indicators. We have developed calcium indicators that minimize facilitated calcium diffusion and are localized to either the nucleus or the cytosol. Using the diffusion coefficient of calcium ions, and measuring the delay between cytosolic and nuclear calcium increases, we show that the nuclear envelope is not a substantial barrier for calcium ions in PC12 (phaeochromocytoma) cells. This suggests that nuclear and cytosolic calcium signals equilibrate rapidly in these cells.

Source of nuclear calcium signals.

Transient increases of Ca2+ concentration in the nucleus regulate gene expression and other nuclear processes. We investigated whether nuclear Ca2+ signals could be regulated independently of the cytoplasm or were controlled by cytoplasmic Ca2+ signals. A fluorescent Ca2+ indicator that is targeted to the nucleus was synthesized by coupling a nuclear localization peptide to Calcium Green dextran, a 70-kDa Ca2+ indicator. Stimulation of rat basophilic leukemia cells by antigen or by photolytic uncaging of inositol 1,4,5-trisphosphate induced transient increases in nuclear and cytosolic Ca2+ concentrations. Elevations in the nuclear Ca2+ concentration followed those in the nearby perinuclear cytosol within 200 ms. Heparin-dextran, an inhibitor of the inositol 1,4,5-trisphosphate receptor that is excluded from the nucleus, was synthesized to specifically block the release of Ca2+ from cytosolic stores. Addition of this inhibitor suppressed Ca2+ transients in the nucleus and the cytosol. We conclude that the Ca2+ level in the nucleus is not independently controlled. Rather, nuclear Ca2+ increases follow cytosolic Ca2+ increases with a short delay most likely due to Ca2+ diffusion from the cytosol through the nuclear pores.