MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity

Background. Perennial ryegrass is a cool-season grass species from the family Poaceae and is widely cultivated in temperate regions because it exhibits rapid growth and establishment, and possesses high forage quality. The extension of the growing season in Ireland in spring and autumn is a breeding target to make farming more profitable since a rass-fed diet based on grazing is the cheapest way of nutrition for ruminants. Methods. Fifty-seven perennial ryegrass accessions were screened for their ability to grow under typical Irish spring conditions as taken from long term emperature records in controlled climate chambers. They were grown in low temperature (8°C/2°C day/night) and control conditions (15°C/8°C day/night) in three consecutive independent experiments. Fresh weight, height, clorophyll content and electrolyte leakage were measured, and these parameters were used to rank plant performance under low temperature growth conditions. Results. The results showed that height, yield and electrolyte leakage are xcellent measures for the impact of cold stress tolerance. Little variation in growth was seen undercold stress, but a wide variety of responses were observed under control conditions. Discussion. Our results suggest that cold stress is under tight physiological control. Interestingly, the various genotypes responded differentially to more amenable control conditions, indicating that a quick response to more amenable growth conditions is a better target for breeding programmes.

BackgroundPerennial ryegrass is a cool-season grass species from the family Poaceae and is widely cultivated in temperate regions because it exhibits rapid growth and establishment, and possesses high forage quality. The extension of the growing season in Ireland in spring and autumn is a breeding target to make farming more profitable since a grass-fed diet based on grazing is the cheapest way of nutrition for ruminants.MethodsFifty-seven perennial ryegrass accessions were screened for their ability to grow under typical Irish spring conditions as taken from long term temperature records in controlled climate chambers. They were grown in low temperature (8 °C/2 °C day/night) and control conditions (15 °C/8 °C day/night) in three consecutive independent experiments. Fresh weight, height, chlorophyll content and electrolyte leakage were measured, and these parameters were used to rank plant performance under low temperature growth conditions.ResultsThe results showed that height, yield and electrolyte leakage are excellent measures for the impact of cold stress tolerance. Little variation in growth was seen under cold stress, but a wide variety of responses were observed under control conditions.DiscussionOur results suggest that cold stress is under tight physiological control. Interestingly, the various genotypes responded differentially to more amenable control conditions, indicating that a quick response to more amenable growth conditions is a better target for breeding programmes.

Mitochondrial Ca2+ ([Ca2+]M) uptake through its Ca2+ uniporter (MCU) is central to many cell functions such as bioenergetics, spatiotemporal organization of Ca2+ signals, and apoptosis. MCU activity is regulated by several intrinsic proteins including MICU1, MICU2, and EMRE. While significant details about the role of MICU1, MICU2, and EMRE in MCU function have emerged recently, a key challenge for the future experiments is to investigate how these regulatory proteins modulate mitochondrial Ca2+ influx through MCU in intact cells under pathophysiological conditions. This is further complicated by the fact that several variables affecting MCU function change dynamically as cell functions. To overcome this void, we develop a data-driven model that closely replicates the behavior of MCU under a wide range of cytosolic Ca2+ ([Ca2+]C), [Ca2+]M, and mitochondrial membrane potential values in WT, MICU1 knockout (KO), and MICU2 KO cells at the single mitochondrion and whole-cell levels. The model is extended to investigate how MICU1 or MICU2 KO affect mitochondrial function. Moreover, we show how Ca2+ buffering proteins, the separation between mitochondrion and Ca2+-releasing stores, and the duration of opening of Ca2+-releasing channels affect mitochondrial function under different conditions. Finally, we demonstrate an easy extension of the model to single channel function of MCU.

TRPM7/ChaK1 is a unique channel/kinase that contains a TRPM channel domain with 6 transmembrane segments fused to a novel serine-threonine kinase domain at its C terminus. The goal of this study was to investigate a possible role of kinase activity and autophosphorylation in regulation of channel activity of TRPM7/ChaK1. Residues essential for kinase activity were identified by site-directed mutagenesis. Two major sites of autophosphorylation were identified in vitro by mass spectrometry at Ser(1511) and Ser(1567), and these sites were found to be phosphorylated in intact cells. TRPM7/ChaK1 is a cation-selective channel that exhibits strong outward rectification and inhibition by millimolar levels of internal [Mg(2+)]. Mutation of the two autophosphorylation sites or of a key catalytic site that abolished kinase activity did not alter channel activity measured by whole-cell recording or Ca(2+) influx. Inhibition by internal Mg(2+) was also unaffected in the autophosphorylation site or "kinase-dead" mutants. Moreover, kinase activity was enhanced by Mg(2+), was decreased by Zn(2+), and was unaffected by Ca(2+). In contrast, channel activity was inhibited by all three of these divalent cations. However, deletion of much of C-terminal kinase domain resulted in expression of an apparently inactive channel. We conclude that neither current activity nor regulation by internal Mg(2+) is affected by kinase activity or autophosphorylation but that the kinase domain may play a structural role in channel assembly or subcellular localization.

The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.

Vertebrate oocytes are arrested in G(2) phase of the cell cycle at the prophase border of meiosis I. Progesterone treatment of Xenopus oocytes releases the G(2) block and promotes entry into the M phases of meiosis I and II. Substantial evidence indicates that the release of the G(2) arrest requires a decrease in cAMP and reduced activity of the cAMP-dependent protein kinase (PKAc). It has been reported and we confirm here that microinjection of either wild type or kinase-dead K72R PKAc inhibits progesterone-dependent release of the G(2) arrest with equal potency and that inhibition can be reversed by a second injection of the heat-stable inhibitor of PKAc, PKI. However, a mutant enzyme predicted to be completely kinase-dead from the crystal structure of PKAc, K72H PKAc, was much less inhibitory when carrying additional mutations that block interaction with either type I or type II regulatory subunit. Moreover, inhibition by K72H PKAc was reversed by PKI at a 30-fold lower concentration and with more rapid kinetics compared with wild type PKAc. K72R PKAc was found to have low but detectable activity after incubation in an oocyte extract. These results indicate that inhibition of the progesterone-dependent G(2)/M transition in oocytes after microinjection of dead PKAc reflects either low residual activity or binding to regulatory subunits with a resulting net increase in the level of endogenous wild type PKAc. Consistent with this hypothesis, the induction of mitosis in Xenopus egg extracts by the addition of cyclin B was blocked by wild type PKAc but not by K72H PKAc. The identification of substrates for PKAc that maintain cell cycle arrest in G(2) remains an important goal for future work.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Data availability References Decision letter Author response Article and author information Abstract Kinase activity in signaling networks frequently depends on regulatory subunits that can both inhibit activity by interacting with the catalytic subunits and target the kinase to distinct molecular partners and subcellular compartments. Here, using a new synthetic molecular interaction system, we show that translocation of a regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane has a paradoxical effect on the membrane kinase activity. It can both enhance it at lower translocation levels, even in the absence of signaling inputs, and inhibit it at higher translocation levels, suggesting its role as a linker that can both couple and decouple signaling processes in a concentration-dependent manner. We further demonstrate that superposition of gradients of PKA-R abundance across single cells can control the directionality of cell migration, reversing it at high enough input levels. Thus, complex in vivo patterns of PKA-R localization can drive complex phenotypes, including cell migration. Editor's evaluation This is a very thorough and important study demonstrating quantitative control of signaling through changes in the abundance and localization of a regulatory kinase subunit. The authors use live imaging experiments in microfluidic devices to reveal nonmonotonic dependence of PKA activity on the level of its regulatory subunit and provide evidence that it translates into corresponding changes of cell polarization and cell migration. Moreover, they provide a mathematical model that explains the underlying mechanism. https://doi.org/10.7554/eLife.66869.sa0 Decision letter eLife's review process Introduction In intracellular signal transduction, the information is encoded in molecular interactions involving recognition of diverse substrates by specific enzymes. These interactions are facilitated by large sets of adapter and scaffold proteins linking the activated enzymes to substrates within specific subcellular compartments, controlling dynamic modifications of protein localization and local concentrations of effector molecules (Langeberg and Scott, 2015). Furthermore, the enzymes, such as the diverse and abundant kinases involved in cell signaling and other functions, frequently contain covalently linked regulatory and catalytic subunits, with the regulatory subunits controlling both the activity of the enzyme and its interactions with other enzymes and substrates. However, the enzymes belonging to the family of protein kinase A (PKA) serine-threonine kinases do not follow this covalent linkage rule. Instead, a PKA molecule is a complex of two catalytic subunits (PKA-C) and a regulatory subunit dimer (PKA-R) that can dissociate following binding of two cyclic AMP (cAMP) molecules to each of the PKA-R subunits (Taylor et al., 2013). This intricate organization of the kinase complex is further complicated by dynamically shifting subcellular pools of cAMP, tethering of the kinase and its substrates to a large family of differentially localized A-kinase anchoring proteins (AKAPs), localized phosphatase and phosphodiesterase activity, and the intrinsic inhibitory function of PKA-R (Baillie, 2009; Wong and Scott, 2004; Zhang et al., 2012). What emerges is a picture of structural and functional complexity of PKA signaling that is still incompletely understood in spite of decades of research and analysis. Given the complexity of intermolecular interactions, the stoichiometry of multi-molecular complexes can have profound effects on the outcome of signaling processes. A particularly striking effect is observed if a linker protein, such as a scaffold molecule, can vary in its abundance. It has been shown, for example, for the MAPK signaling pathways, that a scaffold protein can enhance the pathway activity at an optimal level but can also inhibit it if its concentration exceeds the optimum (Chapman and Asthagiri, 2009; Levchenko et al., 2000). This ‘combinatorial inhibition’ effect (Good et al., 2011) suggests that variation of the relative abundance of the pathway components can modulate the pathway activity even if the input levels do not change. Given the structural complexity of PKA signaling, it is not clear whether and how the relative abundance of various signaling pathway components in different subcellular compartments may modulate the signaling outcomes. More specifically, it is not clear if PKA-R subunits would serve purely as inhibitors of PKA signaling (as expected due to their intrinsic inhibitory role, relieved only in the presence of high cAMP concentrations) or would potentially elevate the kinase activity by enhancing localization of the PKA holoenzyme to subcellular areas with increased signaling inputs. Compartmentalized PKA signaling is important in a number of contexts including glucose homeostasis, cardiomyocyte contractility, cell cycle regulation, and cell migration (Howe, 2004; Langeberg and Scott, 2005; Mauban et al., 2009; Wong and Scott, 2004). In regulating cell migration, PKA is known to positively affect the activity of some cytoskeletal regulators (e.g., Rac1, Cdc42, α4β1 integrin) and negatively affect others (e.g., RhoA). Additionally, both inhibition and activation of PKA have been shown to have inhibitory effects on cell migration (Howe, 2004). As a result, PKA’s role in regulation of cell migration is still unclear. Several studies utilizing FRET biosensors have identified gradients of PKA activity in migrating cells, with relatively high PKA activity at the cell front and relatively low activity at the rear, suggesting that spatial control of the kinase is involved in this process (Lim et al., 2008; Paulucci-Holthauzen et al., 2009; Tkachenko et al., 2011). Furthermore, the regulatory but not the catalytic subunit has been shown to be enriched in the pseudopods of cells in culture, suggesting that regulatory subunit localization may play a role in the regulation of cell migration (Howe et al., 2005). Since PKA-R mediates anchoring of PKA to diverse intracellular locations, in large part due to its interactions with AKAPs, it is particularly important to explore whether and how its abundance at specific subcellular locations modulates the output of signaling activity both under the basal conditions and in response to specific stimulation. This analysis can benefit from a tool that can permit acute localization of PKA-R to a predefined cell location in the absence of direct pathway stimulation. We developed such a tool based on chemically inducible dimerization with the small, cell-permeable molecule rapamycin (Banaszynski et al., 2005). Rapamycin induces dimerization of two small, intracellularly transduced molecular components: FK506-binding protein (FKBP) and the FKPB-rapamycin domain (FRB), which can be tethered to proteins of interest as well as specific subcellular compartments. This technique has been used to study biochemical activity of different proteins (Chu et al., 2014; Dagliyan et al., 2017; Dagliyan et al., 2013; Inoue et al., 2005; Karginov et al., 2010). Previously, we demonstrated that this dimerization strategy, when combined with the use of a microfluidic device controlling spatial rapamycin distribution, can be used to study biomolecular systems controlling cell polarity and migration (Lin et al., 2012). For the current study, we have tethered FKBP to fluorescently labeled PKA-R, while anchoring FRB to the plasma membrane (PM), to enable dynamically controlled localization of PKA-R to the PM in a rapamycin-dependent fashion. Using this tool, we find that PKA-R can have unexpectedly complex regulatory effects on the activity and function of PKA at the PM, elucidating the potential role of PM PKA-R localization in normal and pathological cell function. Results Design and characterization of an inducible PKA-R translocation system Our aim was to develop a synthetic tool to control PKA-R abundance in a specific subcellular location, with the ability to assess the real-time local subunit abundance. We found that of the four PKA-R isoforms (PKAR-Iα/β, PKAR-IIα, and PKAR-IIβ) in a standard HeLa cell line, the expression of PKAR-IIβ was particularly low (Figure 1—figure supplement 1A and D, rightmost lane), prompting us to use this isoform as the basis for development of the chemical dimerization-based tool for acutely controlling local PKA-R abundance. Specifically, to induce rapid and spatially controlled translocation of PKA-R to the PM, we utilized a rapamycin-based dimerization strategy combined with in-dish or in-chip control of rapamycin concentration similar to the strategy we previously used to control Rac1 function (Lin et al., 2012). We linked one binding partner of rapamycin, FKBP, to a fluorescently tagged PKA-RIIβ (PKAR-FKBP-FP) and the other, FRB, to the membrane-targeting sequence of Lyn kinase (Lyn11-FRB, also referred to as Lyn-FRB throughout the text) (Figure 1A). Two color variants of the PKA-R construct were created to facilitate co-imaging with fluorescent biosensors and dyes (Figure 1B and Figure 1—figure supplement 2). In addition to our transient expression vectors, lentiviral Gateway expression vectors for PKAR-FKBP-FP and Lyn-FRB were created and integrated into the genome of HeLa cells for ease of experimentation. We will refer to these cell lines as HeLa PFM (mCherry variant) and HeLa PFY (YFP variant), respectively. Although, expectedly, transfection of the modified PKAR-IIβ changed the expression level of this subunit in both transiently transfected (to a lower degree) cells and stably transfected clones (to a greater degree), the expression of PKA-C and most other isoforms was not affected (Figure 1—figure supplement 1), the localization of the modified PKAR-IIβ was confined to the cytosol prior to stimulation (Figure 1C) and there was no detectable phenotypic effect of this induced expression. Figure 1 with 2 supplements see all Download asset Open asset Design of regulatory subunit of the protein kinase A (PKA-R) translocation system. (A) Schematic of PKA-R translocation system. Rapamycin induces heterodimerization of FKBP and FRB, resulting in translocation of PKA-R to the plasma membrane (FKBP = FK506-binding protein, FRB = FKBP-rapamycin-binding domain, R = rapamycin, PKA-R = PKA regulatory subunit, C = PKA catalytic subunit). (B) DNA construct design. Two versions of recombinant PKA-R were created with different fluorescent labels. (C) Subcellular localization of PKAR-FKBP-YFP (green) within a transiently transfected HeLa cell at 0 and 10 min after addition of 100 nM rapamycin. Scale bar, 10 µm. mCherry protein (red) co-expressed for visualization. (D) PKA-R translocation in HeLa PFM cells quantified as cytoplasmic intensity drop in mCherry channel following addition of DMSO or 100 nM rapamycin. p = 0.0039 at t = 24 min post-rapamycin addition; two-tailed Student’s t-test. (E) Catalytic subunit of the protein kinase A (PKA-C) translocation in HeLa cells transiently transfected with PKAR-FKBP-YFP, Lyn-FRB, and mCherry-PKA-C, quantified as a cytoplasmic intensity drop in mCherry channel following addition of 100 nM rapamycin. Cells transfected with mCherry protein instead of mCherry-PKA-C were used as a control. p = 0.037 at t = 24 min post-rapamycin addition; two-tailed Student’s t-test. Graphs display the mean of each data set with standard error of the mean (SEM) indicated by shaded region. Number of cells in each data set is as indicated in the figure. Data is the result of one (D) and three (E) independent experiments, respectively. Mean and SEM values for each condition and time point are provided in Figure 1—source data 1. Arrows indicate the timing of drug addition. Figure 1—source data 1 Characterization of regulatory subunit of the protein kinase A (PKA-R) translocation system. (a) Sheet 1, Figure 1D Time Course. Change in mCherry cytoplasmic intensity following addition of 0.1% DMSO or 100 nM rapamycin. Mean, standard error of the mean (SEM), and number of cells given for each time point and condition. (b) Sheet 2, Figure 1E Time Course. Change in mCherry cytoplasmic intensity following addition of 100 nM rapamycin. Mean, SEM, and number of cells given for each time point and condition. https://cdn.elifesciences.org/articles/66869/elife-66869-fig1-data1-v1.xlsx Download elife-66869-fig1-data1-v1.xlsx To assay translocation of PKA-R to the membrane, HeLa cells transiently expressing the mCherry-tagged translocation system were treated with 100 nM rapamycin and imaged for 30 min. Addition of rapamycin resulted in rapid PKA-R translocation to the PM evaluated as a significant decrease in the cytoplasmic fluorescence intensity over the first 3 min of stimulation (Figure 1C and D). No significant translocation occurred after this initial period. To determine whether PKA-C translocated to the membrane along with the exogenous PKA-R, we co-transfected HeLa cells with PKAR-FKBP-YFP, Lyn-FRB, and the constructs encoding either recombinant mCherry protein or mCherry-tagged PKA-C. Following addition of rapamycin, PKA-C indeed translocated to the cell membrane whereas mCherry alone underwent no significant change in localization, demonstrating that our synthetic system can bring the intact PKA holoenzyme to the PM (Figure 1E and Videos 1 and 2). Furthermore, in contrast to absence of any detectable phenotypic effect of PKAR-FKBP-FP expression in unstimulated HeLa cells, these cells treated with rapamycin displayed a rapid change in cell morphology (spreading) and an increase in filopodia formation (not observed in untransfected cells treated with the same rapamycin dose) (Figure 1C and Video 1), pointing to a pronounced effect of a change in the local PKA-R levels at the PM. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Rapamycin-induced translocation of regulatory subunit of the protein kinase A (PKA-R). Translocation of PKA-R (green) to the plasma membrane of HeLa cells following treatment with 100 nM rapamycin at time zero as indicated in the video. Cells were co-transfected with mCherry protein (red) as a counterstain for visualization. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Co-recruitment of catalytic subunit of the protein kinase A (PKA-C) and regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane. Co-localization of PKA-C (red) and PKA-R (green) before and after treatment with 100 nM rapamycin at time zero as indicated in the video. Characterization of cell response to PKA-R translocation The dissociation of PKA-C from PKA-R is commonly seen as a relief of PKA-C inhibition within the holoenzyme, with the regulatory subunit thus treated as a negative regulator of the kinase activity. To test whether membrane translocation of PKA-R would indeed inhibit the basal PKA activity in this compartment, we transfected HeLa PFM cells with Lyn-AKAR4, a membrane-bound FRET probe for PKA activity (Depry et al., 2011). The dynamic range of the intracellular Lyn-AKAR4 responses in this cell line was determined to be 20.9% ± 2.0% (n = 12) (mean ± standard error of the mean [SEM] [n = number of cells]) following cell treatment with a combination of an adenylyl cyclase activator forskolin (Fsk, 50 μM) and a competitive non-selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM) to maximally increase the intracellular cAMP levels (Figure 2—figure supplement 1). Surprisingly, we found that rapamycin-induced PKA-R translation alone (without any additional stimulation) was able to induce a significant increase in PKA activity, which was transient in some cells and sustained in others (Figure 2A and B). To determine whether the variability in the concentration of PKA-R could account for this cell-cell variation of response, we examined the dependence of the maximum levels of PKA activity on the estimated PKA-R abundance, using mCherry fluorescence intensity as a proxy. Interestingly, we observed that the increase in activity was greatest for cells with intermediate PKA-R concentrations, decreasing when PKA-R was either higher or lower than this optimal level (Figure 2C). Furthermore, in the cells with the highest PKA-R expression, PKA activity, following the initial rise, later not only decreased vs. the maximum, but dropped below the basal level, indicating active inhibition of the PKA activity (Figure 2D and E). Notably, even in the cells in which the PKA activity was inhibited vs. the basal levels, this activity transiently increased, suggesting that the PKA-R translocation was gradual, and thus first reached activating levels at the PM, but ultimately exceeded these levels and reached inhibitory concentrations. Figure 2 with 1 supplement see all Download asset Open asset Characterization of protein kinase A (PKA) activity response to regulatory subunit of the PKA (PKA-R) translocation. (A) Effect of rapamycin-induced PKA-R translocation on PKA activity at the plasma membrane (PM) as detected by Lyn-AKAR4. 100 nM rapamycin or 0.1% DMSO added at time = 0. p = 9.83 x 10-12 at t = 4 min post-rapamycin addition; two-tailed Student’s t-test. (B) Single cell PKA activity dynamics at the PM following addition of 100 nM rapamycin (a subset of data used for (A) is shown for clarity). (C) Relationship between PKA-R concentration, as estimated by mCherry fluorescence intensity, and maximal PM PKA activity increase following PKA-R translocation (n = 44 cells). Envelope overlaid for visualization. (D) Classification of cells into ‘low’ and ‘high’ expressors of PKA-R. (E) Average PM PKA activity over time for ‘low’ vs. ‘high’ expressors of PKA-R (defined in panel D). p = 0.024 at t = 58 min post-rapamycin addition; two-tailed Student’s t-test. (F) PM PKA activity response to two different rapamycin doses. p = 0.023 at t = 60 min post-rapamycin addition; two-tailed Student’s t-test. Graphs in (A, E, F) display the mean of each data set with standard error of the mean (SEM) indicated by shaded region. Number of cells in each data set is as indicated in the figure. Data is the result of three (A–E) and two (F) independent experiments, respectively. Mean and SEM values for each condition and time point, as well as single cell measurements for data presented in (A–E), are provided in Figure 2—source data 1. Arrows indicate the timing of drug addition. All experiments completed in HeLa PFM cells transiently expressing Lyn-AKAR4. Figure 2—source data 1 Lyn-AKAR4 data following regulatory subunit of the protein kinase A (PKA-R) translocation. (a) Sheet 1, Figure 2A Time Course. Lyn-AKAR4 response following addition of 0.1% DMSO or 100 nM rapamycin. Mean, standard error of the mean (SEM), and number of cells given for each time point and condition. (b) Sheet 2, Single cell data. Single cell time courses from Figure 2A–E. Cells are labeled as having low or high PKA-R expression. (c) Sheet 3, Figure 2E Time Course. Lyn-AKAR4 response following addition of 100 nM rapamycin for cells with low vs. high PKA-R expression. Mean, SEM, and number of cells given for each time point and condition. (d) Sheet 4, Figure 2F Time Course. Lyn-AKAR4 response following addition of 2 or 20 nM rapamycin. Mean, SEM, and number of cells given for each time point and condition. https://cdn.elifesciences.org/articles/66869/elife-66869-fig2-data1-v1.xlsx Download elife-66869-fig2-data1-v1.xlsx To further investigate whether the local concentration of PKA-R played a role in determining the magnitude and duration of the response at the PM, we treated cells with two different lower concentrations of rapamycin – 2 and 20 nM. We again found that treatment of cells with 20 nM of rapamycin led to PKA activation that was, on average, transient, consistent with the gradual accumulation of PKA-R at the PM, first to optimal and then inhibitory levels. Importantly, we found that inducing a decreased level of PKA-R translocation with a lower dose of rapamycin (2 nM) resulted in a slower but much more sustained increase in PKA activity, reaching, on average, much higher levels than those seen for the higher dose (Figure 2F), suggesting that the lower PKA-R levels achieved at this rapamycin concentration were close to optimal. These results collectively suggested that PKA-R translocation to the PM can the PKA activity to an optimal level of this subunit but can also inhibit PKA when the PKA-R levels the optimal level in the PM To account for this paradoxical effect of the local PKA-R abundance, we that similar to linker or scaffold this subunit can both enhance and inhibit signaling on its concentration relative to the concentrations of the other components of the holoenzyme (e.g., cAMP or PKA-C of this molecule can other complex components thus negative whereas the lower levels of the linker are for the complex We a new mathematical model of this along with our prior et al., suggested that there is indeed an optimal level of concentration expected for PKA-R, if it as such a linker molecule 1). Furthermore, an increase in the local PKA-R abundance was to potentially drive the signaling to below levels, if the initial concentration of PKA-R at the PM was high prior to PKA-R translocation. the combination of and mathematical suggested that rapamycin-based PKA-R translocation to the PM to a gradual accumulation of this PKA subunit in the specific subcellular compartment, it to at and levels, and thus enhance and then inhibit signaling by of the role of a linker in the holoenzyme translocation of PKA-R induces a of cell polarity We the functional effects of induced PM localization of PKA-R. PKA activity and PKA-R abundance have been shown to be at the front of migrating cells in but the role that spatial localization of PKA in regulating cell migration is not well understood (Lim et al., 2008; Paulucci-Holthauzen et al., We used our translocation system to probe the effect of intracellular PKA activity induced by gradients of PKA-R PM on cell polarization and migration. To we of the that intracellular gradients can be induced by rapamycin gradients controlled within a microfluidic device (Lin et al., and and 2015). We used a of the microfluidic of chemical gradients by which was previously developed in our (Lin et al., 2015). We increased the to enable of to cells in for 1D cell migration (Figure and Appendix 2). A of rapamycin in the was induced through between and and using a of similar molecular The cells in the to the not because of a much higher within the relative to a much lower in and HeLa PFY cells were into the and to into to directionality of cell most cells an initial polarity of migration, resulting in the migration to as labeled in Figure before rapamycin was Figure 3 with 1 supplement see all Download asset Open asset translocation of regulatory subunit of the protein kinase A (PKA-R) induces a of cell (A) Schematic of the microfluidic device used to gradients of rapamycin across HeLa PFY or PFM (B) Single cell response to 20 nM rapamycin show time in = PKAR-FKBP-YFP = = (C) Average to t = for HeLa PFY cells in 20 nM rapamycin vs. rapamycin control cells stably expressing PKAR-FKBP-YFP but not error of the mean (SEM) indicated by shaded p = at min post-rapamycin addition; two-tailed Student’s t-test. Number of cells in each data set as indicated in the figure. (D) Single cell data for rapamycin control cells in 20 nM rapamycin Data from one independent (E) Single cell data for HeLa PFY cells in 20 nM rapamycin Data from three independent Data from (D) in (F) of intracellular PKA activity in HeLa PFM cells using the transiently FRET probe Lyn-AKAR4. Rapamycin at t = 0. FRET as indicated by color Mean intracellular plasma membrane (PM) PKA activity along the cell from the high of the rapamycin in panel F) to the low in panel Data the mean of = HeLa PFM cells from two independent experiments with SEM indicated by shaded region. Cells were into 20 with the FRET for Arrows in indicate addition of rapamycin. Scale in (B) and 10 µm. Mean and SEM values for each time point and condition in (C) and each time point and in are provided in Figure data 1. Figure data 1 and Lyn-AKAR4 data in microfluidic (a) Sheet 1, Figure and Figure supplement 1 Time Course. data for cells in 20 nM rapamycin with or the membrane subunit of the regulatory subunit of the protein kinase A (PKA-R) translocation system Results also shown for cells in a DMSO as displayed in Figure supplement 1. Mean, standard error of the mean (SEM), and number of cells given for each time point and condition. (b) Sheet 2, Figure PKA Mean intracellular PM PKA activity along the cell from the high of the rapamycin to the low for three time Mean, SEM, and number of cells given for all 20 and each time Download of a rapamycin we found a pronounced of the directionality of the migration in the to the initial with the new thus to the of the rapamycin (Figure and and Video This was in contrast to cells expression of Lyn-FRB or cells to a DMSO that not PKA-R translocation and migrating in the (Figure and D, Figure supplement 1, and Video This result was and in with the that an increase in PKA-R at the cell front would enhance the cell polarization the rapamycin However, it was consistent with our prior suggesting that in cells, the translocation of PKA-R induced by a high rapamycin concentration could have an inhibitory than activating effect on This inhibitory effect was for a 20 nM spatially dose of rapamycin, as by the experiments and analysis (Figure Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Single cell response to regulatory subunit of the protein kinase A (PKA-R) Single cell response to 20 nM rapamycin at time zero as indicated in the video. = PKAR-FKBP-YFP = = Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Single cell response to DMSO Single cell response to DMSO at time zero as indicated in the video. = PKAR-FKBP-YFP = = To determine whether PKA activity was indeed affected by a

The thesis work started with the test of specific antibodies used for MsRBR1/OsRBR1 and phospho-MsRBR1/OsRBRs detection. With these antibodies, cell-cycle dependent RBR and phospho- RBR proteins variation in alfalfa and rice cultured cells were checked. Total amount of MsRBR1/OsRBR1 barely changed throughout the cell cycle, while phosphorylated forms of MsRBR1/OsRBR1 proteins showed clear cell cycle dependent changes. In my study, I found that the OsPP2A B” regulatory subunit associates with OsRBR1 but not with OsRBR2. The B pocket of OsRBR1 is essential and sufficient for the interaction between OsPP2A B”. As for B”, none of the truncated version can associate with OsRBR1; the C-terminal of B” is essential but not sufficient for the interacting between these two proteins. Three putative CDK phosphorylation sites of the OsPP2A B” regulatory subunit were verified through LC-MS/MS analysis and the set up of a series of site-directed mutagenesis. The B” regulatory subunit interact with PSTAIRE-motif containing kinases directly and phosphorylate by them, the conclusion comes from the results of co-immunoprecipitation. Elimination of phosphorylation sites in B” did not affect the binding to the PP2A catalytic subunit but did weaken the binding to OsRBR1; the effect was more significant in in vivo yeast two-hybrid system. The phosphorylation elimination of B” regulatory subunit down-regulated the activity of B” containing PP2A heterotrimeric holoenzyme. In contrast, phosphorylation mimicking of B” regulatory subunit up-regulated the activity of B” containing PP2A heterotrimeric holoenzyme. Neither eliminated nor mimicked the phosphorylation of B” regulatory subuint changed the binding strength to PP2A catalytic subunit. The phosphorylation of B” subunit just stimulated the activity of the PP2A complex. It can be postulated that the free Ca2+ ions has the role in regulate the activity of B” subunit since it contains two EF-hand domain. The postulation was verified by the experiments which indicated that the presence of Ca2+ increased the activity of the PP2A holoenzyme and conversely, the absence of Ca2+ (with the chelator, EGTA) inhibited the phosphatase activity of PP2A complex.

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca(2+) uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca(2+) uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca(2+) uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.

Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Delta psi) and chemical (Delta pH) component of the mitochondrial transmembrane proton electrochemical gradient (Delta mu(H(+))) was investigated. The overall O(2) consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Delta psi and Delta pH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C(IV) = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C(IV) = 0.85) was also measured in the presence of nigericin, i.e. when Delta psi is maximal, and in the presence of nigericin and valinomycin (C(IV) = 0.77), when Delta mu(H(+)) is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C(IV) = 0.30) upon addition of valinomycin, when Delta psi is converted into Delta pH. These results show that Delta psi is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.

Protein S-palmitoylation, the reversible thioester linkage of a 16-carbon palmitate lipid to an intracellular cysteine residue, is rapidly emerging as a fundamental, dynamic, and widespread post-translational mechanism to control the properties and function of ligand- and voltage-gated ion channels. Palmitoylation controls multiple stages in the ion channel life cycle, from maturation to trafficking and regulation. An emerging concept is that palmitoylation is an important determinant of channel regulation by other signaling pathways. The elucidation of enzymes controlling palmitoylation and developments in proteomics tools now promise to revolutionize our understanding of this fundamental post-translational mechanism in regulating ion channel physiology.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIalpha and Calpha subunits of PKA type I, but not the RIIalpha and Calpha subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.

Modulation of ion channels: a "current" view of AKAPs.

Perhydroderivatives of polyene antibiotics have a much lower activity against eukaryotic cells than the polyene antibiotics itself. Bacterial cells are normally resistant against most polyene antibiotics and their perhydroderivatives. In earlier experiments with wall less L-form cells of Escherichia coli we have shown that the bacterial cell wall may be responsible for the resistance of the intact bacterial cells against polyene antibiotics and their perhydroderivatives by masking internal target sites. In the present paper we studied the effect of polyene antibiotics and their perhydroderivatives on intact cells and protoplasts of Candida guilliermondii. Our experiments have shown that most of the perhydroderivatives studied had a lower activity against intact cells as well as protoplasts than the corresponding polyene antibiotics. This means that in the case of eukaryotic cells the cell wall as a penetration barrier cannot mainly be responsible for the low activity of perhydroderivatives. The results are compared with those obtained previously with intact cells and protoplast type L-form cells of E. coli.