We consequently examined whether or not it is possible to exploit the large-affinity cAMPbinding portions of the regulatory subunits of protein kinase A (PKA) as a molecular approach for controlling intracellular elevations of cAMP

The capacity to “buffer” cAMP in the cytosol Eglumegadand in particular microdomains may enable to take care of these problems. We for that reason examined no matter if it is possible to exploit the significant-affinity cAMPbinding portions of the regulatory subunits of protein kinase A (PKA) as a molecular tactic for managing intracellular elevations of cAMP. PKA is the major effector of the cAMP signal, and consists of two catalytic subunits (PKA-C) bound noncovalently to a dimer of regulatory subunits (PKA-R). Cyclic AMP binding to PKA-R qualified prospects to dissociation of the holoenzyme into a PKA-R subunit dimer (with four cAMP molecules sure) and two active C monomers. There are two lessons of PKA regulatory subunits (RI and RII) and just about every of these exist as two subtypes, a and b. The RI subunits have the optimum affinity for cAMP and therefore give rise to PKA holoenzymes with reduce thresholds of activation as when compared to the PKA-RII holoenzymes [two,nine]. The first 100 amino acids (aa) of PKA-RI have the biologically lively domains responsible for homo-dimerization and binding to the PKA-C subunit even though the two cAMP binding domains are located in the carboxy terminus [two,ten]. In the present examine we describe a focused large-affinity cAMP buffer primarily based on the carboxy-terminal cAMP-binding fragment of the regulatory subunit RIb. Over-expression of this “cAMP sponge” was in a position to buffer agonist-induced cAMP alerts as measured at the single cell degree and also blocked the downstream activation of PKA. Ultimately we utilized this software to show that cells devoid of the buffer provide as a source of cAMP when coupled by way of hole junctions to cells harboring the cAMP sponge, and create added cAMP to compensate for the further buffering electric power supplied by the sponge build transiently expressing these chimeras. Bands of the anticipated molecular weights (<60 kD) were detected using either a PKA-RIb specific antibody (Figure 2a), or one that recognized mCherry (Figure 2b). We noted that the PKA-RIb antibody also reacted with a second set of bands (<35 kD) likely attributed to extraction-dependent proteolysis of the full-length expressed protein[14]. Confocal imaging of live NCM460 cells expressing the three different cAMP sponge constructs showed similar expression levels as measured by mCherry intensity and the expected subcellular distribution (i.e. non-targeted vs. nuclear exclusion Figure 2c).The PKA-RI cAMP binding domains are known to be stable structures that bind cAMP when separated from the rest of the protein[2,10,11]. In order to confirm that the ability to bind cAMP was retained in the chimeric sponge proteins, we performed immunoprecipitation experiments using agarose beads coated with a cAMP analog, Sp-2-AEA-cAMPS-Agarose (Biolog) (see Methods). We used lysates from NCM460 cells transfected with our sponge constructs, or as controls, untransfected cells. As shown in figure 3a the cAMP sponge construct was enriched in the precipitates (lane 6), while as expected, no binding was detected for its mutant version (lane 5) or the untransfected cells (lane 4). We also tested the cAMP-binding specificity in pull down assays where increasing doses of exogenous cAMP competed with the Sp2-AEA-cAMPS coating the agarose beads. Both NCM460 and HeLa cells were used because the latter express endogenous PKARIb, making possible the comparison of our RIb-based chimeras to the endogenous protein. Low concentrations of cAMP (0.5 mM2.5 mM) drastically reduced the binding of both the endogenous PKA-RIb, and of cAMP sponge, which was completely abolished at concentrations above 10 mM (Figure 3b and supplementary Figure S1). In contrast, addition of 1 mM or 5 mM of guanosine 39, 59-cyclic monophosphate (cGMP) did not displace our constructs (or the endogenous PKA-RIb) from the beads (supplementary Figure S2). These experiments confirmed that our construct specifically bound cAMP in vitro with roughly submicromolar affinity, and that the mutant version lacked this ability.We cloned the PKA-RIb C-terminus (AA 13380), purposely omitting the PKA catalytic inhibitory domain located at Nterminus (AA 9000). This construct binds cAMP with high affinity, but is unable to generate dimers or bind PKA-C [11]. By labeling our chimera with the red fluorescent protein, mCherry (a gift of Roger Tsien[12]) we generated a non-targeted ``cAMP sponge'' construct. The addition of targeting motifs permitted localization to nuclear, plasma membrane, and cytosolic (i.e. nonnuclear) compartments. We extensively characterized this latter cytosolic construct, bearing the N-terminal nuclear exclusion signal (NES: ALPPLERTLTL). As a control, we also generated a mutant version of this protein unable to bind cAMP called ``mutNES-cAMP sponge'' in which four point mutations were introduced, two per each of the cAMP binding sites[2] (Figure 1). We assessed the expression of our constructs by western blots from total lysates of NCM460[13] cells (a human colonic epithelial schematic overview of the strategy used for the generation of cAMP sponge constructs. We cloned the PKA-RIb C-terminus (AA 13380), purposely omitting the PKA catalytic inhibitory domain located at N-terminus (AA 9000). This construct was tagged at its C-terminus with the improved far-red fluorescent protein, mCherry (DNH2PKARIb-mCherry). In order to generate a cAMP sponge that was specifically localized to the cytoplasm, we appended the nuclear exclusion signal sequence (NES: ALPPLERTLTL) at the N-terminus, generating NESDNH2PKARIb-mCherry (NES-cAMP sponge). Finally, in order to obtain a cAMP-resistant sponge we mutated the four critical cAMP-binding amino acids in the construct NESDNH2 E202G, R211G, E226G, R335G PKARIb-mCherry, which we called mut-NES-cAMP sponge.Expression and subcellular localization of cAMP sponge constructs. Western blot analysis using: (A) PKA-RIb specific antibody, and (B) Ds-Red antibody that recognizes mCherry. (C) Confocal photomicrographs of NCM460 cells co-expressing cAMP ``sponges'' (mCherry) and a nuclear-targeted EYFP (nuc-EYFP). The chimera named cAMP sponge was present throughout the cell without a specific subcellular localization. The addition of an amino terminus nuclear exclusion signal sequence caused the constructs NES-cAMP sponge and its mutant (mut-NES-cAMP sponge) version to be confined within the cytoplasm. Figures are representative of three biological replicates, and the observed localization efficiency was always more that 85% of the cells.Cyclic AMP sponge is able to bind cAMP in vitro. (A) NCM460 cell lysates immunoprecipitated (IP) using Sp-2-AEA-cAMPS-Agarose beads (Sp-cAMPS): lanes 1: input, 4: IP, 4: untransfected, 5: mut-NES-cAMP sponge, 6: NES-cAMP sponge. (B) cAMP competitive assay, HeLa cell lysates, lanes 1: input, 44: IP, 4: untranfected, 9: mut-NES-cAMP sponge, 104: NES-cAMP sponge, lanes 8 and 14: beads only. Loading control: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).We used the FRET-based cAMP sensor ``Epac H30'' which is built around the native cAMP-binding protein Epac in order to assess the effectiveness of our buffers at the single cell level[15,16]. These experiments were conducted in NCM460 and HEK293 cell lines stably expressing the Epac H30 sensor (see Methods). These cells were transiently transfected with cAMP sponge, and cAMP responses of single, isolated sponge-transfected cells (identified by mCherry fluorescence) were directly compared to neighboring control cells in the same microscope field. As shown in Figure 4a all controls responded to prostaglandin E2 (PGE2, black line), while the sponge-expressing cells (red line) typically gave no response. Of 19 isolated sponge-expressing cells examined in 11 experiments, there were four cells that did respond weakly to PGE2, but with a .3-fold time delay as compared to the controls (supplementary Figure S3). Supra-maximal doses of forskolin (FSK a nonspecific adenylyl cyclase activator) combined with the general phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), caused the cAMP sponge to eventually become saturated, yielding a response similar to isolated control cells. Similar experiments were performed using HEK293 cells (supplementary Figure S4). In contrast, isolated HEK293 cells (Figure 4b typical of 33 control, 6 cAMP sponge cells in 5 experiments), and NCM460 cells (supplementary Figure S5 74 controls, 10 cAMP sponge cells in 6 experiments) expressing the mutant cAMP sponge showed no significant differences in the amplitude or timing of the response as compared to the controls. As a further control to confirm that the Epac H30 FRET sensor was competent to respond to cAMP in the sponge-expressing cells, we used a cell-permeable Epac-specific cAMP analog, 8CPT2Me-cAMP (8-(4-chloro-phenylthio)-29-O-methyladenosine-39,59cyclic monophosphate)[17]. This compound binds to native Epac and the Epac H30 sensor, but not to the PKA-RIb. We therefore expected that the PKA-RIb-based cAMP sponge would not recognize 8CPT-2Me-cAMP. In fact, no differences between control and sponge-expressing cells were observed when the cells were treated with the Epac-specific analog, whereas the response to an elevation in native cAMP was clearly affected (Figure 4c 41 controls, 10 cAMP sponge cells in 5 experiments).We next examined whether cAMP sponge, by damping free [cAMP], would also attenuate the activation of PKA during agonist stimulation. For this purpose, we used two genetically encoded sensors, AKAR2 and AKAR3 (gifts of Roger Tsien and cAMP sponge abolishes agonist-induced cAMP signals and downstream activation of PKA. (A) Experiments in NCM460 cells stably expressing cAMP sensor EpacH30. Cells transiently expressing NES-cAMP sponge (identified by mCherry red trace) showed significant attenuation of PGE2-induced cAMP signals as compared to control cells in same field (black trace mean 6 SEM of 6 cells), typical of 78 controls, 19 cAMP sponge cells in 11 experiments. (B) HEK293 cells expressing mut-NES-cAMP sponge (red line) showed no significant differences as compared to control cells (black trace mean of 4 cells). Inset: time to peak of PGE2 response paired data of 33 controls, 6 mut-NES-cAMP sponge 5 experiments, n.s. (C) NCM460 cells treated with the cell permeable EPAC-specific cAMP analog (8-(4-chloro-phenylthio)-29-O-methyladenosine-39,59-cyclic monophosphate. (D) NCM460 cells expressing AKAR3 plus NES-cAMP sponge (red trace) showed no PKA activation due to PGE2 challenge, in contrast to controls expressing AKAR3 alone in the same field (black and gray traces)that report phosphorylation by PKA via a change in FRET. These sensors do not bind cAMP directly. As shown in Figure 4d, NCM460 cells transfected with AKAR3 alone responded normally to PGE2 stimulation, while neighboring cells co-expressing AKAR3 and the cAMP sponge showed no significant FRET response, indicating a lack of PKA activity. As expected FSK plus IBMX eventually saturated the buffer, restoring the PKA activity in the sponge-expressing cells (typical of 10 controls, 10 sponge cells in 6 experiments). These data provide independent confirmation that cAMP sponge can effectively dampen cAMP signaling, measured as a loss of activation of the major downstream target of the cAMP signal, PKA.The coordinated physiological activity of many tissues relies on cell-to-cell transfer of metabolites, electrical signals, and second messengers (including cAMP) via gap junctions[20,21]. Imaging studies using FRET-based sensors have shown that cAMP levels in individual cells follow those of the surrounding cells due to diffusion through these junctions[20,21]. We questioned how the presence of the cAMP buffer would affect the agonist-stimulated cAMP signal when buffer-expressing cells were physically connected to non-transfected controls. To this aim we sought out couplets of NCM460 cells in the microscope field in which one of the cells expressed the cAMP sponge construct and the other did not (control cell). Time-lapse images of the Epac H30 FRET ratio during agonist stimulation suggested that the control cells were acting as a source of cAMP, while the connected buffer-expressing cells served as a sink for the second messenger (supplementary Movie S1). As shown in Figure 5a, under these conditions there was a significant delay in the PGE2 response, averaging ,50 seconds in the cAMP sponge cells, compared to untransfected controls (49 controls, 11 mCherry cells, 9 experiments p,0.0005). Pretreatment with the reversible gap junction blocker 18aglycyrrhetinic acid[22] inhibited the cell-to-cell transfer of cAMP, and this was translated into a doubling of the delay in the agonist response observed in cAMP sponge-expressing cells. When 18aglycyrrhetinic acid was rinsed away, the cAMP transfer from controls to buffer expressing cells was rescued, with a significantly shorter delay in the response (Figure 5b 46 controls, 10 cAMP sponge cells in 6 experiments). It is noteworthy that the amplitude and time course of the cAMP response in the control cell was the same independent of whether cAMP was permitted to diffuse into the buffer-expressing cell via the 18a-glycyrrhetinic acid-dependent pathway.9422796 This would suggest that second messenger produced in one cell is able to compensate for a lagging cell, otherwise the cAMP response in the control cell would have been larger in the presence of the gap junction inhibitor.Transfer of cAMP from control cells to connected buffer expressing cells through gap junctions. (a) NCM460 cells expressing NES-cAMP sponge (red line) connected to control cells (black line mean of 4 cells), showed a small delay (time to peak) in the PGE2 response (inset: mean 6 s.e.m. of 49 controls, 11 NES-cAMP sponge in 9 experiments). (b) Pre-incubation with the gap-junction inhibitor 18a-glycyrrhetinic acid (18aGRA) significantly increased the time to peak of the buffer expressing cells. Inset: summary of 46 controls, 10 cAMP sponge cells in 6 experiments ( p,0.05 p,0.001 p,0.0001). See also supplementary Movie S1.The introduction of cell-permeant calcium chelators such as BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid, acetoxymethyl ester) by Roger Tsien in the early 1980’s [23,24] revolutionized the study of Ca2+ signaling. This high affinity Ca2+ chelator can be loaded non-invasively into living cells, and used to rapidly clamp [Ca2+] in the cytosol to resting levels during agonist activation. This invaluable tool permitted investigators to dissect out the relative importance of the Ca2+ spike in complex systems involving concurrent activation of multiple signaling pathways. Low-affinity Ca2+ buffers such as N,N,N’,N’tetrakis (2-pyridylmethyl)ethylene diamine or TPEN, used previously to clamp [Ca2+] within endoplasmic reticulum Ca2+ stores[25], have also proven useful for reversibly manipulating free [Ca2+] within subcellular compartments. Uchiyama and colleagues extended this concept by generating the first genetically encoded buffer for inositol 1,4,5-trisphosphate (IP3).