Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signalling

Abstract

cAMP is a ubiquitous second messenger responsible for the cellular effects of multiple hormones and neurotransmitters via activation of its main effector, protein kinase A (PKA). Multiple studies have shown that the basal concentration of cAMP in several cell types is about 1 μM. This value is well above the reported concentration of cAMP required to half-maximally activate PKA, which measures in the 100-300 nM range. Several hypotheses have been suggested to explain this apparent discrepancy including inaccurate measurements of intracellular free cAMP, inaccurate measurement of the apparent activation constant of PKA or shielding of PKA from bulk cytosolic cAMP via localization of the enzyme to microdomains with lower basal cAMP concentration. However, direct experimental evidence in support of any of these models is limited and a firm conclusion is missing. In this study we use multiple FRET-based reporters for the detection of cAMP and PKA activity in intact cells and we establish that the sensitivity of PKA to cAMP is almost twenty times lower when measured in cell than when measured in vitro. Our findings have important implications for the understanding of compartmentalized cAMP signalling.

Figure 1

FRET sensors used in this study. Schematic representation of the different sensors illustrating the working principle. EPAC-S^H187 and C9H6 are “loss of FRET” sensors and show decreasing FRET with increasing cAMP concentrations. CUTie, AKAP-CUTie and AKAR3 are “gain of FRET” sensors, showing increased FRET with increasing cAMP concentrations. CNBD = cyclic nucleotide binding domain, cAMP is shown in green, red star indicates the PKA anchoring site. Throughout this study the change in fluorescence intensity ratio (FRET change) is calculated so that an increase in cAMP or PKA activity corresponds to an increase in the ratio value (indicated on the right).

Figure 2

In cell determination of basal intracellular cAMP concentration. cAMP-FRET concentration-dependency curves generated by microinfusion of known cAMP concentrations in CHO cells stably expressing the cytosolic cAMP FRET-reporters CUTie or EPAC-S^H187. The calculated x-crossings are 0.71 µM for EPAC-S^H187 and 1.14 µM for CUTie. Inset shows the x-crossings in more detail. Best fit values were: −13.73% (bottom), 100% (top), 1.071 (Hill coefficient), 7.385 (EC₅₀) for CUTie, and −1.650% (bottom), 100% (top), 1.750 (Hill-coefficient), 7.406 (EC₅₀) for EPAC-S^H187. Light grey shaded areas represent 95% confidence intervals and dark grey areas show the overlap. For curves including individual concentration data points see Supplementary Figure S3a,b).

Figure 3

Comparison of the cAMP levels detected by cytosolic and targeted CUTie sensors. Images of (a) CHO-cells stably expressing cytosolic CUTie and (b) membrane targeted AKAP79-CUTie showing the sensor localization. Scale bars are 10 µm. (c) cAMP-FRET concentration-dependency curves generated by microinfusion of known cAMP concentrations in CHO cells stably expressing CUTie or AKAP79-CUTie. Inset shows a magnification of the x-crossings of the curves. Calculated x-crossing for the targeted AKAP 79-CUTie was 1.01 µM. Best fit values for AKAP 79-CUTie were: −9.080% (bottom), 100% (top), 1.230 (Hill-coefficient), 7.179 µM (EC₅₀). For CUTie values refer to Fig. 2. Light grey shaded areas represent the 95% confidence intervals, dark grey areas show the overlap. For curves including individual concentration data points see Supplementary Figure S3b,c.

Figure 4

In cell determination of the apparent activation constant of overexpressed PKA. (a) Representative kinetics of FRET change recorded in CHO cells stably expressing the PKA-based sensor C9H6 and microinfused with different cAMP concentrations (as indicated). The arrow shows establishment of the whole-cell configuration and start of microinfusion. (b) Concentration-dependency curve for C9H6. N ≥ 3 independent experiments for each concentration. Inset shows a magnification of the x-crossing of the curve. The calculated x-crossing is at 1.26 µM. Best fit values were: −0.657% (bottom), 14.47% (top), 2.191 (Hill-coefficient), 5.231 µM (EC₅₀). Gray shaded areas represent the 95% confidence interval for the curve.

Figure 5

In cell determination of endogenous PKA activity. (a) upper panel: trace showing the avarage FRET change recorded in CHO cells stably expressing AKAR3 and treated with forskolin (1 µM) prior to the addition of H89 (10 µM). Lower panel: Effect of H89 (10 µM) on the FRET signal recorded at basal conditions. Subsequent application of 10 µM forskolin shows no FRET change confirming complete blockage of PKA activity. N = 31 cells for both traces. (b) Average kinetics of FRET change detected in CHO cells expressing AKAR3 and treated with Calyculin (10 nM,) or Cyclosporin A (200 nM), as indicated. Forskolin (10 µM) was applied to confirm that the AKAR sensor is responsive to PKA phosphorylation. Arrows indicate time of application. N ≥ 27 cells for both traces. Traces in a and b are representative for at least 3 independent experiments. (c) Representative kinetics of FRET changes recorded in CHO cells stably expressing the PKA-activity reporter AKAR3. The arrow indicates establishment of the whole-cell configuration and start of microinfusion. cAMP concentrations in the pipette are indicated close to the respective curve. (d) Concentration-dependency curve for the AKAR3-sensor. Inset shows a magnification of the x-crossing of the curve. The calculated x-crossing is at 0.57 µM. Best fit values were: −0.4348% (bottom), 20.74% (top), 2.971 (Hill-coefficient), 2.086 µM (EC₅₀). Grey shaded areas represent the 95% confidence interval for the curve. N ≥ 3 independent experiments for each concentration.

Figure 6

PKA activity in CHO cells lysates. (a) Western blotting of lysates from CHO cells expressing a GFP-tagged version of the PKA target protein troponin I and probed with a phospho-troponin I specific antibody (p-TPNI) and a GFP specific antibody for total troponin I. (b) Western blotting of endogenous protein in lysates from CHO cells probed with phospho-CREB antibody (p-CREB) and total CREB antibodies. Blocker mix consists of phosphatase and PDE inhibitors. Blots are representative of at least 3 independent experiments. Shown are the relevant bands. Full-size blots are presented in Supplementary Figure S4.

Figure 7

Effect of different concentrations of bath-applied cAMP on the FRET signal generated by C9H6 PKA in cell lysates. Representative trace showing the time course of FRET change detected in a cell lysates from CHO cells stably expressing the sensor C9H6 PKA and challenged with different concentrations of cAMP as indicated. Insert: mean FRET change (± SEM) of 3 independent experiments at the indicated concentrations.

Figure 8

Comparison of the concentration-dependent activities of PKA (right ordinate) and the absolute activities of selected PDEs (left ordinate). “High” and “low” denote high and low affinity states of the respective PDE. Note that the activity of all PDE4 isoforms, except PDE4D in its high affinity state, are too low to be distinguishable at this scaling factor. All PDE curves are calculated according to the corresponding K_M values reported in the literature (see Table 1) and assuming, as a first approximation, that K_d ≈ K_M ≅ EC₅₀. The respective absolute activity was calculated from V_max and used to set the “top” of the curves (see Table 1). “Bottom” was set to zero. As cAMP-degradation by PDEs is a simple dual molecule reaction without cooperativity a maximal Hill-coefficient of 1 was assumed. In vitro PKA curves (green) are recalculated according to, in cell PKA activity (blue) is calculated according to values found in this study. Also shown is the production rate of adenylyl cyclase. All activities are calculated as number of cAMP-molecules degraded/generated in one second by one PDE/AC molecule.