AKAP79 enables calcineurin to directly suppress protein kinase A activity
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Evaluation Summary:
This manuscript will be of interest to neuroscientists as well as a broad audience of cell biologists, as it provides new insight into the myriad of cellular functions regulated by the well-studied cAMP-dependent protein kinase, PKA. Rigorous biochemical data supports a model for PKA inactivation wherein dephosphorylation of the PKA regulatory subunit within a multiprotein complex leads to rapid capture of the PKA catalytic subunit limiting signaling duration. Overall, the biochemical data and modeling support the conclusions although a few details can be addressed further and the in vivo data remains preliminary. The work nevertheless presents exciting findings that provide a tantalizing mechanism to selectively modulate PKA activity at precise subcellular locations.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Interplay between the second messengers cAMP and Ca 2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca 2+ -sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes toward LTD. This non-canonical mode of PKA regulation may underlie many other cellular processes.
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Joint Pubic Review:
Church et al. carry out a mechanistic study focused on regulation of PKA activity at a specific multiprotein complex nucleated by the scaffolding protein AKAP79. The manuscript presents a rigorous biochemical approach combined with computational modeling to address fundamental issues related to PKA signaling. This is a very important but complex system and the authors have nicely addressed it using in vitro approaches. The in vitro data provide evidence that suggests that the phosphatase calcineurin (CaN), by dephosphorylating the PKA regulatory subunit type II (RII), promotes rapid re-association of the PKA catalytic subunit (C) to RII, leading to PKA inactivation. The model proposed is that this modality of PKA inactivation takes place selectively at the multiprotein complex organized by AKAP79, where CaN, PKA and …
Joint Pubic Review:
Church et al. carry out a mechanistic study focused on regulation of PKA activity at a specific multiprotein complex nucleated by the scaffolding protein AKAP79. The manuscript presents a rigorous biochemical approach combined with computational modeling to address fundamental issues related to PKA signaling. This is a very important but complex system and the authors have nicely addressed it using in vitro approaches. The in vitro data provide evidence that suggests that the phosphatase calcineurin (CaN), by dephosphorylating the PKA regulatory subunit type II (RII), promotes rapid re-association of the PKA catalytic subunit (C) to RII, leading to PKA inactivation. The model proposed is that this modality of PKA inactivation takes place selectively at the multiprotein complex organized by AKAP79, where CaN, PKA and PKA phosphorylation targets are co-localized: the proximity of CaN to RII at the AKAP79 complex would enhances the efficiency of RII dephosphorylation by one order of magnitude, allowing fast re-association of C and RII subunits. This would reduce the proportion of free C subunits and therefore the level of local PKA substrate phosphorylation. Using purified the FRET reporter AKAR4 as a reporter for PKA activity, they further confirm that the level of phosphorylation of this PKA target at a given cAMP concentration depends on the ability of CaN to interact with AKAP79. Based on these findings the authors conclude that CaN anchored to AKAP79 dephosphorylates AKAP79 anchored RII, leading to fast recapturing on C and inhibition of PKA catalytic activity. They then create a kinetic model for this process where cAMP and calcium are working in opposing ways. Notably, the authors also provide an estimate for the concentration of RII subunits in the hippocampal CA1 neuropil layer and find that this falls within the range at which CaM efficiently dephosphorylated RII in vitro.
In the context of compartmentalized cAMP/PKA signaling, this mechanism would provide yet another regulatory feature to ensure specific control of target phosphorylation at individual subcellular locations. For example, in dendritic spines PKA regulates long-term depression (LTD) of CA3-CA1 hyppocampal synapses via phosphorylation of AMPA-type glutamate receptors, which is facilitated by simultaneous interaction of receptor and kinase with AKAP79. In this context, at a given cAMP concentration, CaN-dependent inhibition of PKA activity would selectively attenuate AMPA phosphorylation and LTD, while PKA may still be able to phosphorylate targets at other sites.
The paper presents very clear biochemical data but can be further strengthened by some additional attention to the following:
While the in vitro data convincingly demonstrate the requirement for CaN to be anchored to AKAP79 for efficient dephosphorylation of RII and confirm that phosphorylation of RII at S98 results in more active PKA, the requirement for RII to be anchored to AKAP-79 for this regulation is not investigated, leaving open the possibility that the more efficient dephosphorylation of RII in vitro may be due increased catalytic activity of CaN when the phosphatase is associated to AKAP79c97.
The authors show convincingly that the pRII subunits are better substrates when the AKAP scaffold is present. However, they need to address the relevance of having the enzyme (CN) and the substrate (pRIIb holoenzyme) scaffolded to the same complex so that diffusion is no longer a rate-limiting factor in the catalytic event. Are MM kinetics relevant for this process? This is a single molecule event that does not necessarily require that the product be released. Instead the product is returned to the active site of the cleft of the C-subunit in the holoenzyme:CN complex where in the cell it is rapidly re-phosphorylated. Also the authors could show what happens when you have a 1:1 concentration of CN and pRIIb. Following this single transfer event does not require dissociation of the holoenzyme and is likely to be more physiologically relevant.
Do the authors know if calcium vs. Mg influences this process? Calcium stabilizes the product whereas Mg stabilizes the substrate in the case of the kinase. If calcium levels are high following release of the phosphate, would this tend to keep the phosphorylated holoenzyme in a more inhibited state until calcium went down and cAMP went up?
This process will take place at membranes which may play a significant role in determining whether the A-subunit is released into solution or not.
Another important question to consider is whether it is even necessary to dissociate the holoenzyme complex at all. Is it sufficient, for example, to simply unleash the linker region of the RII subunit and thereby open up the active site cleft of the C-subunit? Since the tail of the channel is also tethered nearby, it is perfectly reasonable to catalyze this event without dissociating the complex especially given earlier data by Wang, et al showing that the holoenzyme is very stable even when the key arginines in the inhibitor site are mutated. The same motif has access either to the active site of the C-subunit or to the active site of calcineurin in a cAMP/Ca++ dependent cycle. This leaves the phosphorylated tail of the channel free to be dephosphorylated by other phosphatases that are also tethered to AKAP79 and leaves CN committed to recycling of the RII holoenzyme. In principle this does not require dissociation of the RII holoenzyme if CN is tethered nearby. This is a very fundamental question.
One point that is not addressed in the study and is important for the interpretation of the results is whether interaction of CaN with AKAP79c97 increases CaN activity per se, such that the more effective dephosphorylation of RII is not due to the physical proximity of CaN to RII on the AKAP but to a more active CaN. This could be addressed by testing the dephosphorylation rate of a phospho-substrate other than 32P-RII, in the absence and in the presence of AKAP79c97 or by repeating the experiments shown in Fig 1 in the presence of the AKAP79c97 variant where the PKA (391-400) anchoring site has been removed.
AKAR4 is a reversible reporter of PKA activity, so it is surprising that the authors find that its phosphorylation is not affected by CaN. One possibility is that AKAR4 is not a good substrate for CaN. However, multiple studies have shown that AKAP4 can effectively be dephosphorylated. The ability of CaN to dephosphorylate AKAR4 should be investigated further to demonstrate more robustly that, in the in vitro experimental conditions used, the observed reduced phosphorylation of AKAR4 is due to less active PKA rather than more active CaN. This could be done, for example, by repeating the experiments summarized in Figure 3-figure supplement 1C & D using a different phosphatase, to ascertain that the experimental conditions allow for detection of AKAR4 dephosphorylation.
One limitation of the in vitro work is that only AKAR4 is used to measure the level of PKA dependent phosphorylation. AKAR4 is not a natural substrate for either PKA or CaN and the accessibility of the phosphorylation site to these enzymes may be different than for physiological targets. In addition, AKAR4 is not anchored to AKAP79 and may not be the ideal reporter to investigate the effects of CaN-dependent regulation of PKA targets associated to AKAP79.
Stoichiometry of free RII subunits. The authors have shown convincingly that the RII subunits in particular are present in excess of the C-subunits, and this has led to some new concepts for PKA signaling. There are two questions that need to be addressed here. Perhaps in the discussion is adequate but they do need to be addressed. First is whether there are separate pools of free RII subunits and holoenzymes within single cells. This is essential for the model of PKA signaling taking place in the presence of a 10-fold excess free RII-subunits. Are the dissociated R-subunits in the same subcellular location? Second is whether the free RII subunits are bound to cAMP. The cAMP-free subunits are noticeably less stable and degraded more rapidly that the holoenzymes so are these free R-subunits bound to cAMP? If not, are they bound to something else that keeps them stable? RII subunits do not form membrane-less puncta as was recently reported in Cell by Zhang but is there some other mechanism that allows for the sequestration of large amounts of free RII subunits?
Do you need to saturate all four sites to have an active C-subunit that can phosphorylate the tail of a channel? This relates to the question above. Perhaps this would not be measured by the AKAR4 reporter but could it be sensed if AKAR4 were fused to the tail of AKAP79 so that it would be tethered close by similar to the tail of the channel.
Stoichiometry of two calcineurins vs. one RII holoenzyme or one? The authors need to address this stoichiometry question more rigorously. It is quite fundamental for their assays. Does the computational model provide any ability to ascertain stoichiometry of the productive complex?
While it is true that neither S/A or S/E will be substrates for CN, they will in fact have a different effect on the RII holoenzyme. Ser/Ala and Ser/Glu mutants are, in principle, quite different in terms of their accessibility to the active site of the C-subunit vs. the active site of CN. The Ser/Ala mutant, for example, should be locked into the active site of the C-subunit, and this would be presumably strengthened by ATP since this is a pseudosubstrate. Does the affinity for C-subunit change in an ATP-dependent manner? The Ser/Ala mutant should be a good inhibitor that cannot be regulated by phosphorylation. It could be activated by high concentrations of cAMP but not by the cAMP signaling that is being described here. The Ser/Glu mutation would favor docking into the active site of CN but would be trapped in this state as it also could not be dephosphorylated. Is this consistent with the models proposed by the authors?
The in vivo work to assess the physiological relevance on this proposed new modality of PKA regulation is very preliminary. By overexpressing S97A and S97E mutants of RII in hippocampal neurons the authors confirm that modulation of PKA sensitivity to cAMP via RII phosphorylation affects spine density. However, no experimental data directly assess the role of CaN-dependent dephosphorylation of RII at the AKAP79 complex and there is no evidence that this mechanism regulates AMPA phosphorylation or phosphorylation of other physiologically relevant targets. Thus, the caveats that are associated with the system and in particular the physiological relevance of the analyses needs to be addressed. Conclusions based on the preliminary 'in cell' data on physiological relevance should be appropriately tempered.
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Evaluation Summary:
This manuscript will be of interest to neuroscientists as well as a broad audience of cell biologists, as it provides new insight into the myriad of cellular functions regulated by the well-studied cAMP-dependent protein kinase, PKA. Rigorous biochemical data supports a model for PKA inactivation wherein dephosphorylation of the PKA regulatory subunit within a multiprotein complex leads to rapid capture of the PKA catalytic subunit limiting signaling duration. Overall, the biochemical data and modeling support the conclusions although a few details can be addressed further and the in vivo data remains preliminary. The work nevertheless presents exciting findings that provide a tantalizing mechanism to selectively modulate PKA activity at precise subcellular locations.
(This preprint has been reviewed by eLife. We …
Evaluation Summary:
This manuscript will be of interest to neuroscientists as well as a broad audience of cell biologists, as it provides new insight into the myriad of cellular functions regulated by the well-studied cAMP-dependent protein kinase, PKA. Rigorous biochemical data supports a model for PKA inactivation wherein dephosphorylation of the PKA regulatory subunit within a multiprotein complex leads to rapid capture of the PKA catalytic subunit limiting signaling duration. Overall, the biochemical data and modeling support the conclusions although a few details can be addressed further and the in vivo data remains preliminary. The work nevertheless presents exciting findings that provide a tantalizing mechanism to selectively modulate PKA activity at precise subcellular locations.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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