CaMKII autophosphorylation can occur between holoenzymes without subunit exchange

Article activity feed

1. 

   Version published to 10.7554/elife.86090 on eLife

   Aug 11, 2023
2. 

   eLife

   Aug 10, 2023

   Author response

   Reviewer #1 (Public Review):

   The potential role of the CaMKII holoenzyme in synaptic information processing, storage, and spread has fascinated neuroscientists ever since it has been described that self-phosphorylation of CaMKII at T286 (pT286) can maintain the kinase in an activated state beyond the initial Ca2+ stimulus that induced kinase activation and pT286. The current study by Lučić et al utilizes biochemical and biophysical methods to re-examine two pT286 mechanisms and finds:

   (1) that a previously proposed activation-induced subunit exchange within the holoenzyme can not provide pT286 maintenance or propagation; and

   (2) that pT286 can occur not only within a holoenzyme but also between two holoenzymes, at least at sufficiently high concentrations.

   For the observation regarding the subunit …

   Author response

   Reviewer #1 (Public Review):

   The potential role of the CaMKII holoenzyme in synaptic information processing, storage, and spread has fascinated neuroscientists ever since it has been described that self-phosphorylation of CaMKII at T286 (pT286) can maintain the kinase in an activated state beyond the initial Ca2+ stimulus that induced kinase activation and pT286. The current study by Lučić et al utilizes biochemical and biophysical methods to re-examine two pT286 mechanisms and finds:

   (1) that a previously proposed activation-induced subunit exchange within the holoenzyme can not provide pT286 maintenance or propagation; and

   (2) that pT286 can occur not only within a holoenzyme but also between two holoenzymes, at least at sufficiently high concentrations.

   For the observation regarding the subunit exchange, the authors go above and beyond to demonstrate that a previously proposed activation-induced subunit exchange does not actually occur in their hands and that the previous appearance of such a subunit exchange may instead be due to activation-induced interactions between the kinase domains of separate holoenzymes. This provides important clarification, as the imagination about the possible functions of this subunit exchange has been running wild in the literature.

   By contrast, pT286 between holoenzymes at sufficiently high concentrations was largely predicted by the previously reported concentration-dependence of pT286 between monomeric truncated CaMKII (although these previous experiments did not rule out that such pT286 could have been excluded for intact full-length holoenzymes). Notably, the reaction rate reported here for pT286 between two holoenzymes is more than two orders of magnitude slower compared to the previously described rate of the pT286 reaction within a holoenzyme.

   The only point on which we disagree (and we think it’s unarguable) is that the current consensus is that inter-holoenzyme phosphorylation simply doesn’t happen (whether or not monomers can phosphorylate each other). The reviewer is of course right that this view seems now less and less likely. We now performed new experiments to investigate this critical point further (see below).

   The probable reason for the discrepancy in reported half-time of phosphorylation measured in earlier reports and in our paper is the fact that earlier reports (for example Bradshaw et al., 2002) measured autophosphorylation rate of wild-type CaMKII holoenzymes, at catalytically-competent enzyme concentrations of 0.1-5 µM. We are reporting the phosphorylation rate of 4 µM kinase-dead CaMKII, which is only a substrate, by 10 nM catalytically competent enzyme (CaMKII wild-type). There is up to 500 times less catalytically competent enzyme in our reactions, which is probably the reason why the reaction itself is several orders of magnitude slower.

   In summary, this study contains two somewhat disparate parts: (1) one technical tour-de-force to provide evidence that argues against activation-induced subunit exchange, which was a tremendous effort that provides influential novel information, and (2) another set of experiments showing the somewhat predictable potential for pT286 between holoenzymes, but without indication for the functional relevance of this rather slow reaction. Unfortunately, in the current/initial title of the manuscript, the authors chose to emphasize the weaker part of their findings.

   We agree with the reviewer that the title should be modified to emphasize both findings of our study. We also hope that our new experiments do bolster our findings with regard to pT286 between holoenzymes, as the reviewer puts it.

   The seemingly slow inter-holoenzyme phosphorylation is only slow under conditions in which one of the proteins is kinase-dead. In situation in which all CaMKII holoenzymes are wild-type and therefore capable of performing phosphorylation (both intra- and inter-holoenzyme) the reaction rates for pT286 are expected to be orders of magnitudes faster, than those reported here for the phosphorylation of T286 on kinase-dead protein.

   Reviewer #2 (Public Review):

   This well-written manuscript provides a technical tour-de-force to provide a novel mechanism for sustaining CaMKII autophosphorylation through an interholoenzyme reaction mechanism the authors term inter-holoenzyme phosphorylation (IHP). The authors use molecular engineering to create designer molecules that permit detailed testing of the proposed interholoenzyme reaction mechanism. By catalytically inactivating one population of enzymes, they show using standard assays that the inactive enzyme can be phosphorylated by active holoenzymes. They go on to show that in cells, the inactive enzyme is phosphorylated only in the presence of co-expressed active CaMKII and that this does not appear to be due to active and inactive subunits mixing within the same holoenzyme. The authors suggest reasons for why previous experiments failed to expose IHP and in some experiments provide evidence that reproduces and then extends earlier studies. Some noted differences from earlier experiments are the reaction temperature, the time course of the reactions, and that significantly higher concentrations of the inactive (substrate) kinase in the present study amplify the IHP. These are plausible reasons for earlier studies not finding significant evidence for IHP and the presented data is well-controlled and of high quality.

   The authors then take on the idea of subunit exchange employing multiple strategies. Using genetic expansion, they engineer an unnatural amino acid into the hub domain of the kinase (residue 384). In the presence of the photoactivatable crosslinker BZF and UV illumination, a ladder of subunits was generated indicating intraholoenzyme crosslinks were established. Using this cross-linked enzyme, presumably incapable of subunit exchange, the authors show significant phosphorylation of the kinase-dead mutant. This further supports that IHP is the cause of phosphorylation and not subunit exchange. Extending these experiments, they could not find evidence when CaMKIIF394BZF was mixed with the kinase-dead mutant and exposed to UV light, that there was evidence of the kinasedead subunits exchanged into CaMKIIF394 (active) enzymes.

   Just a note, instead of residue 384, this should read 394.

   With an entirely different approach, the authors use isotopic labeling of different pools of wt CaMKII (N14 or N15) followed by bifunctional cross-linking and mass spec to assess potential intra- and interholoenzyme contacts. Several interesting findings came of these studies detailed in Figure 4, mapped in detail in Figure 5, and extensively documented in supplementary tables. Critically, numerous crosslinks were found between different domains of the enzyme (catalytic, regulatory, hub) that are themselves a nice database of proximity measurements, but critical to the hypothesis, no heterotypic cross-links were found in the hub domains at any activated state or time point of incubation. This data supports two findings, that catalytic domains come into close proximity between holoenzymes when activated, supporting the potential for IHP, but that no subunit exchange occurs.

   The authors then pursue the approach used originally to provide evidence of subunit mixing, single molecule-based fluorescence imaging. Using pools of CaMKII labeled with spectrally separable dyes, the authors reproduce the earlier findings (Stratton et al, 2016) showing that under activating conditions, but not basal conditions, colocalized spots were detected. Numerous controls were done that confirm the need for full activation (Ca2+/CaM + Mg2+/ATP) to visualize co-localized CaMKII holoenzymes. Extending these studies, the authors mix holoenzymes, fully activate them, and after sufficient time for subunit exchange (if it occurs), the reactions were quenched, and then samples were analyzed. The result was that no evidence of dual-colored holoenzymes was present; if subunits had mixed between holoenzymes, dual-colored spots should have been evident after quenching the reactions. This was not the case. Further, experiments repeated with pools of differentially labeled kinase dead enzymes produced no colocalization, as predicted, if activation of the catalytic domains is necessary to establish IHP.

   Finally, the authors employ mass photometry to investigate the potential for interholoenzyme interactions. At basal conditions, only a mass peak consistent with CaMKII dodecamers was evident. Upon activation, a small fraction of dimeric complexes was evident (with Ca2+/CaM bound) but the majority of the peak was a dodecamer with 12 associated CaM molecules, and importantly, a significant fraction of a mass population was found consistent with a pair of holoenzymes with associated CaM. As an aside, the holoenzyme population appeared to be modestly destabilized as evidence of a minor fraction of dimers appeared as the authors diluted the enzyme, but the pools of holoenzyme and pairs of holoenzymes (with CaM) remained the dominant species when activated under all three enzyme concentrations assessed. Supporting the importance of activation for interactions between holoenzymes, the catalytically dead kinase even under activating conditions, shows no evidence of dimers of holoenzymes.

   Each of the approaches is well-controlled, the data is of uniformly high quality, and the authors' interpretations are generally well-supported.

   We are very grateful for these supportive comments.

   Reviewer #3 (Public Review):

   CaMKII is a multimeric kinase of great biologic interest due to its crucial roles in long-term memory, cardiac pacemaking, and fertilization. CaMKII subunits organize into holoenzymes comprised of 1214 subunits, adopting a donut-like, double-ringed structure. In this manuscript, Lucic et al challenge two models in the CaMKII field, which are somewhat related. The first is a longstanding topic in the field about whether the autophosphorylation of a crucial residue, Thr286, can be phosphorylated between intact holoenzymes (inter-holoenzyme phosphorylation). The second is a more recent biochemical finding, which tested the long-running theory that CaMKII exchanges subunits between holoenzymes to create mixed oligomers. These two models are connected by the idea that subunit exchange could facilitate phosphorylation between subunits of different holoenzymes by allowing subunits to integrate into a different holoenzyme and driving transphosphorylation within the CaMKII ring. Here, the authors attempt to show that one intact holoenzyme phosphorylates another intact holoenzyme at Thr286. The authors also provide evidence suggesting that subunit exchange is not occurring under their conditions, and therefore not driving this phosphorylation event. The authors propose a model where instead of exchanging subunits, two holoenzymes interact via their kinase domains to enable transphosphorylation at Thr286 without integrating into the holoenzyme structure. In order for the authors to successfully convince readers of all three facets of this new model, they need to provide evidence that 1) transphosphorylation at Thr286 happens when subunit exchange is blocked, 2) subunit exchange does not occur under their conditions, and 3) there are interactions between kinases of different holoenzymes that lead to productive autophosphorylation at Thr286.

   Strengths:

   The authors have designed and performed a battery of cleverly designed and orthogonal experiments to test these models. Using mutagenesis, they mixed a kinase-dead mutant with an active kinase to ask whether transphosphorylation occurs. They observe phosphorylation of the kinase-dead variant in this experiment, which indicates that the active kinase must have phosphorylated it. A few key questions arise here: 1) whether this phosphorylation occurred within a single CaMKII holoenzyme ring (which is the canonical mechanism for Thr286 phosphorylation), 2) whether the phosphorylation occurred between two separate holoenzyme rings, and 3) why was this not observed in previous literature? To address questions 1 and 2, the authors implemented an innovative strategy introducing a geneticallyencoded photocrosslinker in the oligomerization domain, which when crosslinked using UV light, should lock the holoenzyme in place. The rate of phosphorylation was the same when comparing uncrosslinked and crosslinked CaMKII variants, indicating that phosphorylation is occurring between holoenzymes, rather than through a subunit exchange mechanism that would require some type of disassembly and reassembly (presumably blocked by crosslinking). The 3rd question remains as to why this has not been previously observed, as it has not been for lack of effort. The authors mention low temperature and low concentration as culprits, however, Bradshaw et al, JBC v. 277, 2002 carry out a series of careful experiments that indicated that autophosphorylation at T286 is not concentration-dependent (meaning that the majority of phosphorylation occurs via intra-holoenzyme), and this is done over a concentration and temperature range. It is possible that due to the mutants used in the current manuscript, it allows for the different behavior of the kinase-dead domains, which will have an empty nucleotide-binding pocket. Further studies will need to elucidate these details, and importantly, understand what physiological conditions facilitate this mechanism.

   We thank the reviewer for their assessment of our work.

   The paper cited by the reviewer (Bradshaw et al, JBC v. 277, 2002) is indeed a carefully designed biochemical investigation of CaMKII activity. As the reviewer pointed out, one of the conclusions of the paper is that the autophosphorylation of CaMKII is not concentration dependent, implying that it has to occur exclusively intra-holoenzyme. However, there are some limitations which colour the interpretation of this classic paper. Bradshaw and colleagues used only CaMKII wild-type protein, so the autophosphorylation which is taking place in their reactions is possible both within holoenzymes and between holoenzymes, but this is impossible to distinguish. The authors of the cited paper then used “Autonomous activity assay” (not any measurement of pT286 on CaMKII itself) in which they first stopped the initial autophosphorylation reaction at T286 by adding a quench solution which contained a mixture of EDTA and EGTA, and then measured phosphorylation of the peptide-substrate of CaMKII (autocamtide-2), in the absence of Calmodulin binding (autonomous activity). They also diluted the autophosphorylation reaction to 10 nM CaMKII before adding it to the “Autonomous activity assay”.

   As a side point, each reaction was quenched and diluted to the same final CaMKII concentration of 10 nM. They measured the activity of this dilution with phosphorylation of a peptide-substrate (autocamptide-2), in the absence of CaM binding. The authors contend that autonomous activity reported in this way reflects the amount of pT286, which is not impossible, but it is not a direct measure of pT286.

   All this adds up to allowing the autophosphorylation of wild-type CaMKII at various concentrations ranging from 0.1 to 4.6 µM in the presence of 10 µM Ca/CaM and 500 µM Mg/ATP. This is a very fast reaction, concentrations of enzyme (CaMKII wild-type), activator (Ca/CaM) and ATP/Mg are all high at the beginning of the autophosphorylation reaction and would expect to allow for maximal autophosphorylation in very short times (seconds). Most importantly, this experiment does not exclude a inter-holoenzyme reaction slower than the intra-holoenzyme one. It certainly could not detect it.

   In any case, to relate these concepts to our experiments and current understanding of CaMKII, we performed a new set of experiments modelled on the Bradshaw paper. Critically, we used CaMKII wild-type as the enzyme, and CaMKII kinase-dead, as the substrate. Intraholoenzyme phosphorylation cannot occur in this reaction, which was designed to detect a concentration-dependent phosphorylation reaction. We used a fixed concentration of the substrate kinase (4 µM), and 4 different concentrations of CaMKIIWT ranging from 0.5 -100 nM. In our assay, the level of phosphorylation on substrate CaMKII(CaMKIIKD) was dependent on concentration of enzyme CaMKII (CaMKIIWT) (Figure 1-figure supplement 3), adding more evidence to the hypothesis that CaMKII autophosphorylation can occur inter-holoenzyme.

   The possibility that empty nucleotide binding pocket is influencing the phosphorylation status of T286 in the regulatory domain of kinase-dead CaMKII is highly unlikely. One could maybe envision that empty nucleotide binding pocket might expose the regulatory domain in kinase-dead CaMKII for phosphorylation, which would be prevented in CaMKIIWT, but in all available structures of CaMKII (Chao et al, 2011; Myers et al., 2017, Buonarati et al., 2021), the regulatory domain is docked to the kinase domain of CaMKII, although the nucleotide binding pocket is empty (either by mutation of residue K42 and/or simply by not adding the ATP/Mg to reduce chemical dispersity of the sample). The only time the regulatory domain was not docked on the kinase domain is when CaMKII was in complex with Calmodulin (Rellos et al., 2010). Finally, in our crosslinking mass spectrometry experiments, we used both heavy and light forms of CaMKII wild-type, and there we can clearly see interactions between kinase/regulatory domains of two different species of CaMKIIWT, which are dependent on activation.

   The most convincing data that subunit exchange does not occur is from the crosslinking mass spectrometry experiment. The authors created mixtures of 'light' and 'heavy' CaMKII holoenzymes, either activated or not and then used a Lys-Lys crosslinker (DSS) to trap the enzyme in its final state. The results of this experiment indicate that subunit exchange is not occurring under their conditions. A caveat here is that there are not many lysines at hub-hub interfaces, which is the crux of this experiment. If there is no subunit exchange under their conditions, how does transphosphorylation occur between holoenzymes? The authors show very nice mass photometry data indicating that there are populations of 24-mers, which corresponds to a double-holoenzyme. Paired with the data from their crosslinking mass spectrometry which shows crosslinks between kinase domains of different holoenzymes, this indicates that perhaps kinases between holoenzymes do interact, and they do so in a competent manner to allow transphosphorylation to occur.

   It is true that there are “only” 6 Lysines in the hub domain of CaMKII. However, it is clear from our crosslinking mass spectrometry data that we can detect hub:hub peptides coming from the same holoenzymes (homocrosslinks, either 14N: 14N or 15N: 15N species), but never between holoenzymes (14N with 15N). The fact that peptides can be detected in the homocrosslinks speaks to the validity of using Lysine crosslinkers in this experiment.

   Weaknesses:

   The authors should be commended for performing three orthogonal experiments to test whether CaMKII holoenzymes exchange subunits to form heterooligomers. However, there are technical issues that dampen the strength of the results shown here. For simplicity, let's consider that CaMKII holoenzymes are comprised of two stacked hexameric rings. It has been proposed that the stable unit of CaMKII assembly and perhaps also disassembly and subunit exchange is a vertical dimer unit (comprised of one subunit from each hexameric ring). In the UV crosslinking data shown in this paper, the authors have a significant number of monomers, some crosslinked dimers (of which there are two populations), and fewer higher-order oligomers. To effectively block subunit exchange, robust crosslinking into hexamers is necessary, which the authors have not done. Incomplete crosslinking results in smaller species that can still exchange (and/or dissociate), confounding the results of this experiment. In addition, Figure 3 shows a trapping experiment, where if the exchange was occurring, there would be an oligomeric band in Lane 8, which is visible and highlighted with a blue arrow by the authors. This result is explained by nonspecific UV effects, however by eye it is not clear if there is an equivalent band in lane 10. The overall issue here is inefficient crosslinking.

   We agree with the reviewer that the robustness of the UV-induced crosslinking is not extremely high. However we do observe higher order oligomers on the gel (Figure 2 and Figure 3B, pT286 blot), which states that at least a portion of the holoenzymes is crosslinked. On the other hand, the UVinduced crosslinking is not slowing down the trans-phosphorylation reaction, which would be expected if the subunit exchange would be the prevailing mechanism for spread of kinase activity between holoenzymes.

   In figure 3, lanes 8 and 10 show a small portion of dimers (less than 5% by densitometry), and at the absolute limit of detection. This dimer band is most likely due to unspecific UV-induced disulfide bridging (we already lessened it by adding 50 mM TCEP prior to UV treatment (Figure 3-figure supplement 1B and C). Previous reviewers of this manuscript criticized the small dimer band in lane 8, and we wanted to address this transparently in the submission to eLife.

   Unfortunately, if we absolutely crank up the contrast to see this band in lane 10, we start to see other features in the noise as well. We have now edited the image in Figure 3B to highlight these minor bands more clearly, but this is also not ideal.

   With regard to the trapping experiment, the overall problem is not inefficient crosslinking, because we see that P-T286 signal is quite nicely represented in higher order bands from F394BzF protein, but kinase dead protein (Avi-tagged signal in Figure 3) is almost entirely absent. Any crosslinking of Avitagged protein (possibly corresponding to subunit exchange) is a minor process at the limit of detection on WB.

   Unfortunately we did not yet find any better crosslinking sites than the two we report (we have tried about 10). But the results we did obtain encouraged us to employ other techniques to probe subunit exchange (for example, the MS X-linking).

   The authors also employ a single-molecule TIRF experiment to further interrogate subunit exchange. Upon inspection of the TIRF images, it is not clear that the authors are achieving single molecule resolution (there are evident overlapping and distorted particles). The analysis employed here is Pearson's correlation coefficient, which is not sufficient for single molecule analysis and would not account for particle overlap, particles that are too bright, and/or particles that are too dim. For example, an alternative explanation for the authors' results is that activation results in aggregation (high correlation), and subsequent EGTA treatment leads to dissociation at these low concentrations (low correlation). However, further experimentation and analysis are necessary.

   In the manuscript we present raw images, not processed. As we wrote in the material and methods, we thresholded the images for further processing. All colocalization methods have drawbacks, but we found that our thresholding combined with the Pearson coefficient was highly reproducible. We did also look at Manders coefficients, but these are less straightforward to understand, whilst still giving in our hands the same answer. We agree, there are more experiments that can be done, with particular predictions based on our new mechanism. And we are doing them and will report them when they are ready.

   At the risk of repeating ourselves, the reversible loss of overlap of the two labelled populations is the key result and cannot be explained by spurious dim or bright particles, or by a few overlapping profiles.

   Taken together, the authors have provided important food for thought regarding inter-holoenzyme phosphorylation and subunit exchange. However, given the shortcomings discussed here, it remains unclear exactly what mechanisms are at play within and between CaMKII holoenzymes once activated.

   We thank the reviewer for their critical assessment of our manuscript. We will continue to investigate the relevant points and refine the overall picture of CaMKII, to better clarify the mechanisms.

   Read the original source
3. 

   eLife

   Aug 6, 2023

   eLife assessment

   This manuscript reports the fundamental finding that an oligomeric protein kinase, CaMKII, can be phosphorylated by another molecule of the holoenzyme in a manner that does not involve subunit exchange. The evidence for the main conclusion is compelling, supported by several independent experiments. If independently confirmed in future, the study will stand as having provided a novel regulatory mechanism for the autophosphorylation of this kinase. The work will be of broad interest to molecular and cellular neuroscientists as well as biochemists.

   Read the original source
4. 

   eLife

   Aug 6, 2023

   Reviewer #1 (Public Review):

   The potential role of the CaMKII holoenzyme in synaptic information processing, storage, and spread has fascinated neuroscientists ever since it has been described that self-phosphorylation of CaMKII at T286 (pT286) can maintain the kinase in an activated state beyond the initial Ca2+ stimulus that induced kinase activation and pT286. The current study by Lučić et al utilizes biochemical and biophysical methods to re-examine two pT286 mechanisms and finds:
   (1) that a previously proposed activation-induced subunit exchange within the holoenzyme can not provide pT286 maintenance or propagation; and
   (2) that pT286 can occur not only within a holoenzyme but also between two holoenzymes, at least at sufficiently high concentrations.

   For the observation regarding the subunit exchange, the authors go above and …

   Reviewer #1 (Public Review):

   The potential role of the CaMKII holoenzyme in synaptic information processing, storage, and spread has fascinated neuroscientists ever since it has been described that self-phosphorylation of CaMKII at T286 (pT286) can maintain the kinase in an activated state beyond the initial Ca2+ stimulus that induced kinase activation and pT286. The current study by Lučić et al utilizes biochemical and biophysical methods to re-examine two pT286 mechanisms and finds:
   (1) that a previously proposed activation-induced subunit exchange within the holoenzyme can not provide pT286 maintenance or propagation; and
   (2) that pT286 can occur not only within a holoenzyme but also between two holoenzymes, at least at sufficiently high concentrations.

   For the observation regarding the subunit exchange, the authors go above and beyond to demonstrate that a previously proposed activation-induced subunit exchange does not actually occur in their hands and that the previous appearance of such a subunit exchange may instead be due to activation-induced interactions between the kinase domains of separate holoenzymes. This provides important clarification, as the imagination about the possible functions of this subunit exchange has been running wild in the literature.

   By contrast, pT286 between holoenzymes at sufficiently high concentrations was largely predicted by the previously reported concentration-dependence of pT286 between monomeric truncated CaMKII (although these previous experiments did not rule out that such pT286 could have been excluded for intact full-length holoenzymes). Notably, the reaction rate reported here for pT286 between two holoenzymes is more than two orders of magnitude slower compared to the previously described rate of the pT286 reaction within a holoenzyme.

   In summary, this study contains two somewhat disparate parts: (1) one technical tour-de-force to provide evidence that argues against activation-induced subunit exchange, which was a tremendous effort that provides influential novel information, and (2) another set of experiments showing the somewhat predictable potential for pT286 between holoenzymes, but without indication for the functional relevance of this rather slow reaction. Unfortunately, in the current/initial title of the manuscript, the authors chose to emphasize the weaker part of their findings.

   Read the original source
5. 

   eLife

   Aug 6, 2023

   Reviewer #2 (Public Review):

   This well-written manuscript provides a technical tour-de-force to provide a novel mechanism for sustaining CaMKII autophosphorylation through an interholoenzyme reaction mechanism the authors term inter-holoenzyme phosphorylation (IHP). The authors use molecular engineering to create designer molecules that permit detailed testing of the proposed interholoenzyme reaction mechanism. By catalytically inactivating one population of enzymes, they show using standard assays that the inactive enzyme can be phosphorylated by active holoenzymes. They go on to show that in cells, the inactive enzyme is phosphorylated only in the presence of co-expressed active CaMKII and that this does not appear to be due to active and inactive subunits mixing within the same holoenzyme. The authors suggest reasons for why previous …

   Reviewer #2 (Public Review):

   This well-written manuscript provides a technical tour-de-force to provide a novel mechanism for sustaining CaMKII autophosphorylation through an interholoenzyme reaction mechanism the authors term inter-holoenzyme phosphorylation (IHP). The authors use molecular engineering to create designer molecules that permit detailed testing of the proposed interholoenzyme reaction mechanism. By catalytically inactivating one population of enzymes, they show using standard assays that the inactive enzyme can be phosphorylated by active holoenzymes. They go on to show that in cells, the inactive enzyme is phosphorylated only in the presence of co-expressed active CaMKII and that this does not appear to be due to active and inactive subunits mixing within the same holoenzyme. The authors suggest reasons for why previous experiments failed to expose IHP and in some experiments provide evidence that reproduces and then extends earlier studies. Some noted differences from earlier experiments are the reaction temperature, the time course of the reactions, and that significantly higher concentrations of the inactive (substrate) kinase in the present study amplify the IHP. These are plausible reasons for earlier studies not finding significant evidence for IHP and the presented data is well-controlled and of high quality.

   The authors then take on the idea of subunit exchange employing multiple strategies. Using genetic expansion, they engineer an unnatural amino acid into the hub domain of the kinase (residue 384). In the presence of the photoactivatable crosslinker BZF and UV illumination, a ladder of subunits was generated indicating intraholoenzyme crosslinks were established. Using this cross-linked enzyme, presumably incapable of subunit exchange, the authors show significant phosphorylation of the kinase-dead mutant. This further supports that IHP is the cause of phosphorylation and not subunit exchange. Extending these experiments, they could not find evidence when CaMKIIF394BZF was mixed with the kinase-dead mutant and exposed to UV light, that there was evidence of the kinase-dead subunits exchanged into CaMKIIF394 (active) enzymes.

   With an entirely different approach, the authors use isotopic labeling of different pools of wt CaMKII (N14 or N15) followed by bifunctional cross-linking and mass spec to assess potential intra- and inter-holoenzyme contacts. Several interesting findings came of these studies detailed in Figure 4, mapped in detail in Figure 5, and extensively documented in supplementary tables. Critically, numerous cross-links were found between different domains of the enzyme (catalytic, regulatory, hub) that are themselves a nice database of proximity measurements, but critical to the hypothesis, no heterotypic cross-links were found in the hub domains at any activated state or time point of incubation. This data supports two findings, that catalytic domains come into close proximity between holoenzymes when activated, supporting the potential for IHP, but that no subunit exchange occurs.

   The authors then pursue the approach used originally to provide evidence of subunit mixing, single molecule-based fluorescence imaging. Using pools of CaMKII labeled with spectrally separable dyes, the authors reproduce the earlier findings (Stratton et al, 2016) showing that under activating conditions, but not basal conditions, colocalized spots were detected. Numerous controls were done that confirm the need for full activation (Ca2+/CaM + Mg2+/ATP) to visualize co-localized CaMKII holoenzymes. Extending these studies, the authors mix holoenzymes, fully activate them, and after sufficient time for subunit exchange (if it occurs), the reactions were quenched, and then samples were analyzed. The result was that no evidence of dual-colored holoenzymes was present; if subunits had mixed between holoenzymes, dual-colored spots should have been evident after quenching the reactions. This was not the case. Further, experiments repeated with pools of differentially labeled kinase dead enzymes produced no colocalization, as predicted, if activation of the catalytic domains is necessary to establish IHP.

   Finally, the authors employ mass photometry to investigate the potential for interholoenzyme interactions. At basal conditions, only a mass peak consistent with CaMKII dodecamers was evident. Upon activation, a small fraction of dimeric complexes was evident (with Ca2+/CaM bound) but the majority of the peak was a dodecamer with 12 associated CaM molecules, and importantly, a significant fraction of a mass population was found consistent with a pair of holoenzymes with associated CaM. As an aside, the holoenzyme population appeared to be modestly destabilized as evidence of a minor fraction of dimers appeared as the authors diluted the enzyme, but the pools of holoenzyme and pairs of holoenzymes (with CaM) remained the dominant species when activated under all three enzyme concentrations assessed. Supporting the importance of activation for interactions between holoenzymes, the catalytically dead kinase even under activating conditions, shows no evidence of dimers of holoenzymes.

   Each of the approaches is well-controlled, the data is of uniformly high quality, and the authors' interpretations are generally well-supported.

   Read the original source
6. 

   eLife

   Aug 6, 2023

   Reviewer #3 (Public Review):

   CaMKII is a multimeric kinase of great biologic interest due to its crucial roles in long-term memory, cardiac pacemaking, and fertilization. CaMKII subunits organize into holoenzymes comprised of 12-14 subunits, adopting a donut-like, double-ringed structure. In this manuscript, Lucic et al challenge two models in the CaMKII field, which are somewhat related. The first is a longstanding topic in the field about whether the autophosphorylation of a crucial residue, Thr286, can be phosphorylated between intact holoenzymes (inter-holoenzyme phosphorylation). The second is a more recent biochemical finding, which tested the long-running theory that CaMKII exchanges subunits between holoenzymes to create mixed oligomers. These two models are connected by the idea that subunit exchange could facilitate …

   Reviewer #3 (Public Review):

   CaMKII is a multimeric kinase of great biologic interest due to its crucial roles in long-term memory, cardiac pacemaking, and fertilization. CaMKII subunits organize into holoenzymes comprised of 12-14 subunits, adopting a donut-like, double-ringed structure. In this manuscript, Lucic et al challenge two models in the CaMKII field, which are somewhat related. The first is a longstanding topic in the field about whether the autophosphorylation of a crucial residue, Thr286, can be phosphorylated between intact holoenzymes (inter-holoenzyme phosphorylation). The second is a more recent biochemical finding, which tested the long-running theory that CaMKII exchanges subunits between holoenzymes to create mixed oligomers. These two models are connected by the idea that subunit exchange could facilitate phosphorylation between subunits of different holoenzymes by allowing subunits to integrate into a different holoenzyme and driving transphosphorylation within the CaMKII ring. Here, the authors attempt to show that one intact holoenzyme phosphorylates another intact holoenzyme at Thr286. The authors also provide evidence suggesting that subunit exchange is not occurring under their conditions, and therefore not driving this phosphorylation event. The authors propose a model where instead of exchanging subunits, two holoenzymes interact via their kinase domains to enable transphosphorylation at Thr286 without integrating into the holoenzyme structure. In order for the authors to successfully convince readers of all three facets of this new model, they need to provide evidence that 1) transphosphorylation at Thr286 happens when subunit exchange is blocked, 2) subunit exchange does not occur under their conditions, and 3) there are interactions between kinases of different holoenzymes that lead to productive autophosphorylation at Thr286.

   Strengths:
   The authors have designed and performed a battery of cleverly designed and orthogonal experiments to test these models. Using mutagenesis, they mixed a kinase-dead mutant with an active kinase to ask whether transphosphorylation occurs. They observe phosphorylation of the kinase-dead variant in this experiment, which indicates that the active kinase must have phosphorylated it. A few key questions arise here: 1) whether this phosphorylation occurred within a single CaMKII holoenzyme ring (which is the canonical mechanism for Thr286 phosphorylation), 2) whether the phosphorylation occurred between two separate holoenzyme rings, and 3) why was this not observed in previous literature? To address questions 1 and 2, the authors implemented an innovative strategy introducing a genetically-encoded photocrosslinker in the oligomerization domain, which when crosslinked using UV light, should lock the holoenzyme in place. The rate of phosphorylation was the same when comparing uncrosslinked and crosslinked CaMKII variants, indicating that phosphorylation is occurring between holoenzymes, rather than through a subunit exchange mechanism that would require some type of disassembly and reassembly (presumably blocked by crosslinking). The 3rd question remains as to why this has not been previously observed, as it has not been for lack of effort. The authors mention low temperature and low concentration as culprits, however, Bradshaw et al, JBC v. 277, 2002 carry out a series of careful experiments that indicated that autophosphorylation at T286 is not concentration-dependent (meaning that the majority of phosphorylation occurs via intra-holoenzyme), and this is done over a concentration and temperature range. It is possible that due to the mutants used in the current manuscript, it allows for the different behavior of the kinase-dead domains, which will have an empty nucleotide-binding pocket. Further studies will need to elucidate these details, and importantly, understand what physiological conditions facilitate this mechanism.

   The most convincing data that subunit exchange does not occur is from the crosslinking mass spectrometry experiment. The authors created mixtures of 'light' and 'heavy' CaMKII holoenzymes, either activated or not and then used a Lys-Lys crosslinker (DSS) to trap the enzyme in its final state. The results of this experiment indicate that subunit exchange is not occurring under their conditions. A caveat here is that there are not many lysines at hub-hub interfaces, which is the crux of this experiment. If there is no subunit exchange under their conditions, how does transphosphorylation occur between holoenzymes? The authors show very nice mass photometry data indicating that there are populations of 24-mers, which corresponds to a double-holoenzyme. Paired with the data from their crosslinking mass spectrometry which shows crosslinks between kinase domains of different holoenzymes, this indicates that perhaps kinases between holoenzymes do interact, and they do so in a competent manner to allow transphosphorylation to occur.

   Weaknesses:
   The authors should be commended for performing three orthogonal experiments to test whether CaMKII holoenzymes exchange subunits to form heterooligomers. However, there are technical issues that dampen the strength of the results shown here. For simplicity, let's consider that CaMKII holoenzymes are comprised of two stacked hexameric rings. It has been proposed that the stable unit of CaMKII assembly and perhaps also disassembly and subunit exchange is a vertical dimer unit (comprised of one subunit from each hexameric ring). In the UV crosslinking data shown in this paper, the authors have a significant number of monomers, some crosslinked dimers (of which there are two populations), and fewer higher-order oligomers. To effectively block subunit exchange, robust crosslinking into hexamers is necessary, which the authors have not done. Incomplete crosslinking results in smaller species that can still exchange (and/or dissociate), confounding the results of this experiment. In addition, Figure 3 shows a trapping experiment, where if the exchange was occurring, there would be an oligomeric band in Lane 8, which is visible and highlighted with a blue arrow by the authors. This result is explained by nonspecific UV effects, however by eye it is not clear if there is an equivalent band in lane 10. The overall issue here is inefficient crosslinking.

   The authors also employ a single-molecule TIRF experiment to further interrogate subunit exchange. Upon inspection of the TIRF images, it is not clear that the authors are achieving single molecule resolution (there are evident overlapping and distorted particles). The analysis employed here is Pearson's correlation coefficient, which is not sufficient for single molecule analysis and would not account for particle overlap, particles that are too bright, and/or particles that are too dim. For example, an alternative explanation for the authors' results is that activation results in aggregation (high correlation), and subsequent EGTA treatment leads to dissociation at these low concentrations (low correlation). However, further experimentation and analysis are necessary.

   Taken together, the authors have provided important food for thought regarding inter-holoenzyme phosphorylation and subunit exchange. However, given the shortcomings discussed here, it remains unclear exactly what mechanisms are at play within and between CaMKII holoenzymes once activated.

   Read the original source
7. 

   Version published to 10.1101/2022.08.03.502606v2 on bioRxiv

   Sep 7, 2022
8. 

   Version published to 10.1101/2022.08.03.502606v1 on bioRxiv

   Aug 5, 2022