ATP-independent phosphate recycling on AGC kinase activation loops induced by alkali metal ions

  1. Koji Kubouchi
  2. Hideyuki Mukai

  • Curated by eLife

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    eLife Assessment

    AGC kinases, such as PKN1, are regulated by activation loop phosphorylation. This paper reports that exposing cells to high concentrations of monovalent cations induces rapid activation loop dephosphorylation, with rapid re-phosphorylation when physiological salt is restored. Re-phosphorylation is apparently independent of ATP or candidate kinases, and the paper presents an extraordinary and unconventional mechanism involving phosphate exchange between the activation loop and an unknown acceptor molecule. The findings are intriguing and the approach is logical, but the evidence is incomplete and the significance unclear until the biochemical mechanism is identified.

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Abstract

Changes in extracellular Na⁺ and K⁺ concentrations have traditionally been considered to influence intracellular signal transduction through alterations in cell volume or membrane potential. However, whether intracellular ion concentration changes directly regulate signaling molecules, independent of these conventional pathways, remains largely unexplored. In this study, we demonstrate that even in the absence of cellular membranes, an increase in Na⁺ or K⁺ concentration rapidly reduces activation-loop phosphorylation of multiple AGC kinases, including PKN, PKCζ/λ, and p70 S6 kinase. When ion concentrations were reduced, the activation-loop phosphorylation, which had initially decreased, recovered within a short period. Notably, this recovery occurred in the absence of PDK1, a known kinase responsible for the phosphorylation of these activation loops, and did not require ATP or Mg²⁺ in lysate assays. ³²P tracing experiments revealed a novel ‘reacquisition of phosphate group’ mechanism, in which phosphate groups transiently dissociate from the activation loops under high Na⁺ or K⁺ conditions and are subsequently re-incorporated into the activation loops when ion concentrations are reduced. These findings indicate that elevated Na⁺ or K⁺ concentrations directly and rapidly reduce the activity of multiple AGC kinases, and that the activity rapidly recovers upon reduction in ion concentrations, through an unconventional phosphate transfer mechanism distinct from canonical protein phosphorylation reactions. Our study suggests the existence of a robust phosphorylation homeostasis mechanism independent of conventional kinase-phosphatase systems, providing new insights into signaling pathways regulated by intracellular Na⁺/K⁺ ion dynamics.

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  1. eLife

    eLife Assessment

    AGC kinases, such as PKN1, are regulated by activation loop phosphorylation. This paper reports that exposing cells to high concentrations of monovalent cations induces rapid activation loop dephosphorylation, with rapid re-phosphorylation when physiological salt is restored. Re-phosphorylation is apparently independent of ATP or candidate kinases, and the paper presents an extraordinary and unconventional mechanism involving phosphate exchange between the activation loop and an unknown acceptor molecule. The findings are intriguing and the approach is logical, but the evidence is incomplete and the significance unclear until the biochemical mechanism is identified.

  2. eLife

    Reviewer #1 (Public review):

    The authors found that high concentrations of a series of monovalent cations, NaCl, KCl, RbCl, and CsCl (although not LiCl), but not equal high osmolarity treatment of cultured cells induced rapid loss of phosphate from pT774 in the activation loop (AL) of the PKN1 Ser/Thr protein kinase, as well the cognate AL phosphoresidue in other related AGC family kinases, including PKCζ, PKCλ, and p70 S6 kinase. Focusing on PKN1, they showed that restoration of the extracellular salt concentration to physiological levels resulted in equally rapid recovery of AL phosphorylation. Using both okadaic acid PP1/PP2A inhibitor, and a selective PP2A inhibitor, PP2A was implicated as the protein phosphatase required for the rapid dephosphorylation of PIN1 pT774 in response to high salt. By making PKN1 T778A knock-in mouse fibroblast cells and re-expressing WT and a kinase-dead mutant PKN1, as well as use of PDK1 KO MEFs, they showed that recovery of T774 phosphorylation did not require PDK1, the protein kinase known to phosphorylate this site in cells, or the kinase activity of PKN1 itself. Surprisingly, they found that dephosphorylation of the PKN1 AL site also occurred when cell lysates were adjusted to high salt, with re-phosphorylation of T774 occurring rapidly when physiological salt level was restored by dilution. Their in vitro lysate experiments also demonstrated that depletion of ATP by apyrase treatment or sequestration of Mg2+ by EDTA did not prevent T744 re-phosphorylation, which would rule out a conventional protein kinase. Various GST-tagged fragments of PKN1, including a 767-780 AL 14-mer peptide,e exhibited the same curious de- and re-phosphorylation effect when mixed with cell lysates and exposed to high KCl followed by dilution. Using 32P γ-ATP and PDK1 to generate 32P-labeled phospho-GST-PKN1 (767-788). They showed the 32P signal was lost from GST-PKN1 (767-788) in lysates exposed to high salt, and restored again upon dilution. Similar results were obtained with unlabeled samples using PhosTag analysis to resolve phosphospecies.

    They went on to test three possible models to explain their data:

    (1) Model 1. Intramolecular transfer of the pT774 phosphate group, where the pT774 phosphate is reversibly transferred onto another residue in the same PKN1 molecule in response to high and normal salt concentrations. They attempted to rule out this model by mutating possible noncanonical phosphate acceptors in the 776GYGDRTSTFCGTPE788 peptide, making C776, D770A, R771A, and E780A mutant peptides, without observing any effect on the dephosphorylation/re-phosphorylation phenomenon.

    (2) Model 2. Re-phosphorylation of T774 involves an unidentified phosphate donor, distinct from ATP or phospho-PKN1. This model was ruled out in several ways, including by demonstrating that added 32P-labeled PKN1 lost its 32P signal in high salt-exposed lysates, with the 32P signal being recovered upon dilution even in the presence of excess unlabeled ATP.

    (3) Model 3. Reversible transfer of the pT774 phosphate group onto an intermediary factor (X) in the presence of high salt and re-phosphorylation in cis by phospho-X upon dilution, which is the model they favored. In support of this model, they showed that the pT774 phosphate could not be transferred onto another PKN1 fragment of a different size, nor did GST-PKN1 767-788 pretreated with λ-phosphatase regain phosphate. In the end, however, they were unable to identify the hypothetical factor X, and no 32P-labeled protein was observed in the experiment with 32P-labeled PKN1 upon high salt-induced dephosphorylation.

    This is an intriguing and unexpected set of findings that could herald a new protein kinase regulatory mechanism, but ultimately, we are left with an intriguing observation without a clear-cut explanation. The authors have been very methodical in their analysis of this odd phenomenon, and their data and conclusions, for the most part, seem convincing, although some of the blot signals are rather weak. However, despite all their efforts, the identity of the hypothetical factor X, which can transiently accept a phosphate from pT774 in the PKN1 activation loop in response to supraphysiological alkali metal cation concentrations and then donate it back again to T774 in cis, when physiological salt concentrations are restored, remains unclear.

    As it stands, there are several unresolved issues that need to be addressed.

    (1) The real conundrum, as their data show, is that phospho-X cannot phosphorylate PKN1 in trans, and therefore has to act in cis, meaning that phospho-X must somehow remain associated with the same dephosphorylated PKN1 molecule that the phosphate came from. Because a small molecule would rapidly diffuse away from PKN1, the only reasonable model is that X is a protein and not a small molecule, such as creatine (the authors considered X unlikely to be a small molecule for other reasons). However, if X were a protein, then it should have been labeled and detectable on the gel in the 32P-experiment shown in Figure 6C, but no other 32P-labeled band was observed in lane 5. Even if phospho-X has a labile phosphate linkage that would be lost upon SDS-gel electrophoresis, it is unclear how phospho-X would remain associated with the very short 14-mer PKN1 activation loop peptide, especially under the extremely dilute conditions of a cell lysate.

    (2) The evidence that PP2A is required in PKN1 dephosphorylation is reasonable, and in the Discussion, the authors consider various scenarios in which PP2A could be involved in generating the hypothetical phospho-X needed for T774 re-phosphorylation, most of which do not seem very plausible. In the end, it remains unclear how free phosphate released from pT774 in PKN1 by PP2A, which does not employ a phosphoenzyme intermediate, ends up covalently attached to molecule X.

    (3) The interpretation of the in vitro data is complicated by the fact that cell lysis results in a massive dilution of both proteins and any small molecules present in the cell (apparently dilution with lysis buffer was at least 10-fold initially, and then a further 2-fold to restore normal salt levels), making it hard to imagine how a large or small molecule would remain tightly associated with a PKN1 molecule, i.e. Model 3 really only works if re-phosphorylation of T774 is a zero order/intramolecular reaction. Moreover, the re-phosphorylation reaction rates would be expected to fall dramatically upon dilution of both the dephosphorylated GST-PKN1 767-788 protein and phospho-X during restoration of normal salt, meaning that the kinetics of T774 re-phosphorylation should be significantly slower in vitro. In this connection, it would be informative if the authors carried out a lysate dilution series to test the extent to which the observed phenomenon is dilution-independent.

    (4) Another issue is that most of the results, apart from the 32P-labeling experiment, are dependent on the specificity of the anti-pT774 PKN1 antibodies they used. The fact that the C776A mutant peptide gave a weaker anti-pT774 signal might be because phospho-Ab binding is, in part, dependent on recognition of Cys776. In turn, this suggests the possibility that reversible oxidation of C776 might cause the loss and regain of the pT774 signal at high and low salt concentrations, as a result of the oxidized form of C776 preventing anti-pT774 antibody binding. The Cell Signaling Technology phospho-PRK1 (Thr774)/PRK2 (Thr816) antibody (#2611) that was used here was generated against a synthetic peptide containing pT774, and while the exact antigenic peptide sequence is not given in the CST catalogue, presumably it had 4 or 5 residues on either side of pT774 (GYGDRTSTFCGTPE) (although C776 might have been substituted in the antigenic peptide because of issues with Cys oxidation).

    (5) Perhaps the most important deficiency is that the target for the monovalent cation that induces PKN1 activation loop dephosphorylation was not established. Is this somehow a direct effect of cations on PKN1 itself - this seems unlikely, since this effect is observed with a 14-mer PKN1 activation loop peptide - or is this an indirect effect? In terms of possible indirect mechanisms, high salt treatment of cells is known to induce elevated ROS as a result of mitochondrial damage, which could lead to oxidative modification of cysteines, such as C776, in the activation loop and might interfere with anti-pT774 antibody recognition.

    In summary, the authors have put a great deal of thought and resources into trying to solve this intriguing puzzle, but despite a lot of effort, have not convincingly elucidated how this dephosphorylation/re-phosphorylation process works. For this, they need to identify phospho-X and define how it remains associated with the original pT774 PKN1 molecule in order to carry out re-phosphorylation.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    This study reports a highly unconventional mechanism by which AGC kinases might undergo reversible activation-loop (T-loop) phosphorylation through an ATP-independent phosphate recycling process that is modulated by alkali metal ions such as Na⁺ and K⁺. The authors propose that these ions trigger phosphate dissociation and subsequent reattachment in the absence of ATP or canonical kinase activity, implying the existence of a novel phosphate-transferring intermediate. If validated, this would represent a radical departure from established models of kinase regulation and signal transduction. I note that this study is personally funded by one of the authors.

    Strengths:

    The study addresses an important and fundamental question in protein phosphorylation biology. The authors have conducted an impressive number of biochemical experiments spanning cellular and in vitro systems, with multiple orthogonal readouts. The idea of an ATP-independent phosphate recycling mechanism is original and thought-provoking, challenging conventional assumptions and inviting further exploration. The manuscript is well organized and written with considerable technical detail.

    Weaknesses:

    The central mechanistic claim contradicts extensive existing evidence on AGC kinase regulation derived from decades of biochemical, mechanistic, pharmacological, genetic, and structural studies. The data, while extensive, do not provide sufficiently direct or quantitative evidence to support the existence of ATP-independent phosphate transfer. Alternative explanations, such as low-level residual ATP-dependent re-phosphorylation or assay artifacts, are not fully excluded. They claim that an unidentified factor-x is involved, but do not provide evidence for the existence of this molecule or characterize this. The physiological relevance of the ion concentrations used is unclear, as the conditions far exceed normal intercellular levels. Overall, the findings are not yet convincing enough to support a paradigm shift in our understanding of AGC kinase activation, in my opinion.

  4. eLife

    Reviewer #3 (Public review):

    This is an intriguing paper that reports a potentially novel mechanism of reversible phosphorylation of AGC kinase activation segments by changes in sodium and potassium ion concentrations. The authors show for a variety of AGC kinases that incubating diverse eukaryotic cell types in 450 and 600 mM NaCl results in dephosphorylation of the activation segment. In contrast, phosphorylation of the activation segment for p38 kinases increases. No dephosphorylation of AGC kinases activation segment occurs with sorbitol, thus dephosphorylation is independent of osmotic pressure. This effect is rapidly reversed when cells are returned to normal media and the AGC kinase is re-phosphorylated. This phenomenon is also observed for eukaryotic cell-free extracts, and is induced by other alkali metal ions but not lithium. Importantly, no dephosphorylation is observed in the E. coli cell extract.

    The authors also make the following observations:

    (1) Dephosphorylation is dependent on PP2A.

    (2) Re-phosphorylation is not dependent on PDK1, ATP, and Mg2+.

    (3) The K/Na-dependent dephosphorylation/phosphorylation is observed even for relatively short protein segments that incorporate the activation segment.

    (4) The phosphorylation observed occurs in cis, i.e., only the activation segment of the protein that is dephosphorylated becomes phosphorylated on reduced KCl. An activation segment from a different length protein is not phosphorylated.

    (5) No evidence for auto(de)phosphorylation.

    (6) The authors propose three models to explain the dephosphorylation/phosphorylation mechanism. Their experimental data suggest that an acceptor molecule is responsible for accepting the phosphate group and then transferring it back to the activation segment.

    Comments on results and experiments:

    (1) Are these results an artefact of their assay? The authors mainly use immunoblotting to assess the phosphorylation status of AGC kinase. However, an assay artefact would not show a difference between control and okadaic-acid-treated cells (Figure 3A). Moreover, the authors show dephosphorylation/phosphorylation using radiolabelling (Figure 6C).

    (2) Preferably, the authors would have a control to test dephosphorylation/phosphorylation does not occur in the absence of cell extract. The E. coli extract shows that dephosphorylation/phosphorylation is specific to eukaryotic cell extracts.

    (3) The authors should show that dephosphorylation/phosphorylation occurs on the same residue of the activation segment (by mass spec).

    (4) Since phosphorylation levels are assessed using immunoblots, the levels of dephosphorylation/phosphorylation are not quantified. What proportion of AGC kinase is phosphorylated initially (before Na/K-induced dephosphorylation)?

    (5) The experiment to test autophosphorylation (Figure 4, Figure supplement 1B) is not completely convincing because the authors use a cell line with a PKN1 mutant knock-in. Possibly PKN2 or another AGC kinase could phosphorylate the proteins expressed from the transfection vector - although the authors do test with AGC kinase inhibitors.

    (6) What are the two bands in Figure 6C (lanes 'Con' and 'diluted)? Only one band disappears with KCl. There is one band in Figure 6 Supplement 2.

    In summary, the results presented in this paper are highly unusual. Generally, the manuscript is well written and the figures are clear. The authors have performed numerous experiments to understand this process. These appear robust, and most of their data lend credence to their model in Figure 6Aiii. The idea that a phosphate group can be transferred by an enzyme onto/between molecule(s) is not unprecedented, i.e., phosphoglycerate mutase catalyses 3-phosphoglycerate isomerisation through a phosphorylenzyme intermediate. It will be important to identify this transfer enzyme. One observation that does not fit easily with their model is the role of PP2A. Since protein dephosphorylation by PP2A does not involve a phosphorylenzyme intermediate, if the initial dephosphorylation reaction is catalysed by PP2A, it is very difficult to envision how the free phosphate is then used to phosphorylate the activation segment.

  5. eLife

    Author response:

    We thank you and the reviewers for the careful assessment and for the thoughtful public reviews of our manuscript. We are encouraged that the novelty of the observations and the systematic nature of our approach are recognised, and we fully appreciate the concerns raised regarding potential artefacts and the incompletely defined mechanism.

    (1) Context for funding (Reviewer #2)

    In response to Reviewer #2’s note that this study is personally funded by one of the authors, we would like to provide some context. When wefirst observed that high-NaCl treatment caused a reversible loss ofactivation-loop phospho-signal for PKN1, we recognised its potential importance and submitted grant applications specifically to investigate this phenomenon. Unfortunately, these applications were not funded. As a result, as Reviewer #2 correctly points out, we have continued this work only modestly, using a personal donation from one of the authors to the university.

    Our initial view that this phenomenon merited detailed study was based mainly on three points:

    (i) Phosphorylation of the activation-loop threonine is critical for the catalytic activity of these kinases.

    (ii) In previous work on PKN, no stress signal had been identified that could induce such a prominent and rapid change in activation-loop threonine phosphorylation.

    (iii) Although the phenomenon was originally detected under high Na⁺ conditions, if it simply reflected the balance between phosphorylation and dephosphorylation, then it seemed plausible that more physiological changes in ion concentrations might drive signals in cells.

    To explore point (iii), we initially attempted to define the ion concentrations that trigger dephosphorylation under conditions where re-phosphorylation was blocked. However, even with potent kinase inhibitors, we were unable to prevent recovery of the phospho-signal.This unexpected result prompted us to investigate the underlying mechanism of this unusual behaviour in more depth.

    (2) Hidden artefacts and mass-spectrometric approaches We fully share the reviewers’ concern expressed as “We remain concerned about hidden artifacts.” Throughout this work, we have repeatedly asked ourselves whether the phenomenon could arise from something as trivial as an artefact inherent to immunoblotting or from an unrecognised flaw in our experimental design, or whether it might ultimately be explainable in terms of conventional rules of protein phosphorylation' and 'dephosphorylation'.

    To capture the phenomenon from an additional, independent angle, we agree with the reviewers’ suggestion to attempt mass spectrometry–based analysis. However, there are several substantial technical hurdles:

    (i) At present, the phenomenon strictly requires the presence of animal cell extracts; we have not been able to reproduce it in their absence.

    (ii) When we attempt to repurify the activation-loop fragments after ion treatment, the phosphate group is re-acquired during the wash steps, even when we use the same high-salt buffer employed for ion treatment.

    (iii) In global phosphoproteomic analyses, reliably detecting a specific change in phosphorylation at a defined site is technically demanding and costly.

    We therefore hope to identify conditions under which we can both (a)preserve the phosphorylation state established by the ion treatmentduring sample handling, and (b) achieve sufficient purification for informative mass spectrometric analysis. Reviewer #3 raised an important question regarding the origin of the two bands observed in Figure 6C. At present, we do not have data that would allow us to address this point in a well-founded manner. We hope that successful mass spectrometric analysis will also enable us to comment more concretely on this issue.

    (3) Role of PP2A and reconstitution experimentsAs emphasised by Reviewers #1 and #3, although PP2A appears to beessential for the phenomenon, we have not yet been able to formulate a mechanistically plausible model that incorporates PP2A in a satisfactory way, and we share the reviewers’ concern on this point. We performed preliminary in vitro reconstitution experiments using recombinant PP2A purified from Sf9 cells (comprising the catalytic C subunit, the scaffold A subunit, and GST-fused PR130 as a B subunit) together with purified PKN1 activation loop fragments, to test whether the phenomenon can be reconstituted under low- and high-KCl conditions. Under the conditions tested so far, we have not yet succeeded in reconstituting the salt-dependent loss and recovery of activation loop phosphorylation. In vivo, PP2A holoenzymes exhibit substantial diversity in their subunit composition, particularly in the B subunit, and it is therefore unclear whether the particular complex we used is the one responsible for the behaviour observed in lysates. We plan to test additional PP2A complexes and, in parallel, to examine the effect of adding bacterial cell extracts—which by themselves do not induce changes in activation-loop phosphorylation in our system—in order to determine whether additional eukaryotic factors are required for reconstitution.

    Through these experiments, we hope to move closer to constructing amechanistic scheme that explicitly includes PP2A and clarifies its role in this unusual process of phosphate loss and reacquisition.

    We are grateful for the constructive feedback and believe these planned revisions will strengthen the clarity, balance, and rigour of our study.

  6. Version published to 10.7554/elife.109457.1 on eLife
  7. Version published to 10.7554/elife.109457 on eLife
  8. Version published to 10.1101/2025.09.04.674365 on bioRxiv