Regulation of ERK2 activity by dynamic S-acylation
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Evaluation Summary:
This manuscript describes the regulation of ERK1/2, two protein kinases that play important roles in cell signaling, by protein cysteine palmitoylation. The intriguing observations reported here could be of broad interest to colleagues in the field of cell signaling and protein post-translational modifications. Mechanistic understanding is, however, still limited and the work would benefit from additional experimental evidence.
(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 agreed to share their name with the authors.)
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Evaluation Summary:
This manuscript describes the regulation of ERK1/2, two protein kinases that play important roles in cell signaling, by protein cysteine palmitoylation. The intriguing observations reported here could be of broad interest to colleagues in the field of cell signaling and protein post-translational modifications. Mechanistic understanding is, however, still limited and the work would benefit from additional experimental evidence.
(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 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
ERK1/2 are two important MAP kinases that play important roles in regulating cell growth, proliferation, and stress responses. Their activities are well known to be regulated by phosphorylation on the activation loops. Here the authors report that ERK1/2 are also regulated by cysteine palmitoylation. This is a new finding and could potentially lead to new ways to control ERK1/2 signaling as new anticancer strategies.
The strengths of the work include: (1) convincing demonstration that ERK1/2 contain cysteine palmitoylation by several complementary methods; (2) identification of the sites of palmitoylation by mutagenesis; (3) showing that cysteine palmitoylation is dynamic and varies with EGF stimulation; (4) showing that the C254A mutation (which lacks palmitoylation on Cys254) in ERK2 promotes pERK2 …
Reviewer #1 (Public Review):
ERK1/2 are two important MAP kinases that play important roles in regulating cell growth, proliferation, and stress responses. Their activities are well known to be regulated by phosphorylation on the activation loops. Here the authors report that ERK1/2 are also regulated by cysteine palmitoylation. This is a new finding and could potentially lead to new ways to control ERK1/2 signaling as new anticancer strategies.
The strengths of the work include: (1) convincing demonstration that ERK1/2 contain cysteine palmitoylation by several complementary methods; (2) identification of the sites of palmitoylation by mutagenesis; (3) showing that cysteine palmitoylation is dynamic and varies with EGF stimulation; (4) showing that the C254A mutation (which lacks palmitoylation on Cys254) in ERK2 promotes pERK2 (Thr185/Tyr187) but inhibits serine phosphorylation of ERK2; (5) Finding that several DHHC proteins as potential writers for this modification; (6) Showing that palmitoylation of ERK is regulated by high-fat diet in vivo in different tissues. These observations are interesting and further understanding of them could provide useful insights to the field.
A major weakness of the work is that even though a lot of interesting observations are made, there is not much mechanistic understanding about these observations. The lack of understanding makes reading the manuscript a little difficult, especially that some observations are contrary to each other. For example, even though the small molecule inhibitor data suggest that APT1 and APT2 are the enzymes that remove the palmitoylation, knockdown of these two enzymes failed to produce the expected effect. In particular, how the palmitoylation on Cys254 affects the two types of phosphorylation differently and how in turn that causes changes in the downstream signaling effects, which DHHCs are the physiological writers and why so many DHHCs can work on ERK1/2 under over-expression conditions. Addressing these questions will significantly enhance the manuscript.
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Reviewer #2 (Public Review):
Azizi et al. examined the regulation of signaling by ERK1/2 via palmitoylation, a post-translational lipid modification by employing a series of cell-based studies and biochemical and molecular studies of ERK1/2 palmitoylation, activation, and downstream gene expression. The authors also investigated ERK1/2 palmitoylation in a mouse model of metabolic syndrome. The authors found that ERK1/2 palmitoylation is induced in response to stimulation with EGF (that activates ERK1/2 downstream of EGFR) and attempted to uncover the kinetics and enzymatic regulation of ERK1/2 palmitoylation by writers (DHHC acyltransferases) and erasers (thioesterases/depalmitoylases). Overall, the data presented clearly establish palmitoylation as a novel regulatory mechanism governing ERK1/2 signaling activity, which is a significant …
Reviewer #2 (Public Review):
Azizi et al. examined the regulation of signaling by ERK1/2 via palmitoylation, a post-translational lipid modification by employing a series of cell-based studies and biochemical and molecular studies of ERK1/2 palmitoylation, activation, and downstream gene expression. The authors also investigated ERK1/2 palmitoylation in a mouse model of metabolic syndrome. The authors found that ERK1/2 palmitoylation is induced in response to stimulation with EGF (that activates ERK1/2 downstream of EGFR) and attempted to uncover the kinetics and enzymatic regulation of ERK1/2 palmitoylation by writers (DHHC acyltransferases) and erasers (thioesterases/depalmitoylases). Overall, the data presented clearly establish palmitoylation as a novel regulatory mechanism governing ERK1/2 signaling activity, which is a significant finding. The precise mechanism by which palmitoylation modulates ERK1/2 activity (i.e. opposing effects on TEY vs. Ser phosphorylation; impact on ERK1/2 localization, stability, etc) and writer/eraser enzymes that definitively control this process are less clear but confounded by the presence of multiple palmtioylation sites on ERK1/2 that may have different and even opposing effects on ERK function and the presence many DHHC writers with different cell-type expression patterns and intracellular membrane localization that could manipulate ERK activity at disparate cellular locations. A major strength of the manuscript is the use of several techniques to decode the enzymatic regulation of ERK2 palmitoylation including metabolic labeling/click chemistry and acyl biotin exchange (ABE) to detect palmitoylation, immunoprecipitation and proximity labeling (TurboID) to assess association of ERK2 with palmitoylases/depalmitoylases, and assessment of the impact of these enzymes on ERK1/2 phosphorylation. Overall, this manuscript provides novel insight on regulation of ERK1/2 by palmitoylation that could be of significance for many diseases including cancer and metabolic syndrome and provides a framework for elucidation of enzymatic regulation of dynamically palmitoylated signaling proteins.
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Reviewer #3 (Public Review):
Azizi and coworkers report an expansive investigation of protein palmitoylation of ERK2 and resulting impact of cell signaling pathways impacted by this kinase. Using several chemical biology approaches, the authors determine the time- and external stimulus-dependence of ERK2 acylation in a number of cell lines. Mutagenesis studies are employed to probe the interdependence of ERK2 acylation and phosphorylation, providing a functional linkage for protein palmitoylation impact on downstream signaling. Pharmacological perturbation of ERK2 palmitoylation is shown to impact gene transcription controlled by ERK2-dependent pathways. The authors attribute ERK2 acylation to a subset of DHHC PATs and deacylation to APT2 based on overexpression studies and detection of protein-protein interactions in cell-based …
Reviewer #3 (Public Review):
Azizi and coworkers report an expansive investigation of protein palmitoylation of ERK2 and resulting impact of cell signaling pathways impacted by this kinase. Using several chemical biology approaches, the authors determine the time- and external stimulus-dependence of ERK2 acylation in a number of cell lines. Mutagenesis studies are employed to probe the interdependence of ERK2 acylation and phosphorylation, providing a functional linkage for protein palmitoylation impact on downstream signaling. Pharmacological perturbation of ERK2 palmitoylation is shown to impact gene transcription controlled by ERK2-dependent pathways. The authors attribute ERK2 acylation to a subset of DHHC PATs and deacylation to APT2 based on overexpression studies and detection of protein-protein interactions in cell-based experiments. Finally, the authors demonstrate ERK1/2 acylation is affected by the cellular and organismal metabolic state. This work illuminates a novel regulatory mechanism for ERK1/2 and supports interplay of complementary posttranslational modifications controlling kinase signaling through this protein.
Overall, the data presented in the paper strongly support the conclusions of the authors with exception of the APT-related experiments. The studies of APT inhibition and associated impact of ERK1/2 acylation and signaling are somewhat problematic. Contrary to the authors assertions in the text, the data presented in Figure 4 and Figure S4C do not compellingly support an increase in ERK1/2 palmitoylation when cells are treated with APT inhibitors. In the absence of further quantitation, the data are not sufficient to indicate APT inhibition increases ERK1/2 palmitoylation. This concern is compounded by the lack of any change in ERK1/2 acylation in presence of APT overexpression or siRNA knockdown. The protein-protein interaction of ERK2 with APT2 demonstrated by biotin labeling does not provide sufficient foundation for annotating APT2 as the eraser for ERK1/2 palmitoylation based on the totality of data presented. Given the promiscuous nature of PalmB, it's also unclear the perturbations in downstream gene transcription in Figure 4C can be directly attributed to changes in ERK1/2 palmitoylation. Additional experiments would be needed to support the proposal of APT2 regulation of ERK1/2 palmitoylation.
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