Nuclear translocation of the tagged endogenous MAPK MPK-1 denotes a subset of activation events in C. elegans development

ABSTRACT The extracellular signal-regulated kinases (ERKs) are mitogen-activated protein kinases (MAPKs) that are utilized downstream of Ras to Raf to MEK signaling to control activation of a wide array of targets. Activation of ERKs is elevated in Ras-driven tumors and RASopathies, and thus is a target for pharmacological inhibition. Regulatory mechanisms of ERK activation have been studied extensively in vitro and in cultured cells, but little in living animals. In this study, we tagged the Caenorhabditis elegans ERK-encoding gene, mpk-1. MPK-1 is ubiquitously expressed with elevated expression in certain contexts. We detected cytosol-to-nuclear translocation of MPK-1 in maturing oocytes and hence validated nuclear translocation as a reporter of some activation events. During patterning of vulval precursor cells (VPCs), MPK-1 is necessary and sufficient for the central cell, P6.p, to assume the primary fate. Yet MPK-1 translocates to the nuclei of all six VPCs in a temporal and concentration gradient centered on P6.p. This observation contrasts with previous results using the ERK nuclear kinase translocation reporter of substrate activation, raising questions about mechanisms and indicators of MPK-1 activation. This system and reagent promise to provide critical insights into the regulation of MPK-1 activation within a complex intercellular signaling network.

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Advance summary and potential significance to field
This manuscript reported the nucleus localization of endogenous ERK/MPK-1 MAP Kinase during C. elegans development. The authors used mKate2-3×Flag as a fluorescent and biochemical tag to label endogenous MPK-1 by CRISPR-Cas9 based genome editing and verified that the fusion protein works well by biochemistry and imaging, demonstrating its functionality. They further examined the localization of mKate2-3×Flag tagged endogenous MPK-1 during maturing oocytes and validate its nuclear translocation as a reporter of certain activation events. They also showed that mKate2-3×Flag tagged endogenous MPK-1 is localized in the nucleus of all the six VPCs. This finding is interesting and appears to be different from the previous results using other ERK-nKTR reporter systems. The study provides a useful tool for the community.

Comments for the author
While the localization of MPK-1 is interesting, the current work does not provide a necessary advance for publication in the JCS, which requires mechanistic insights to a certain level. In other words, a simple description of a kinase localization, though valuable, is probably insufficient.
Other major issues: 1. It remains unclear whether mKate-tag works well for tagging each protein functionally. Because of the apparent discrepancy from the previous results, the major conclusion of MPK-1 localization should be backed up by alternative approaches, including immunofluorescence, or GFP-tagging.
2. They should study mKate2-3×Flag tagged endogenous MPK-1 in VPCs localization in the mutants of Ras>Raf>MEK>ERK cascade and LIN-12/Notch signaling pathway which may provide some mechanistic insights into the function or nuclear location of MPK-1.

Reviewer 2
Advance summary and potential significance to field This paper reports what promises to be a highly useful reagent for studying endogenous MAPK signaling in C. elegans. The marker, a knocked in tagged MAPK protein, is validated in not only oogenesis but also the well -studied vulval development. Progress in the analysis of this paradigmatic case of developmental signaling and signal transduction has lagged in recent years due to lack of tools; this new reagent may well juice up the field!

Comments for the author
The methods are clearly explained, and as far as I can tell, all the reagents are clearly defined. The logic is clear and figures are well presented. The results with this reagent differ somewhat, as clearly explained in the text, from previous reagents. The questions raised by the discrepancy seem interesting, and I have no reason to doubt these current results. While there are many possible experiments that could probe the system further, I think they are beyone the scope of this already information dense, paper. I thus recommend publication as is.

First revision
Author response to reviewers' comments Point-by-point response to reviewer comments. Text and table additions in the manuscript are marked in green. Responses to reviewers is written in blue, below. We also took the opportunity to clean up consistency of nomenclature of CRISPR-based tags in the manuscript.
Reviewer #1: While the localization of MPK-1 is interesting, the current work does not provide a necessary advance for publication in the JCS, which requires mechanistic insights to a certain level. In other words, a simple description of a kinase localization, though valuable, is probably insufficient.
We ask that the reviewer consider that we do present a mechanistic insight of a technological nature: the nKTR reporter of phosphorylation substrate can yield a markedly different answer in live animals than does nuclear translocation of endogenous protein. Not only does this observation, like most good science, raise more questions than it answers, it holds methodological importance for the target audience interested in questions of cell signaling.
It remains unclear whether mKate-tag works well for tagging each protein functionally. Because of the apparent discrepancy from the previous results, the major conclusion of MPK-1 localization should be backed up by alternative approaches, including immunofluorescence, or GFP-tagging. This is an excellent point raised by the reviewer! We have quantified, using animals harboring tagged MPK-1, completion of two developmental events governed by MPK-1: induction of the excretory duct cell, whose absence confers rod-like larval lethality at the L1 and L2 stages, and Vulvaless (Vul) or Multivulva (Muv) phenotypes, caused by under-or over-induction during the VPC patterning process. In this we followed the lead of a fine recent study by Gauthier and Rocheleau, Development (2021). Using these assays, the authors found that the re202 tag of endogenous LET-23/EGFR, provided to them by us, caused low-penetrance Vul and Muv phenotypes, consistent with mild reduction of function. The same study found that tags of LIN-2, LIN-7, and LIN-10 did not detectably perturb function. Our own study (Shin et al. Cell Reports, 2018) similarly evaluated functions of tagged RAL-1/Ral, GCK-2/MAP4K and PMK-1/p38 MAP kinase, finding that none detectably changed function. These data for MPK-1::mKate2 were added as Supplementary Tables 5&6, and are supported by a new paragraph with callouts in the first section of the Results. Thus, we conclude that the tagged MPK-1 used in this study is functional.
They should study mKate2-3×Flag tagged endogenous MPK-1 in VPCs localization in the mutants of Ras>Raf>MEK>ERK cascade and LIN-12/Notch signaling pathway, which may provide some mechanistic insights into the function or nuclear location of MPK-1.
We very much agree that the next step is to compare and contrast MPK-1::mKate2 and ERK-nKTR reporters in developing animals in response to genetic manipulations of the signaling system in VPC patterning. Unfortunately, we think that such an undertaking, with extensive spinning disk filming and image analysis, would be extremely demanding and comprise an independent paper. Reviewer #2 appears to agree with this assessment: "While there are many possible experiments that could probe the system further, I think they are beyone <sic> the scope of this, already information dense, paper. I thus recommend publication as is."