Multiple antagonist calcium-dependent mechanisms control CaM kinase-1 subcellular localization in a C. elegans thermal nociceptor
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eLife assessment
This valuable study provides solid results on the molecular signaling mechanisms of CaM kinase kinase-1 (CKK-1) in the context of the nociceptive behaviors of C. elegans. The authors report previously undescribed elements that control the nuclear/cytoplasmic shuttling of CKK-1, suggesting a complex interplay of multiple nuclear localization and export sequences. Therefore, the work will be of broad interest to scientists studying behavior, neuronal signaling, and signal transduction in general.
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Abstract
Nociceptive habituation is a conserved process through which pain sensitivity threshold is adjusted based on past sensory experience and which may be dysregulated in human chronic pain conditions. Noxious heat habituation in Caenorhabditis elegans involves the nuclear translocation of CaM kinase-1 (CMK-1) in the FLP thermo-nociceptors neurons, causing reduced animal heat sensitivity and avoidance responses. The phosphorylation of CMK-1 on T179 by CaM kinase kinase-1 (CKK-1) is required for nuclear entry. Recently, we identified a specific nuclear export sequence (NES) required to maintain CMK-1 in the cytoplasm at rest (20°C) and showed that Ca 2+ /CaM binding is sufficient to enhance CMK-1 affinity for IMA-3 via a specific nuclear localization signal (NLS) in order to promote nuclear entry after persistent heat stimulation (90 min at 28°C) (Ippolito et al., 2021). Here, we identified additional functional NES and NLS on CMK-1, whose activity can counteract previously identified elements. Furthermore, we clarify the relationship between the CaM-binding-dependent and T179-dependent effects. T179 phosphorylation can promote nuclear entry both downstream of CaM binding and as part of an independent/parallel pathway. Moreover, T179 phosphorylation can also produce the opposite effect by promoting nuclear export. Taken together, our studies suggest that multiple calcium-dependent regulatory mechanisms converge to bias the activity pattern across a network of NES/NLS elements, in order to control CMK-1 nucleo-cytoplasmic shuttling, and actuate stimulation-dependent nociceptive plasticity.
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eLife assessment
This valuable study provides solid results on the molecular signaling mechanisms of CaM kinase kinase-1 (CKK-1) in the context of the nociceptive behaviors of C. elegans. The authors report previously undescribed elements that control the nuclear/cytoplasmic shuttling of CKK-1, suggesting a complex interplay of multiple nuclear localization and export sequences. Therefore, the work will be of broad interest to scientists studying behavior, neuronal signaling, and signal transduction in general.
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Reviewer #1 (Public Review):
Habituation to noxious insults is a conserved mechanism that may act through varying pain-sensitivity thresholds based on previous sensory experience. Impaired regulation of nociceptive habituation may lead to a chronic pain condition. In the current manuscript, the authors identified additional structural elements of the CaM kinase-1 that regulate the protein shuttling between the cytosol and nucleus during nociceptive habituation. Based on the presented findings, we get a more complex regulatory model and a better understanding of the CMK-1 protein redistribution during stimulation-dependent nociceptive plasticity.
The data is carefully planned and results conclusively support the claims of the authors. The performed experiments are easy to follow and the results obtained are robust and statistically …
Reviewer #1 (Public Review):
Habituation to noxious insults is a conserved mechanism that may act through varying pain-sensitivity thresholds based on previous sensory experience. Impaired regulation of nociceptive habituation may lead to a chronic pain condition. In the current manuscript, the authors identified additional structural elements of the CaM kinase-1 that regulate the protein shuttling between the cytosol and nucleus during nociceptive habituation. Based on the presented findings, we get a more complex regulatory model and a better understanding of the CMK-1 protein redistribution during stimulation-dependent nociceptive plasticity.
The data is carefully planned and results conclusively support the claims of the authors. The performed experiments are easy to follow and the results obtained are robust and statistically well-powered. The complex regulatory model presented in the manuscript is well supported by the reported data. Finally, the presented data presents a complex and dynamic mechanism of nuclear import and export rates of the CMK-1 protein to control nociceptive plasticity.
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Reviewer #2 (Public Review):
In this study, Ippolito and colleagues elucidated the molecular mechanism of CMK-1 shuttling between the nucleus and cytoplasm and its function in the context of regulated thermosensation in C. elegans. This study is built on their previous work that identified a specific Nuclear Export Sequence (NES) required for CMK-1 cytoplasmic localization at 20{degree sign}C, and a specific Nuclear Localization Signal (NLS) to promote prolonged heat (28{degree sign}C)-induced CMK-1 nuclear entry. Here they show additional functional NES and NLS which counteract previously identified elements: the NLS297-307-dependent nuclear entry pathway and the S325-dependent cytoplasmic accumulation. Combined with their previous study, their work suggests a model: upon prolonged FLP neuron stimulation by noxious heat, CaM binding to …
Reviewer #2 (Public Review):
In this study, Ippolito and colleagues elucidated the molecular mechanism of CMK-1 shuttling between the nucleus and cytoplasm and its function in the context of regulated thermosensation in C. elegans. This study is built on their previous work that identified a specific Nuclear Export Sequence (NES) required for CMK-1 cytoplasmic localization at 20{degree sign}C, and a specific Nuclear Localization Signal (NLS) to promote prolonged heat (28{degree sign}C)-induced CMK-1 nuclear entry. Here they show additional functional NES and NLS which counteract previously identified elements: the NLS297-307-dependent nuclear entry pathway and the S325-dependent cytoplasmic accumulation. Combined with their previous study, their work suggests a model: upon prolonged FLP neuron stimulation by noxious heat, CaM binding to CMK-1 causes CKK-1-dependent phosphorylation of T179, which in turn has a context-dependent dual effect: it is sufficient for nuclear translocation at 20{degree sign}C in an NLS71-78-dependent manner, and it promotes NES288-294-dependent nuclear export at 28{degree sign}C.
The authors thereby established a direct link between the state of a signal transduction pathway and FLP neuronal activity in response to heat stimulation. They used multiple approaches, including transgenics and reporter quantification analysis to characterize CMK-1 nucleo-cytoplasmic dynamic equilibrium. The experiments are well-designed with appropriate controls and appropriate sample sizes. The data analysis is comprehensive and revealing. The findings expand the functionally relevant intrinsic CMK-1 subcellular localization determinants. The new understanding generated in this study will appeal to readers in the fields of cell biology, signal transduction, and physiology.
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