Identification of CMK-1 phosphorylation substrates

A. Schematic of the two approaches used for the large-scale identification of the CMK-1 phosphorlyation substrates in vitro. In vitro kinase assays using a C. elegans peptide library (left) or a C. elegans native protein library (right) were followed by MS-based phosphoproteomics analyses. The activity of a constitutively active mutant (CMK-1(1-295)T179D) was compared to control situations with either kinase dead mutant (CMK-1(1-295)K52A) or without ATP co-substrate during the incubation. B. Comparison of the results with the two approaches, showing the number of overlapping and non-overlapping phosphosites, the number of corresponding proteins, as well as the 15-residue consensus sequence surrounding the phosphosites. Logos created with Seq2Logo. C. Results of a separate in vitro kinase assay in which purified TAX-6/CnA was used as substrate and showing TAX-6/CnA S443 phosphorylation. N.D., not detected D. Diagram presenting the different regions of the C. elegans TAX6/CnA protein and the localization of S443. E. Alignments showing high conservation of a CnA protein region around S443 across C. elegans, Danio rerio, Mus Musculus and Homo sapiens (Uniprot assessions: Q0G819-2|PP2B_CAEEL; A3KGZ6|A3KGZ6_DANRE; P63328|PP2BA_MOUSE; Q08209|PP2BA_HUMAN).

Impact of loss and gain of CMK-1 and TAX-6/CnA function on C. elegans thermo-nociceptive response

A. Schematic of the scoring procedure. Heat-evoked reversals were first scored in naïve adult C. elegans that had never been exposed to thermal stimuli (T0), animals exposed to 4 s heat pulses every 20 s during 60 min, prior to an endpoint scoring after habituation (T60). B-D. Heat-evoked reversal scored in the indicated genotypes. Results as fraction of reversing animals. Each point corresponds to one assay scoring at least 50 animals. Average (grey bars) and s.e.m (error bars) with indicated n representing the number of independent assays. cmk-1(gf) is cmk-1(syb1633). cmk-1(lf) is cmk-1(ok287). tax-6(gf) is tax-(j107). For Calcineurin inhibition, 10 µM Cyclosporin A was used 24 prior to experiments. **, p<.01 and *, p<.05 versus N2(WT) control in the specific condition by Bonferroni-Holm post-hocs tests. ns, not significant.

Functional interactions between CMK-1 and TAX-6/CnA in the regulation of thermo-nociceptive habituation

A-C. Assessment of the impact of joint gain- and loss-of-function manipulations affecting the CMK-1 and TAX-6/CnA pathways. Heat-evoked response in naïve animals (T0) and after 60 min of repeated stimulations (T60), scored and reported as in Fig. 2. **, p<.01 and *, p<.05 versus N2(WT) control in the specific condition by Bonferroni-Holm post-hocs tests. ns, not significant. D. Schematic of a model explaining the multiple antagonistic interactions observed between CMK-1 and TAX-6/CnA signaling.

CMK-1 works in AFD and ASER to control thermo-nociceptive habituation

A-B. Determination of the CMK-1 place of action in the control of thermo-nociceptive habituation, using cell-specific rescue in cmk-1(ok287) (cmk-1(lf)) background. Heat-evoked response in naïve animals (T0) and after 60 min of repeated stimulations (T60), scored and reported as in Fig. 2. The promoters used to restore CMK-1 expression are indicated below each bar. **, p<.01 and *, p<.05 versus cmk-1(lf) by Bonferroni-Holm post-hocs tests. ns, not significant. ##, p<.01 for the specific contrast between ttx-1p and the gcy-5p;ttx-1p combination. N2(WT) data are shown for comparison purpose. C. Impact of genetic manipulation ablating AFD with a caspase construct (AFD(-)) or inhibiting AFD neurotransmission with TeTx heterologous expression. **, p<.01 and *, p<.05 versus N2(WT) control by Bonferroni-Holm post-hocs tests D. Schematic of the hypothetical circuit controlling noxious-heat evoked reversals, including the tax-4-expressing thermo-responsive sensory neurons AFD, AWC, ASER and ASI, the mec-3-expressing FLP thermo-nociceptor, and a subset of downstream interneurons known to mediate reversal response, including the glr-1-expressing RIM, AVA, AVD, and AVE interneurons. The main loci of action of CMK-1 determined through the cell-specific rescue approach are highlighted in blue.

TAX-6/CnA activity in RIM and AVA/AVD/AVE inhibit thermo-nociceptive habituation

A-B. Determination of the TAX-6/CnA place of action in the control of thermo-nociceptive responses, using cell-specific expression of a TAX-6/CnA gain-of-function mutant. Heat-evoked response in naïve animals (T0) and after 60 min of repeated stimulations (T60), scored and reported as in Fig. 2. The promoters used to drive tax-6(gf) cDNA expression are indicated below each bar. **, p<.01 and *, p<.05 versus N2(WT) by Bonferroni-Holm post-hocs tests. ns, not significant. tax-6(gf) mutant data are shown for comparison purpose. C. Schematics of the hypothetical circuit controlling noxious-heat evoked reversals as in Fig. 4D, with the main loci of action of TAX-6/CnA highlighted in red. TAX-6/CnA-evoked up-regulation of thermo-nociceptive response in naïve animals (top) and TAX-6/CnA-evoked inhibition of habituation (bottom).

Comparison of empirically identified CMK-1 phospho-substrates with previously published predictions

Venn diagram showing the number of phosphosites from the indicated lists and the consensus as in Fig. 1.

CMK-1 overactivating mutation T179D accelerates thermo-nociceptive habituation

A. Schematic of the scoring procedure variation, including earlier scoring timepoints after 10 and 30 min of repeated noxious heat stimuli. B. Heat-evoked reversal scored in the indicated genotypes and reported as in Fig. 2. cmk-1(gf) is cmk-1(syb1633) harboring the CMK-1(T179D) overactivating mutation. *, p<.01 versus corresponding timepoint in N2(WT) control.

Model of the multiple antagonistic interactions observed between CMK-1 and TAX-6/CnA signaling.

Illustration of how the model from Fig. 3D is proposed to be altered for each of the single or combined gain- and loss-of-function manipulations used in Fig. 3. We do not rule out that alternative models might also explain our data, but the model presented in Fig. 3D is the simplest that could explain the complex set of interactions.