Nociception is a conserved process serving as a self-protection system alerting animals of potential damage and underpinning different forms of pain in humans. Some chronic pain conditions may arise from maladaptive modulation in the nociceptive pathway, including within nociceptors, the primary nociceptive sensory neurons. We use Caenorhabditis elegans as a model due to its ability to detect noxious stimuli, perform avoidance behaviors in the form of stimulus-evoked reversals and adapt to repeated stimuli causing a desensitized, analgesia-like state. The worm ortholog of mammalian CaMKI/IV, CMK-1 (calcium/calmodulin-dependent kinase-1) mediates cellular responses to increased calcium levels and is crucial in nociceptors for this avoidance behavior plasticity. However, the downstream elements of the CMK-1 pathway remain unclear. Here, we performed in vitro CMK-1 kinase assays on both peptide and protein from total worm isolates in order to identify direct kinase target candidates via shotgun phosphoproteomics. For in vivo direct/indirect target determination, we carried out stable isotope labeling by amino acids (SILAC) using high-throughput quantitative phosphoproteomics. We used duplex SILAC, where ''light'' 12-carbon and ''heavy'' 13-carbon amino acids were incorporated into nematode proteins for measuring the amounts of phosphorylated proteins in wild type and
cmk-1 null animals. By combining results obtained from these different studies, we were able to ascertain CMK-1 phosphorylation consensus and develop a list of potential CMK-1 targets. Mutants for these candidates were then tested for heat avoidance behavior to determine changes in naive sensitivity to noxious heat and/or adaptation. We used a computer-assisted high-throughput analysis pipeline to quantify heat-evoked reversals in naive animals and animals exposed to repeated stimuli. While wild type animal sensitivity decreased in response to the repeated heat stimuli, some mutant animals failed to adapt. In conclusion, our study reveals several potential CMK-1 targets that may have an important role in behavioral plasticity.