Abstract
Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITC-induced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.
Highlights
Proteins with Cdk[5] sites located within extracellular or transmembrane domains were excluded as we focused on potential phosphorylation sites that are accessible to either membrane bound Cyclin-dependent kinase 5 (Cdk5)/p35 or cytoplasmic Cdk5/p25
We focused on Transient Receptor Potential Ankyrin 1 (TRPA1), based on several reasons: First, we saw that TRPA1 has several putative Cdk[5] sites located within ankyrin repeat domains at the N-terminal domain
We further investigated the extent to which Cdk[5] phosphorylation could be a multifactorial regulator of nociceptive inputs by trying to identify new Cdk[5] substrates that are highly expressed in nociceptive afferents, the TRPV1+ set of ganglionic neurons which are critical for the detection of many painful modalities, especially heat[50]
Summary
The N-terminal domain of TRPA1 contains a total of 17 ankyrin repeats that are thought to confer sensitivity to chemical irritants[32] and are believed to be essential for plasma membrane localization[33]. Mice with increased Cdk[5] activity had increased aversion to consumption of AITC, while the converse was true for mice with decreased Cdk[5] activity These data are consistent with the idea that Cdk[5] can modulate noci-responsive ion channel activity of primary afferent neurons through post-translational phosphorylation with subsequent change in the sensitivity of the nociceptive neurons to multiple pain modalities
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