TRPV1 is a substrate for Cdk5 phosphorylation. (A) Schematic representation of the TRPV1 receptor, showing three putative Cdk5 phosphorylation sites (threonine-108, threonine-407, and serine-612) highlighted in green. Protein kinase A (PKA), protein kinase C (PKC), or calcium/calmodulin kinase II phosphorylate overlapping serine (S) or threonine (T) residues are highlighted in purple. ANK, ankyrin repeats. (B) Alignment of human, dog, mouse, and rat TRPV1 amino acid sequences. Green sequences show ankyrin repeats, blue sequences show the six transmembrane domains, and red sequences show the pore loop. Purple sequences show the three putative Cdk5 phosphorylation sites where the threonine-407 is conserved between the species shown. ∗, all residues are identical; :, conserved substitution; ., semiconserved substitution. (C) TRPV1 was immunoprecipitated (IP) from DRG, NIH 3T3+TRPV1 cells, and DRG culture cell lysates and subjected to Western blotting using TRPV1 antibody. TRPV1 was immunoprecipitated from these lysates, as confirmed by immunodepletion experiments performed by using a TRPV1-specific peptide for the pull-down assay as shown on the right side. (D) TRPV1 protein immunoprecipitated from NIH 3T3+TRPV1 and DRG cultures was used as a substrate for the Cdk5 kinase assay. Cdk5 was immunoprecipitated from WT and Cdk5-knockout (KO) brain lysates by using Cdk5 antibody. Autoradiographs are shown in Upper, and corresponding Western blots are shown in Lower. Phospho-TRPV1 is clearly seen in WT but is absent in Cdk5 knockout lanes. (E and F) To confirm the specificity of C-8 immunoprecipitates from Cdk5 WT and KO lysates, histone H1 (E) and NF-H peptide (F) were used as substrates in kinase assays. Phosphorylation of the substrates was observed in WT but not KO brain samples. (E) Shown are an autoradiograph (Upper) and the corresponding Coomassie blue-stained gel (Lower). (F) Data are the mean ± SEM (n = 3). ∗∗, P < 0.01, Student's t test. (G) Peptides (peptide 1, GVETPPRLY for threonine-108; peptide 2, SSETPNRH for threonine-407; and peptide 3, VESPPHK for serine-612) of the three putative Cdk5 phosphorylation sites and their corresponding nonphosphorylated mutants were synthesized. Cdk5 was immunoprecipitated from WT and Cdk5-knockout brain lysates and used as the kinase source in kinase assays. Peptide 2, corresponding to threonine-407, exhibited strong phosphorylation; peptide 1 (threonine-108) exhibited the second-strongest phosphorylation. Peptide 3 (serine-612) did not exhibit any significant phosphorylation. All data are the mean ± SEM (n = 3). ∗∗, P < 0.01; NS, not significant, Student's t test.

Inhibition of Cdk5 activity blocks TRPV1-mediated calcium influx in DRG cultured neurons. DRG neurons were treated with 2 μM capsaicin for 10 min in the presence of ⁴⁵Ca²⁺, with (experimental) or without (control) 2 h of roscovitine before treatment. Cells were washed and lysed to detect the amount of ⁴⁵Ca²⁺ uptake. Control values were considered as 100%, and experimental values were expressed as percentage of control values. Each treatment was performed six times. Inhibition of Cdk5 activity by roscovitine at 6.25–100 μM resulted in a decrease in capsaicin-mediated ⁴⁵Ca²⁺ uptake in DRG neurons. DRG cells that were recovered for 24 h before assay were still viable and responsive to capsaicin treatment. To ensure specificity of the roscovitine effect, NIH 3T3+TRPV1 cells (lacking Cdk5 activity) stably transfected with TRPV1 were treated in an identical manner. Capsaicin-mediated ⁴⁵Ca²⁺ uptake was not affected by the roscovitine treatment in these cells at these concentrations. Data are the mean ± SEM. ∗∗, P < 0.01, one-way ANOVA.

Conditional deletion of Cdk5 in pain-sensing neurons abrogated TRPV1 phosphorylation and induced hypoalgesia. Cdk5-flox mice were crossed with SNS-Cre mice to generate Cdk5-CoKO mice. (A) Genotyping results for Cdk5-CoKO mice as confirmed by PCR for Cdk5-flox and SNS-Cre. (B and C) DRG and trigeminal ganglia tissue lysates were prepared from Cdk5-CoKO and respective wild-type mice and analyzed by Western blotting to check Cdk5 protein levels and Cdk5-mediated TRPV1 phosphorylation. (B) A significant reduction in Cdk5 protein levels is evident in Cdk5-CoKO. Cdk5-knockout brain lysates were run together as an internal control. (C) Specificity of pTRPV1 threonine-407 antibody was confirmed by performing competitive Western blotting with or without phosphopeptide and non-phosphopeptide (0.2 μg of peptide per μg of antibody). (D) Abrogation of TRPV1 threonine-407 phosphorylation in Cdk5-CoKO mice. (E and F) Cdk5-CoKO and respective control mice were subjected to the Hargreaves test (E) and the tail-withdrawal test (F). In all groups, a total of 10 mice were used for each test. Cdk5-CoKO mice exhibited a delayed response against noxious thermal stimulation compared with their controls. Data are mean ± SEM (n = 8–10). ∗∗, P ≤ 0.01, Student's t test.