Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

Abstract

The purinergic P2X2 receptor (P2X2R) is an ATP-gated ion channel widely expressed in the nervous system. Here, we identified a putative Cdk5 phosphorylation site in the full-size variant P2X2aR (372TPKH375), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine-phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5-activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells, and P2X2/3R mediated increases of intracellular Ca2+ in trigeminal neurons were Cdk5-dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.

TNF-α Increases Production of Reactive Oxygen Species through Cdk5 Activation in Nociceptive Neurons

“…HEK293 cells (ATCC#CRL-1573) were grown in Dulbecco Modified Eagle Medium (DMEM) containing 10% of fetal bovine serum (FBS) and penicillin/streptomycin (100 mg/mL) (Invitrogen, Carlsbad CA). HEK293 cells were transiently co-transfected with mouse CMV-p35 (Coddou et al, 2017 ) and HyPer constructs (Evrogen, Moscow, Russia) by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad CA) and treated with roscovitine (30 μM, Sigma-Aldrich, Saint Louis, MO) or VAS2870 (1 μM, Calbiochem, CA USA) during 24 h and 1 h, respectively.…”

“…Cdk5 is an essential kinase in brain development and function (Dhariwala and Rajadhyaksha, 2008 ; Utreras et al, 2009a ; Contreras-Vallejos et al, 2012 ). Interestingly, our group reported earlier that Cdk5 plays a crucial role during inflammatory pain signaling (Utreras et al, 2009a , 2011 ; Prochazkova et al, 2013 ; Rozas et al, 2016 ; Coddou et al, 2017 ). Cdk5 is a proline-directed serine/threonine kinase, mostly active in post-mitotic neurons, where its specific activators p35 and p39 are mainly expressed (Lew et al, 1994 ; Dhariwala and Rajadhyaksha, 2008 ; Utreras et al, 2009c ).…”

“…During tissue damage, several inflammatory mediators as tumor necrosis factor-α (TNF-α) are locally released, activating signaling pathways in sensory neurons that increase peripheral sensitization and pain signaling (Khan et al, 2008 ; Sessle, 2011 ; Rozas et al, 2016 ). Activated protein kinases such as PKC, PKA, and Cdk5 are involved in peripheral sensitization through phosphorylation of several ion channels expressed on nociceptive neurons (Basbaum et al, 2009 ; Rozas et al, 2016 ; Coddou et al, 2017 ). Cdk5 is an essential kinase in brain development and function (Dhariwala and Rajadhyaksha, 2008 ; Utreras et al, 2009a ; Contreras-Vallejos et al, 2012 ).…”

“…Cdk5 is a proline-directed serine/threonine kinase, mostly active in post-mitotic neurons, where its specific activators p35 and p39 are mainly expressed (Lew et al, 1994 ; Dhariwala and Rajadhyaksha, 2008 ; Utreras et al, 2009c ). We reported also that cytokines such as TNF-α or TGF-β1 up-regulate p35 expression and Cdk5 kinase activity with a subsequent phosphorylation of transient receptor potential vaniloid 1 (TRPV1) and purinergic P2X2a receptor (P2X2aR), which are important receptors channels involved during inflammatory pain signaling (Utreras et al, 2009b , 2011 , 2012 , 2013 ; Prochazkova et al, 2013 ; Rozas et al, 2016 ; Coddou et al, 2017 ).…”

Crosstalk between Cdk5/p35 and ERK1/2 signalling mediates spinal astrocyte activity via the PPARγ pathway in a rat model of chronic constriction injury

“…Unlike the activation of other Cdks, the activation of Cdk5 requires the binding of its own specific partners, p35 and p39 (Pareek and Kulkarni ), especially p35, which is more capable of binding and activating Cdk5 than p39 (Utreras et al ). Increasing evidence has shown that Cdk5 activation may be involved in pathological pain conditions (Jendryke et al ; Coddou et al ; Moutal et al ) and contribute to neurodegeneration (Yousuf et al ). Administration of the Cdk5/p35 inhibitor roscovitine alleviates neuropathic pain (Yang et al ), inflammation‐induced pain (Zhang et al ) and cancer‐induced pain (Zhang et al ), implying that the activation of Cdk5 is critical for the formation of pain.…”