Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception

Abstract

1Cyclin-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). AITCinduced 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.Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that can modulate pain signaling 1,2 . Cdk5 is unlike other cyclin-dependent kinases because it is mostly active in post-mitotic neurons. Additionally, Cdk5 is not activated by a cyclin, but, instead, binds to two regulatory subunits, either p35 or p39, that are primarily restricted to neurons. Cdk5 activity is upregulated following peripheral administration of inflammatory agents such as carrageenan and Complete Freund's Adjuvant (CFA) 1,3 . Increased Cdk5 activity can, in turn, promote both mechanical and heat hyperalgesia 1,2 . Cdk5 activity can also affect morphine tolerance, where reduced Cdk5 activity correlates with delayed tolerance 4 . Therapeutically, intrathecal injection of the Cdk5 inhibitor roscovitine appears to attenuate CFA-induced heat hyperalgesia but not mechanical allodynia 3 while also reducing formalin-induced nociceptive behavior 5 . Cdk5 substrates include P/Q-type voltage-dependent calcium channels, which control calcium influx during nociception, and N-methyl-D-aspartate (NMDA) receptors, which relay pain signaling via the excitatory neurotransmitter glutamate 4 . Cdk5, however, is most directly linked to pain signaling by phosphorylating the pain transducing ion channel Transient Receptor Potential Vanilloid 1

Proximal C-Terminus Serves as a Signaling Hub for TRPA1 Channel Regulation via Its Interacting Molecules and Supramolecular Complexes

“…It has been demonstrated that TRPA1 can be sensitized by protein kinase A (PKA), protein kinase C (PKC), cyclin-dependent kinase 5, and by early signaling events linked to Ca 2+ -dependent phosphoinositide-specific phospholipase C (PLC) enzymes that hydrolyze PIP 2 in the inner membrane leaflet (Bandell et al, 2004;Bautista et al, 2006;Dai et al, 2007;Wang et al, 2008;Hynkova et al, 2016;Brackley et al, 2017;Meents et al, 2017;Hall et al, 2018;Sulak et al, 2018; Figure 2B). For effective phosphorylation by PKA and PKC, TRPA1 needs the presence of a scaffolding protein, AKAP (Brackley et al, 2017), that is also required for the PKA phosphorylation of TRPV1 (Zhang et al, 2008).…”

Phosphorylation, Dephosphorylation, and Multiprotein Assemblies Regulate Dynamic Behavior of Neuronal Cytoskeleton: A Mini-Review

“…Ankyrin G vaccination is tested for the reduction of Aβ and lowering toxicity ( Santuccione et al, 2013 ). Ankyrin repeats are interaction sites of phosphorylation dependent dynamic assembly of proteins and nucleic acids ( Hall et al, 2018 ) including those involved in transcription regulation ( Kohda et al, 2016 ) and signaling, and present promising targets for the design of new drugs.…”

Phospho-Mimetic Mutation at Ser602 Inactivates Human TRPA1 Channel

“…Among such agents are extracellular proteases, prostaglandins, bradykinin, serotonin, substance P, calcitonin gene related peptide, nerve growth factor (NGF), tumor necrosis factor α (TNFα), and many more [17][18][19][20]. These mediators can directly activate TRPA1 [21][22][23] or stimulate protein kinase A (PKA) [24][25][26][27], protein kinase C (PKC) [25], p38 mitogen-activated protein kinases [28], cyclin dependent kinase 5 [29,30], or phospholipase C (PLC) pathways [24,31] to induce phosphorylation of TRPA1. Phosphorylation, in turn, significantly alters the activation threshold and/or membrane expression of TRPA1, amplifying the inflammation and rendering nociceptors more sensitive to noxious stimulation.…”