Disruption of Cdk5-associated phosphorylation of residue threonine-161 of the delta-opioid receptor: impaired receptor function and attenuated morphine antinociceptive tolerance.

Morphine is the most commonly used and most effective analgesic in the clinic. However, its use is limited by the tolerance. Evidence indicates that the delta-opioid receptor (DOR) is essential for morphine antinociceptive tolerance; however, their underlying mechanisms are poorly understood. Here, we show that cyclin-dependent kinase 5 (Cdk5), activated in morphine antinociceptive tolerance, directly phosphorylates DOR at Thr-161 in DRG neurons. Cdk5 was found to phosphorylate Thr-161 in the second loop of DOR, but not the corresponding residue in the mu-opioid receptor (MOR). Phosphorylation at Thr-161 is required for normal cell surface expression of DOR, and the formation of DOR-MOR heterodimers. Our studies indicated that inhibition of Cdk5 activity or overexpression of a DOR mutant lacking the Cdk5 phosphorylation site displayed relatively low cell surface expression and relatively low abilities to form heterodimers of DOR and MOR; intrathecal delivery of a construct expressing the T161A mutant of DOR attenuated morphine antinociceptive tolerance in rats, suggesting that Thr-161 phosphorylation of DOR contributed to Cdk5-mediated morphine antinociceptive tolerance. Furthermore, an engineered Tat fusion-interfering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L), reduced the cell surface expression of DOR, disrupted the formation of DOR-MOR heterodimers, and significantly attenuated the development of morphine antinociceptive tolerance after intrathecal injection. The present study indicates that Cdk5-mediated phosphorylation of DOR at Thr-161 plays a crucial role in the development of morphine tolerance and suggests the possibility of targeting DOR phosphorylation at Thr-161 to attenuate morphine antinociceptive tolerance during pain management.

Disruption of δ-opioid receptor phosphorylation at threonine 161 attenuates morphine tolerance in rats with CFA-induced inflammatory hypersensitivity.

OBJECTIVE: Our previous study identified Threonine 161 (Thr-161), located in the second intracellular loop of the δ-opioid receptor (DOR), as the only consensus phosphorylation site for cyclin-dependent kinase 5 (Cdk5). The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund's adjuvant (CFA)-induced inflammatory pain and morphine tolerance. METHODS: Dorsal root ganglion (DRG) neurons of rats with CFA-induced inflammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 (pThr-161-DOR), and paw withdrawal latency was measured after intrathecal delivery of drugs or Tat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. RESULTS: Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. Intrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L) increased inflammatory hypersensitivity, and inhibited DOR- but not µ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. CONCLUSION: Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr-161 attenuates morphine tolerance.

The presence of active Cdk5 associated with p35 in astrocytes and its important role in process elongation of scratched astrocyte.

Cyclin-dependent kinase 5 (Cdk5) is a unique member of the Cdk family; its kinase activity requires association with its activator, p35 or p39. p35 is the strongest and best characterized activator. Previous studies showed that p35 is a neuron-specific protein that restricts Cdk5 activity in neurons. However, a high expression level of Cdk5 is found in astrocytes, which raises the possibility that astrocytic Cdk5 is functional. Here we show the presence of functional Cdk5 associated with p35 in astrocytes and demonstrate its important role in process elongation of scratched astrocytes. We found that p35 and glial fibrillary acidic protein (GFAP) were co-localized in primary cultured and acute isolated brain cells. Cdk5 could form an immunocomplex with p35 and its activity was shown in pure primary cultured astrocytes. p35 was upregulated in astrocytes injured by scratching, concomitantly with upregulation of Cdk5 kinase activity. Pretreatment of the scratched astrocytes with a Cdk5 inhibitor, roscovitine, could delay wound healing by inhibiting the reorganization of tubulin, GFAP, and the extension of hypertrophic processes. Moreover, overexpression of dominant negative Cdk5 could shorten the length of extending protrusion of reactive astrocytes. Thus, our findings demonstrated that functional Cdk5, associated with p35, was expressed in astrocytes and its activity could be upregulated in reactive astrocytes, a new role of Cdk5 that has never been reported in the nervous system. The present study may provide new insight for understanding the multifunctional protein complex Cdk5/p35 in the nervous system.

Role of CDK5 in neuroprotection from serum deprivation by mu-opioid receptor agonist.

Cyclin-dependent kinase 5 (CDK5), a unique member of the CDK family of cyclin-dependent kinases, is predominantly expressed in postmitotic neurons with proposed roles in both cell survival and programmed cell death. To understand how CDK5 participates in such disparate cellular outcomes, we investigated whether activation of CDK5 could mediate neuroprotection from serum deprivation by mu-opioid receptor agonist in differentiated SH-SY5Y cells and primary hippocampal neurons. We found that CDK5 kinase activity decreased following serum deprivation in differentiated SH-SY5Y cells coincident with increased cell loss and activation of caspases cascade activation, which was reversed by opioid antagonist. Overexpression of CDK5 in serum-free medium reversed activation of caspase cascade and augmented DAMGO neuroprotection. Blocking CDK5 activity by pharmacologic inhibitor, roscovitine or overexpression of dominant negative CDK5 augmented activation of cell death markers and diminished mu-opioid receptor agonist protection. Reduction in CDK5 activity corresponded to reduction in protein levels of CDK5 activator p35 during serum deprivation which was also reversed by mu-opioid receptor agonist. Phosphorylation of STAT3 at Serine 727 by CDK5 decreased during serum deprivation, and partly recovered by mu-opioid agonist. PI3K signaling pathway was not required for CDK5-mediated mu-opioid neuroprotection against serum deprivation. These findings indicate that neuroprotection by mu-opioid receptor agonist against serum deprivation is mediated by activation of CDK5 through up-regulation of p35 and phosphorylation of STAT3 by CDK5 may contribute to the neuroprotection.