Cyclin-Dependent Kinase 5 Inhibitors: Inhibition of Dopamine Transporter Activity

Abstract

Cyclin-dependent kinase (Cdk) 5 reduces the rewarding properties of psychostimulants by dampening postsynaptic dopamine (DA) receptor signaling. Cdk5 is also present in midbrain DA neurons, where the DA transporter (DAT) is localized and limits DA neurotransmission by removing extracellular DA. Here, we tested the hypothesis that Cdk5 could also affect the disposition of DA by regulating DAT activity. Incubation of rat dorsal striatal (dSTR) synaptosomes with the Cdk5 inhibitors roscovitine, olomoucine, and 4-{ [(7-oxo-6,7-dihydro-8H-[1,3] To explore the involvement of Cdk5 in roscovitine-induced down-regulation of DAT activity, Cdk5 protein was knocked down via Cdk5-small interfering RNA by as much as 86% in porcine aortic endothelial cells stably expressing human (h)DATs. However, Cdk5 depletion did not alter hDAT activity. Taken together, our results suggest that roscovitine inhibits DAT activity independently of Cdk5; therefore, results obtained with such inhibitors should be interpreted with caution. Our study is the first to demonstrate that Cdk5 inhibitors reduce brain DAT activity via a mechanism that is independent of DAT trafficking and reverse-transport. Dopamine (DA) neurotransmission and its related neural functions underlie psychomotor control, affect, and rewarding behaviors (Schultz, 2007). It is important that dysfunctional DA neurotransmission contributes, in part, to the clinical manifestations of Parkinson's disease, schizophrenia, and drug addiction. The DA transporter (DAT) is a plasmalemmal membrane carrier protein that contributes to spatialtemporal regulation of DA signaling. This is accomplished by the active removal of extracellular DA through functional DAT molecules, which are located on the cell surface of DA neuronal axons, varicosities, and dendrites (Nirenberg et al., 1996). In addition to helping maintain DA homeostasis, the DAT is a molecular target of psychostimulants such as cocaine and amphetamine (AMPH) (Giros et al., 1996;Chen et al., 2006).Significant progress during the last decade has demonstrated that DATs are rapidly regulated via trafficking-dependent and -independent mechanisms (Blakely and Bauman, 2000;Zahniser and Sorkin, 2009). In addition to DAT regulation by psychostimulants, several protein kinases influence DAT activity. These include cAMP-dependent protein kinase A (Pristupa et al., 1998), protein kinase C (Daniels and Amara, 1999;Melikian and Buckley, 1999;Chen et al., 2009) Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.056978.ABBREVIATIONS: DA, dopamine; DAT, dopamine transporter; AMPH, amphetamine; MAPK, p44/42 mitogen-activated protein kinase; Cdk, cyclin-dependent kinase; DARPP-32, dopamine and cAMP-regulated phospho-protein of relative molecular mass 32,000; dSTR, dorsal striatal/ striatum; PAE, porcine aortic endothelial; YFP, yellow fluorescent protein; HA, hemagglutinin epitope; h, human; siRNA, small interfering RNA; PMA, 12-myristate 13-acetate; DMSO, dimethyl sul...

Locomotor conditioning by amphetamine requires cyclin-dependent kinase 5 signaling in the nucleus accumbens

“…However, no evidence that roscovitine enhanced locomotor responding to amphetamine was found in the present experiments (see Figure 1). Similarly, roscovitine was found in experiments using different in vitro preparations to increase DA overflow and potentiate the ability of amphetamine to do so (Price et al , 2009). Yet again, our findings do not support this possibility in the NAcc as infusions of roscovitine into this site did not induce locomotion or enhance amphetamine-induced locomotion (Figure 1).…”

Inhibiting cyclin-dependent kinase 5 in the nucleus accumbens enhances the expression of amphetamine-induced locomotor conditioning

“…In addition to inhibiting Cdk5, Ros has also been shown to produce other effects with lower potency, including slowing of the deactivation of N-type calcium channels [18] and increasing dopamine overflow in vitro [19], effects that could also enhance behavioral responding to psychostimulants [20–21]. In these cases as well, however, it remains to be determined by which signaling pathway these effects might have selectively modulated the expression of conditioned locomotion in the present experiment as no detectible effects of NAcc-Ros were observed in Unpaired and Control rats.…”

Regulation of monoamine transporters: Role of transporter phosphorylation

“…However, changes in surface expression are not always associated with kinase-induced alterations in DAT activity. For example, Cdk5 inhibition reduces DAT activity without affecting the levels of surface DAT protein (Price et al, 2009). Another study provided evidence that PKC induced internalized DAT can be recycled back to the cell surface by PKA-dependent mechanisms (Pristupa et al, 1998).…”