Phospho-specific antibodies targeting the amino terminus of the human dopamine transporter

Abstract

The dopamine transporter (DAT), which mediates the inactivation of released dopamine through its reuptake, is the primary molecular target for the actions of psychostimulants. An increasing number of studies support an essential role for phosphorylation of serines (Ser) in the distal amino (N) terminus of DAT in regulating its function. Still, the molecular details of the regulation of phosphorylation and its impact on function are not fully understood. To address this, we have developed and characterized two distinct phospho-antibodies that recognize human DAT when it is phosphorylated at Ser7 or Ser12. Our data show that treatment of cells with phorbol 12-myristate 13-acetate (PMA), amphetamine (AMPH) or okadaic acid (OA) leads to an increase in the phosphorylation of DAT at both residues and that these responses are dependent on the activity of protein kinase C. We also show that AMPH-induced and OA-induced phosphorylation of DAT are also dependent on Ca2+/calmodulin-dependent protein kinase α. Our data further suggest that the lipid raft localization of DAT is necessary for efficient N-terminal phosphorylation and for the associated behavioral effects of AMPH, demonstrating the potential of these novel antibodies as powerful tools to study DAT regulation and function in vivo.

Phosphorylation of the Amino Terminus of the Dopamine Transporter: Regulatory Mechanisms and Implications for Amphetamine Action

“…Early studies demonstrating the ability of DAT to undergo phosphorylation were performed using 32 P metabolic labeling in heterologous expression systems (Granas, Ferrer, Loland, Javitch, & Gether, 2003; Huff, Vaughan, Kuhar, & Uhl, 1997) as well as in rat and mouse striatal tissue (Foster, Pananusorn, & Vaughan, 2002; Vaughan, Huff, Uhl, & Kuhar, 1997). More recently, our group developed phosphospecific anti-DAT antibodies to probe the phosphorylation of individual serine resides in DAT in response to various stimuli (Karam, Sen, & Javitch, 2017). These studies showed that DAT phosphorylation is rapidly elevated by activation of protein kinase C (PKC) with phorbol 12-myristate, 13-acetate (PMA) (Huff et al, 1997; Karam et al, 2017; Vaughan et al, 1997), diacylglycerol analogs (Vaughan et al, 1997), or G q activation by G protein-coupled receptor agonists (Granas et al, 2003).…”

“…More recently, our group developed phosphospecific anti-DAT antibodies to probe the phosphorylation of individual serine resides in DAT in response to various stimuli (Karam, Sen, & Javitch, 2017). These studies showed that DAT phosphorylation is rapidly elevated by activation of protein kinase C (PKC) with phorbol 12-myristate, 13-acetate (PMA) (Huff et al, 1997; Karam et al, 2017; Vaughan et al, 1997), diacylglycerol analogs (Vaughan et al, 1997), or G q activation by G protein-coupled receptor agonists (Granas et al, 2003). DAT phosphorylation was also shown to be strongly enhanced by treating cells with protein phosphatase inhibitors such as okadaic acid (OA), indicating that the transporter is subject to ongoing kinase activity even in the absence of exogenous kinase activators (Foster, Pananusorn, Cervinski, Holden, & Vaughan, 2003; Foster et al, 2002; Huff et al, 1997; Karam et al, 2017; Vaughan et al, 1997).…”

“…These studies showed that DAT phosphorylation is rapidly elevated by activation of protein kinase C (PKC) with phorbol 12-myristate, 13-acetate (PMA) (Huff et al, 1997; Karam et al, 2017; Vaughan et al, 1997), diacylglycerol analogs (Vaughan et al, 1997), or G q activation by G protein-coupled receptor agonists (Granas et al, 2003). DAT phosphorylation was also shown to be strongly enhanced by treating cells with protein phosphatase inhibitors such as okadaic acid (OA), indicating that the transporter is subject to ongoing kinase activity even in the absence of exogenous kinase activators (Foster, Pananusorn, Cervinski, Holden, & Vaughan, 2003; Foster et al, 2002; Huff et al, 1997; Karam et al, 2017; Vaughan et al, 1997). Critically, treatment with psychostimulants such as AMPH and methamphetamine (METH) stimulates an increase in DAT phosphorylation in both heterologously expressing cells and in rat brain tissue (Cervinski, Foster, & Vaughan, 2005; Karam et al, 2017).…”

“…DAT phosphorylation was also shown to be strongly enhanced by treating cells with protein phosphatase inhibitors such as okadaic acid (OA), indicating that the transporter is subject to ongoing kinase activity even in the absence of exogenous kinase activators (Foster, Pananusorn, Cervinski, Holden, & Vaughan, 2003; Foster et al, 2002; Huff et al, 1997; Karam et al, 2017; Vaughan et al, 1997). Critically, treatment with psychostimulants such as AMPH and methamphetamine (METH) stimulates an increase in DAT phosphorylation in both heterologously expressing cells and in rat brain tissue (Cervinski, Foster, & Vaughan, 2005; Karam et al, 2017). In contrast, the DAT blocker cocaine does not stimulate DAT phosphorylation, but it does prevent the phosphorylation increase induced by METH (Cervinski et al, 2005).…”

“…PKC activators, such as PMA, stimulate N-terminal phosphorylation of DAT in cells in culture (Granas et al, 2003; Huff et al, 1997; Karam et al, 2017) and in rat striatum (Cowell, Kantor, Hewlett, Frey, & Gnegy, 2000; Foster et al, 2002; Granas et al, 2003), and treatment with PKC inhibitors attenuates OA-induced (Foster et al, 2002; Gorentla, Moritz, Foster, & Vaughan, 2009; Huff et al, 1997; Karam et al, 2017; Vaughan et al, 1997) and AMPH-induced DAT phosphorylation (Cervinski et al, 2005; Karam et al, 2017), suggesting a role for PKC in both basal and stimulant-induced DAT phosphorylation. Treatment with a PKC activator was also shown to elicit release of 3 [ H ]dopamine from striatal synaptosomes that is cocaine-sensitive and independent of extracellular Ca 2+ (Davis & Patrick, 1990).…”