Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization

Abstract

Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32 Plabeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (␤-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. ␤-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the G␣ q -coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to ␤-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by ␤-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by ␤-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following ␤-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization. The norepinephrine (NE)2 transporter (NET) regulates noradrenergic signaling by mediating the clearance of NE and is an important target for antidepressants and psychostimulants (2-5). NE signaling is linked to behavioral arousal (6) and is affected in stress-related paradigms linked to depression (7,8). NE acutely inhibits nociceptive transmission, including that mediated by SP (NK1), potentiates opioid analgesia, and underlies part of antinociceptive effects of tricyclic antidepressants (9). Various biologic stimuli regulate NE signaling, and alterations in NE signaling, including NE clearance and NET density, are observed in cardiovascular diseases and brain disorders (10 -13). Recent studies provided evidence for protein kinase C (PKC)-mediated regulation of NET function attributed to alterations in NET surface redistribution (1, 14, 15). Signals mediated through G-protein-coupled receptors are a likely trigger for PKC-mediated regulation of NET, and such receptors are abundant on neuronal and non-neuronal cells.Human placenta expresses both SERT and NET (16 -18), and we have developed trophoblast cultures from the rat placenta that robustly express endogenous NET (19). Placenta also expresses several receptors, including receptors for peptides such as insulin, SP, and NKB, neurotransmitters,. Our recent study using rat placental trophobla...

Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization

“…Our previous studies have demonstrated that NET in localization in lipid rafts and PKC activation either directly by phorbol ester or via SP activation of NK1R stimulates NET internalization (10,14). We have identified a trafficking motif involved in NK1R/PKC-mediated NET down-regulation and established a close relationship between transporter phosphorylation and internalization (10,14).…”

“…We have identified a trafficking motif involved in NK1R/PKC-mediated NET down-regulation and established a close relationship between transporter phosphorylation and internalization (10,14). Many GPCRs including NK1R mediate PKC signaling, and are known to redistribute signaling partner proteins when activated.…”

“…Some of these NET interacting proteins modulate PKC-mediated NET regulation (5)(6)(7)(8). Signals linked to G protein-coupled receptor (GPCR) activation are also known to regulate NET (9,10). Studies have shown evidence for receptor activation regulating transporter and transporter modulation regulating receptor function (4,(11)(12)(13).…”

A Role for p38 Mitogen-activated Protein Kinase-mediated Threonine 30-dependent Norepinephrine Transporter Regulation in Cocaine Sensitization and Conditioned Place Preference

“…We have documented that phosphorylation of NET may be a common mechanism dictating NET expression and thus NE transport function in maintaining NE homeostasis (1,17,18). In particular, our recent in vitro and in vivo studies documented that cocaine increases NET function and surface expression in rodent PFC and NAc via p38 MAPKmediated NET phosphorylation (1).…”

Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A

“…1). Although NET is known to be phosphorylated at T258 in response to PMA treatments, an event to link to internalization (Jayanthi et al, 2006), it is not clear yet whether Ca 2+ -dependent NET trafficking or dissociation of the NET/SYN1A complex observed in this study involves NET phosphorylation at the same site. Ca 2+ dependent phosphorylation of SYN1A at S188 by the Death-associated protein kinase, a Ca 2+ /calmodulin-dependent serine/threonine protein kinase, is known to regulate SYN1A interactions with Munc18 (Tian et al, 2003) and R151 in SYN1A has been reported to be important for Ca 2+ -sensitive interactions with CaMKII (Ohyama et al, 2002).…”