Abstract
The norepinephrine transporter (NET) mediates the clearance of norepinephrine (NE) from the extracellular space and is a target of therapeutic antidepressants and psychostimulants. Previously we identified a MAP kinase phosphatase 3 (MKP3), as an important modulator of protein kinase C (PKC) mediated internalization of the related dopamine transporter (DAT). Here we show that MKP3 decreases PKC-mediated down regulation of NET expressed in PC12 cells. We demonstrate that this process involves a PKC-stimulated decrease of NET surface expression that is dependent on dynamin. Surprisingly, MAP kinase inhibitors have no effect on the PKC-mediated regulation of NET activity, suggesting that, like PKC-mediated regulation of the DAT, the acute activation of MAP kinases is not likely to be involved. To elucidate potential mechanisms we used a substrate trap-based assay to identify extracellular-signal-regulated kinase (ERK)1/2 as the predominant substrate of MKP3. Furthermore we also established that brief chemical stabilization of a modified destabilized MKP3 does not alter PKC-mediated down regulation of NET. Finally, the expression of a dominant negative version of H-Ras, an upstream activator of ERK1/2, abolishes phorbol 12-myristate 13-acetate (PMA)-mediated down regulation of NET in a manner similar to MKP3. Taken together we propose that chronic MKP3 expression regulates surface NET through the sustained inhibition of ERK1/2 MAP kinase signaling that alters gene expression in PC12 cells. This is supported by gene expression data from naïve and MKP3-expressing PC12 cells that reveal robust decreases in gene expression of several genes in the MKP3-tranfected cells. Interestingly, caveolin-1, a protein with a critical role in membrane protein trafficking is down regulated by MKP3 expression. We further show that selective silencing of the caveolin-1 gene in naïve PC12 cells attenuates PKC-mediated downregulation of NET activity, consistent with a potential role for caveolins in regulating NET surface expression. In summary, these results suggest that chronic MKP3 expression alters the expression of genes in PC12 cells that are involved in the regulation of NET surface expression.
Highlights
The norepinephrine transporter (NET) belongs to the SLC6A family of Na/Cl-dependent neurotransmitter transporters (Kristensen et al, 2011)
This was triggered by our previous study on the closely related dopamine transporter (DAT) in which we found that protein kinase C (PKC)-mediated and dynamin-dependent internalization of the DAT was attenuated by MAP kinase phosphatase 3 (MKP3) (Mortensen et al, 2008)
Because a previous study showed that direct phosphorylation of NET could affect NET activity in human placental trophoblast cells (Jayanthi et al, 2006) we examined the effect of PKC on a double mutant of NET in which PKC-mediated NET phosphorylation sites were eliminated
Summary
The norepinephrine transporter (NET) belongs to the SLC6A family of Na/Cl-dependent neurotransmitter transporters (Kristensen et al, 2011). Previous studies of NET regulation in PC12 cells have demonstrated that protein kinase C (PKC) activation decreased NET activity (Uchida et al, 1998; Mandela and Ordway, 2006) The mechanism of this regulation was not established but the results of one study implied that changes in surface expression might be involved (Mandela and Ordway, 2006). In the present study we have investigated the mechanism of PKC-mediated regulation of NET further and have tested the involvement of MAP kinase phosphatase 3 (MKP3 or DUSP6) in the modulation of endogenously expressed NET in PC12 cells. We provide evidence that the PKC-induced down regulation is not caused by an immediate short-lived downstream activation of MAP kinase pathways, and we provide evidence that suggest a process involving sustained inhibition of MAP kinases including extracellular-signal-regulated kinase (ERK)1/2 resulting in changes in gene expression that mediate the observed regulation of NET
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