Role of Protein Kinase Phosphorylation on SERT Oligomerization: A Novel Mechanistic View

The serotonin transporter (SERT) is the primary mechanism for the termination of synaptic signaling. It is of clinical relevance because it is a high-affinity target for therapeutics such as anti-depressants (SSRIs) and drugs of abuse such as cocaine, amphetamine, and 3,4 methylenedioxymethamphetamine (ecstasy). It is also seen as a protein potentially involved in the etiology underlying disorders such as clinical depression, autism, anxiety-related disorders, obsessive compulsive disorder, and anorexia nervosa among others. SERT has also been found to be a target of protein kinases that exert their effects through phosphorylation. For example, activation of protein kinase C (PKC) decreases SERT uptake of serotonin (5-HT) by increasing internalization of SERT from the plasma membrane. Conversely, phosphorylation of SERT by protein kinase G (PKG) appears to increase SERT uptake of 5-HT from the synapse. It has also been thought that oligomerization of SERT is a necessary component of endoplasmic reticulum (ER) exocytosis and plasma membrane expression. However, there has been no common tie between these two very important processes of SERT regulation. In this study, we examined regulation of SERT phosphorylation\u27s effect on SERT oligomerization. Our results indicated that phosphorylation of SERT by kinases that stimulate 5-HT uptake, p38 mitogen-activated protein kinase (MAPK) and PKG decrease SERT oligomerization in HEK-293 cells. We also identified the putative phosphorylation site of SERT for PKG. PKC however showed the opposite effect by increasing SERT oligomerization upon activation with phorbol esters. In this study we also show that phosphorylation is a potential mechanism for regulating the oligomeric state of SERT using site-directed mutagenesis at a putative site in SERT phosphorylated by PKG. Lastly, we show that human mutant polymorphisms found in patients with obsessive compulsive disorder and other serotonin-related disorders, that have been shown to increase 5-HT uptake by SERT, appear to decrease the homo-oligomeric complex of SERT much like PKG and p38 MAPK. Lastly, we show that oligomerization is necessary for correct protein expression and that physiologically relevant concentrations of cocaine appear to increase SER T oligomerization. These results show for the first time that the oligomerization status of SERT can be regulated by protein kinases