Genetic complementation screen identifies a mitogen-activated protein kinase phosphatase, MKP3, as a regulator of dopamine transporter trafficking.
The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C-induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis.
Mortensen
Ole Valente
OV
Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
Larsen
Mads Breum
MB
Prasad
Balakrishna M
BM
Amara
Susan G
SG
D000818:Animals; D001692:Biological Transport; D045744:Cell Line, Tumor; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D054642:Dual Specificity Phosphatase 6 / Q000235:genetics* / Q000502:physiology; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D006801:Humans; D020935:MAP Kinase Signaling System; D008954:Models, Biological; D014982:Xenopus laevis; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D048051:p38 Mitogen-Activated Protein Kinases; D054642:Dual Specificity Phosphatase 6; D034281:Dynamins; D004298:Dopamine