title: A kinome wide screen identifies novel kinases involved in regulation of monoamine transporter function.
abstract: The high affinity transporters for the monoamine neurotransmitters, dopamine, norepinephrine, and serotonin, play a key role in controlling monoaminergic neurotransmission. It is believed that the transporters (DAT, NET and SERT, respectively) are subject to tight regulation by the cellular signaling machinery to maintain monoaminergic homeostasis. Kinases constitute a pivotal role in cellular signaling, however, the regulation of monoamine transporters by the entire ensemble of kinases is unknown. Here, we perform a whole human kinome RNA interference screen to identify novel kinases involved in regulation of monoamine transporter function and surface expression. A primary screen in HEK 293 cells stably expressing DAT or SERT with siRNAs against 573 human kinases revealed 93 kinases putatively regulating transporter function. All 93 hits, which also included kinases previously implicated in monoamine transporter regulation, such as Protein kinase B (Akt) and mitogen-activated protein kinases (MAPK), were validated with a new set of siRNAs in a secondary screen. In this screen we assessed both changes in uptake and surface expression leading to selection of 11 kinases for further evaluation in HEK 293 cells transiently expressing DAT, SERT or NET. Subsequently, three kinases; salt inducible kinase 3 (SIK3), cAMP-dependent protein kinase catalytic subunit alpha (PKA C-α) and protein kinase X-linked (PrKX); were selected for additional exploration in catecholaminergic CATH.a differentiated cells (CAD) and rat chromocytoma (PC12) cells. Whereas SIK3 likely transcriptionally regulated expression of the three transfected transporters, depletion of PKA C-α was shown to decrease SERT function. Depletion of PrKX caused decreased surface expression and function of DAT without changing protein levels, suggesting that PrKX stabilizes the transporter at the cell surface. Summarized, our data provide novel insight into kinome regulation of the monoamine transporters and identifies PrKX as a yet unappreciated possible regulator of monoamine transporter function.
authors.affiliation: Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark; Department of Pharmacology, Drug Development and Therapeutics, University of Turku, and Unit of Clinical Pharmacology, Turku University Hospital, FI-20014 Turku, Finland.
authors.forename: Anne
authors.initials: A
authors.lastname: Vuorenpää
authors.affiliation: Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Ina
authors.initials: I
authors.lastname: Ammendrup-Johnsen
authors.affiliation: Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Trine N
authors.initials: TN
authors.lastname: Jørgensen
authors.affiliation: Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark. Electronic address: gether@sund.ku.dk.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D018377:Neurotransmitter Agents / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D018377:Neurotransmitter Agents; D050486:Serotonin Plasma Membrane Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D012313:RNA; D011494:Protein Kinases; C109450:PRKX protein, human; D017346:Protein Serine-Threonine Kinases; C509079:SIK3 protein, human; D051571:Protein Kinase C-alpha
keywords: Kinase; Monoamine transporter; PrKX; Surface expression; Uptake; siRNA screen