A kinome wide screen identifies novel kinases involved in regulation of monoamine transporter function. (September 2016)

• Record Type: Journal Article
• Authors: Vuorenpää, Anne; Ammendrup-Johnsen, Ina; Jørgensen, Trine N.; Gether, Ulrik
• Is Part Of: Neurochemistry international. Volume 98(2016)
• Page Start: 103
• Page End: 114
• Publication Date: 2016-09
• Subjects: Monoamine transporter -- Uptake -- Surface expression -- Kinase -- siRNA screen -- PrKX; DAT dopamine transporter -- SERT serotonin transporter -- NET norepinephrine transporter -- DA dopamine -- 5-HT 5-hydroxytryptamine -- NE norepinephrine -- siRNA small interfering RNA -- PMA phorbol 12-myristate 13-acetate -- EGFP enhanced GFP -- β2-AR β2-adrenergic receptor -- SLC solute carrier -- CAD cells Cath.a differentiated cells -- SIK3 salt inducible kinase 3 -- PKA C-α cAMP-dependent protein kinase catalytic subunit alpha -- PrKX protein kinase X-linked; Neurochemistry -- Periodicals; Neurochemistry -- Periodicals; Neurochimie -- Périodiques; Neurochemistry; Periodicals; 612.804205
• DOI: 10.1016/j.neuint.2016.03.013
• Languages: English
• ISSNs: 0197-0186

Abstract

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.