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Activation of Protein Kinase a Stimulates Sumoylation, Expression, and Transport Activity of Organic Anion Transporter 3

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Activation of Protein Kinase a Stimulates Sumoylation, Expression, and Transport Activity of Organic Anion Transporter 3 The AAPS Journal (2019) 21: 30 DOI: 10.1208/s12248-019-0303-4 Research Article Activation of Protein Kinase A Stimulates SUMOylation, Expression, and Transport Activity of Organic Anion Transporter 3 Haoxun Wang,1 Jinghui Zhang,1 and Guofeng You1,2 Received 15 November 2018; accepted 22 January 2019; published online 13 February 2019 Abstract. Organic anion transporter 3 (OAT3) plays a vital role in removing a broad variety of anionic drugs from kidney, thus avoiding their possible toxicity in the body. We earlier established that activation of protein kinase C (PKC) enhances OAT3 ubiquitination, which promotes OAT3 internalization from the cell plasma membrane to intracellular endosomes and consequent degradation. As a result, OAT3 expression and transport activity are reduced. In the current study, we discovered that protein kinase A (PKA) had an opposite effect to PKC on the regulation of OAT3. We showed that activation of PKA by Bt2-cAMP stimulated OAT3 transport activity, which was largely caused by an enhanced plasma membrane expression of the transporter, kinetically reflected as an augmented maximal transport velocity Vmax without notable alteration in substrate-binding affinity Km. Additionally, we showed that PKA activation accelerated the rate of OAT3 recycling from intracellular compartments to the plasma membrane and decelerated the rate of OAT3 degradation. We further showed that OAT3 is subjected to post-translational modification by SUMO-2 and SUMO-3 not by SUMO-1. PKA activation enhanced OAT3 SUMOylation, which was accompanied by a reduced OAT3 ubiquitination. Finally, insulin-like growth factor 1 significantly stimulated OAT3 transport activity and SUMOylation through PKA signaling pathway. In conclusion, this is the first demonstration that PKA stimulated OAT3 expression and transport activity by altering the trafficking kinetics of OAT3 possibly through the crosstalk between SUMOylation and ubiquitination. Our studies are consistent with a remote sensing and signaling model for transporters (Wu et al. in Mol Pharmacol. 79(5):795–805, 2011). KEY WORDS: drug transport; organic anion transporter; protein kinase A; regulation; SUMOylation. INTRODUCTION identified by the elements of cell surface internalization machinery, and triggers OAT internalization from the plasma Organic anion transporter 3 (OAT3) is a member of the membrane to intracellular early endosomes partly through a organic anion transporter family, which plays vital parts in the clathrin-mediated pathway. As soon as in the endosomes, removal of many drugs from the kidney, such as antiviral OAT either becomes deubiquitinated and recycles back to the drugs, anti-tumor therapeutics, antibiotics, and antihyperten- plasma membrane or targets to a proteolytic system for sive and anti-inflammatory drugs, and thereby avoiding their degradation. We further demonstrated that activation of possible toxicity in the body (1–6). protein kinase C (PKC) downregulates OAT transport The transport activity of OAT3 relies on its expression activity by enhancing OAT ubiquitination, which promotes level at the plasma membrane. Our laboratory has earlier an accelerated OAT internalization from the plasma mem- established that OATs naturally internalize from and recycle brane to intracellular early endosomes and subsequent back to the plasma membrane. A crucial event prior to OAT degradation without affecting OAT recycling. Therefore, the internalization is the modification of lysine residues of cell expression level of OAT at the plasma membrane is surface OAT by ubiquitin conjugation. Ubiquitin is an 8-kDa significantly reduced, which results in a significant decrease polypeptide, and, when attached to cell surface OAT, can be in OAT transport activity. Clearly, post-translational modifi- cation of OAT by ubiquitination is a major mechanism that 1 governs PKC-regulated OAT trafficking and transport activity Department of Pharmaceutics, Rutgers, The State University of – New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey 08854, (7 10). fi USA. Another important ubiquitin-like modi er is the small 2 To whom correspondence should be addressed. (e–mail: ubiquitin-related modifier (SUMO) (11,12). The SUMO [email protected]) family consists of three functional isoforms SUMO1–3. All 1550-7416/19/0200-0001/0 # 2019 American Association of Pharmaceutical Scientists 30 Page 2 of 11 The AAPS Journal (2019) 21: 30 three are polypeptides of ~ 12 kDa and are broadly detected Cell Culture and Transfection in many tissues, including brain, liver, and kidney. SUMO2 and SUMO3 are regularly mentioned as SUMO2/3 as they Parental COS-7 cells were cultured in DMEM medium have 97% identity. In contrast, SUMO2/3 is only ~ 50% (Invitrogen, Carlsbad, CA) containing10% fetal bovine identical in the sequence with SUMO-1. Consistent with such serum at 37°C in 5% CO2. Cells stably expressing human sequence differences, SUMO1 and SUMO2/3 modify differ- OAT3 (hOAT3) were maintained in DMEM containing ent substrates in vivo. The modification of substrate proteins 0.2 mg/ml G418 (Invitrogen, Carlsbad, CA), 10% fetal bovine by SUMOylation involves the consecutive steps of the E1 serum. SUMO-activating enzymes and the E2 SUMO-conjugating Transfection with cDNA plasmids was done for 48 h enzyme 9 (Ubc9). This process leads to the formation of an using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), fol- isopeptide bond between the carboxy-terminal glycine of lowing the manufacturer’s instructions. SUMO and lysine residues of specific target proteins. Although chemically stable, modification by SUMO is Transport Measurements reversible by specific isopeptidases that break the bond between SUMO and the lysine residue(s) of target substrate. The uptake solution consisted of PBS/Ca2+/Mg2+ SUMOylation was initially identified as a major regulatory (pH 7.3) and [3H] estrone sulfate (ES) (0.3 μM). The uptake mechanism of protein function on nuclear proteins. Within solution was added to the cells. At an indicated period of the last decade, more and more membrane proteins such as time, uptake was ended by aspirating the uptake solution and channels and receptors were identified as SUMO substrates rapidly washing the cells with ice-cold PBS solution. The cells (13–19). And the roles of SUMOylation in the regulation of were then lysed in 0.2 N NaOH, neutralized in 0.2 N HCl, and these membrane proteins have been investigated. Recently, it aliquoted for liquid scintillation counting. has become obvious that ubiquitin and SUMO, although performing different biological functions, frequently commu- Cell Surface Biotinylation nicate and cooperatively affect the properties of shared substrate proteins, in certain cases by modifying the same The amount of hOAT3 at the cell surface was deter- site in a competitive manner (20,21). Whether OAT is subject mined using the membrane-impermeable biotinylation re- to the regulation by SUMOylation is currently unknown. agent, NHS-SS-biotin as described in our previous Contrary to the inhibitory effect of PKC on OATs, publications (9,26). The hOAT3 in the pool of surface protein kinase A (PKA) has been shown to stimulate OAT proteins was detected by SDS-PAGE and immunoblotting – activity (22 24). However, its mechanism of action is not well using an anti-Myc antibody 9E10. understood. A thorough understanding of PKA-regulated fi OAT activity is of high signi cance because various physio- Recycling Assay logical stimuli such as insulin-like growth factor exert its biological effect through the activation of PKA (25). In the We followed the procedure previously established in our present study, we examined the mechanism of PKA action on laboratory (7). hOAT3-expressing cells were biotinylated with OAT3. We demonstrated that PKA stimulated OAT3 expres- sulfo-NHS-SS-biotin at 4°C to label hOAT3 at the plasma fi sion and transport activity by altering the traf cking kinetics membrane. Then, one set of cells was unceasingly biotinyl- of the transporter possibly through the crosstalk between ated at 4°C. The duplicate set of cells was warmed to 37°C ubiquitination and SUMOylation. and unceasingly biotinylated at 37°C. At the indicated time points, biotinylation was ended and biotin-labeled hOAT3 MATERIALS AND METHODS was examined by SDS-PAGE and immunoblotting as de- scribed above. Recycled hOAT3 was calculated as the Materials difference between hOAT3 biotin-labeled at 37°C and hOAT3 biotin-labeled at 4°C. COS-7 cells were obtained from ATCC (Manassas, VA). [3H]-labeled estrone sulfate (ES) was obtained from Internalization Assay PerkinElmer (Waltham, MA). Membrane-impermeable bio- tinylation reagent NHS-SS-biotin, streptavidin-agarose beads, We followed the procedure established in our laboratory and protein G-agarose beads were obtained from Pierce (7,9) using a biotinylation approach in conjunction with (Rockford, IL). cDNAs for HA-tagged SUMO-1, SUMO-2, immunoblotting. Relative OAT3 internalized was calculated SUMO-3, and Ubc9 were kindly provided by Dr. Jorge A as % of the total initial cell surface OAT3 pool. Iñiguez-Lluhí from the University of Michigan Medical School. Mouse anti-Myc antibody (9E10) was obtained from Degradation Assay Roche (Indianapolis, IN). Rabbit anti-HA antibody and mouse anti-E-Cadherin antibody were obtained from Abcam The procedure previously established in our laboratory was (Cambridge, MA). Mouse anti-ubiquitin antibody and mouse followed (9,27). The hOAT3-expressing cells were subjected to anti-β-actin were obtained from Santa Cruz (Santa Cruz, biotinylation
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