Regulatory Function of Dopamine Transporter Phosphorylation Site Threonine 53 - Research Paper Example

Biology Research Paper December Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-Stimulated Efflux Introduction Dopamine (DA) is a neurotransmitter that takes part in major brain functions, which include motor activities, inspiration, and incentives. Conversely, dopamine transporter (DAT) is involved in the control of the quantities of extra neuronal dopamine in the central nervous system. The transport events are regulated by multiple signaling pathways, substrate efflux, and other DAT roles by mechanisms that have not been well elucidated. DAT is phosphorylated by protein kinase C in two regions. The first location is in a serine group at the far ends of the cytoplasmic N-terminus and proline-directed phosphorylation at membrane-proximal residue threonine Foster et al. (29702). Activities that lead to the reuptake of DAT are crucial for the functionality of the dopaminergic neurons. Dopaminergic disorders, for instance, schizophrenia and depression among others may come about as a result of dysregulation of the transport of DAT, which causes an inequality of dopamine. Commonly abused substances including drugs such as cocaine and amphetamine are often targeted by DAT. In addition, therapeutic agents that treat dopamine complications are also targeted by DAT. Specifically, AMPH and its congeners induce multiple short-term and long-term effects on DAT. The mechanism behind the efflux is related with transporter-generated currents. Various techniques have been used to determine the precise phosphorylation site of DAT. It has been established that that a recombinant peptide containing N-terminal residues 1–65 of rDAT underwent phosphorylation outside living tissues in the presence of proline-guided kinases. It was also established that the precise phosphorylation site in heterologously expressed protein was a threonine residue, which was located at position 53. However, it was vital to ascertain the phosphorylation site using other techniques. The researchers in this paper aimed at determining the role of dopamine transporter phosphorylation site threonine 53 on the reabsorption of the substrate and amphetamine-triggered efflux Foster et al. (29702). The researchers make use of mass spectrometry and a novel antibody that is specific to phosphates to ascertain the presence of DAT phosphorylation at Thr53 in striatal tissue obtained from rodents as well as heterologous expression systems. Experimental Protocol Cell culture and dopamine transporter mutagenesis was done using Lewis carcinoma cells that were stably expressing WTrDAT (rDAT-LLCPK) (31) or T53A or T53D rDAT and were maintained in alpha minimum essential medium. Dulbeccos modified Eagles medium together with 10% FBS and penicillin/streptomycin was used to culture tsA201 cells. The cells were then incubated in a CO2 incubator set at 5% CO2 and 37oC in a humidified environment. Three mutations were introduced to the rDAT pcDNA3.0 template using StratageneQuickChange® kit with codon substitution verified by sequencing. Transfected cells were maintained under selection with 800mg/ml G418 (29) with the aim of producing pooled stable transformants. Thereafter, tsA201 cells were transfected briefly with WT rDAT using the ExGen500 reagent according to the manufacturers instructions. For experiments with T53E, LLC-PK, the cells were briefly transfected with 0.6mg of WT or T53E DNA/FuGENE and assayed for transport activity after 24 h. Tandem mass spectrometry analysis (LC-MS/MS) Rat striatal synaptosomes rDAT LLCPK cells, or tsA201 cells were solubilized in a lysis buffer constituted with 1% Triton x-100, 20mM Tris-HCl (pH 8.0), 150mM NaCl, 1mM EDTA, 1mM sodium orthovanadate, 5 mM sodium fluoride, 5mM sodium pyrophosphate, and a protease inhibitor mixture on a tube rotator for 2 h at 4°C. Centrifugation was performed at 14,000×g for 30 minutes at 4°C and collected the supernatant and incubated it overnight with goat anti-DAT Polyclonal antibody. Protein G beads were used to collect the immune complexes after which they were extensively washed and their elution for bound proteins done in laemmli buffer at 95°C for 3 min. SDS- polyacrylamide gel was used in size fractionation of the eluted proteins and then visualized them using Coomasie Brilliant Blue staining and the indicated bands excised out. 50% acetonitrile was used in the destaining of the gel pieces in 25mM ammonium bicarbonate, and drying was done in a vacuum concentrator. The pieces of gel were then washed and dried. 25mM of ammonium bicarbonate with10ng/ml trypsin was used to rehydrate the gel pieces. The rehydrated gel pieces were then incubated for 18 h at 37°C. 50% acetonitrile in 5% formic acid was used in the extraction of the digested peptide mixtures and concentration done in a speed vacuum concentrator for tandem mass spectrometry. For LC-MS/MS data acquisition, an ion trap mass spectrometer incorporated with an ultimate 300 nano-HPLC was employed. For reverse phase chromatography, a PepMap100 c-18 trap column and PepMap100 c-18 analytical column were used in the presence of two buffers, 0.1% forrmic acid water (buffer A), and 0.08% formic acid acetonitrile (buffer B) with 125 min gradient. The Peptide spectra were recorded by spraying the eluted peptides into the mass spectrometer over a mass to charge ratio between 350 and1500 and MS/MS information-reliant data attainment over a mass to charge ratio that ranged from 100 to 2800. MS spectra were recorded repeatedly. Interpretation of MS spectra was done using Mascot search engine. The following were accepted, three absent cleavage locations, the presence of carbamidomethylation on cysteine, oxidation on the amino acid methionine, deamination on asparagine/glutamine and phosphorylation on serine/threonine. Phosphospecific antibody generation was done by phosphosolutions using a control peptide with non-phosphorylated threonine that was synthesized. A phosphopeptide based on DAT N-terminal amino acid sequence (Thr P) was used to immunize rabbits briefly. Chronological cycles of chromatography against stationary phospho- and dephopeptide was used in the purification of specific polyclonal antibody in rabbit immune serum and was concentrated to 1mg/ml Foster et al. (29709). DAT immunoblot and immunoprecipitation was done with mouse N-terminal Ab16 generated against residues 42-59 to determine total DAT. The determination of Thr Phosphorylation Stoichiometry was done by immunoprecipitating rat striatal lysates with Thr(P) Ab and bound and unbound portions were immunoblotted with Thr (P) to determine the portion of Thr phosphorylated retained in DAT transporters in the pellet. DA Uptake and Cell Surface Biotinylation Assays WT cells were cultured in 24-well plates to 70-89% confluence in alpha minimum essential medium at 37°C. The cells were rinsed twice in 0.5 ml of Krebs-Ringer-HEPES buffer followed by the addition of 0.5ml of warmed KRH buffer at 37°C. Results and Discussion It was found that DAT was phosphorylated at Thr 53 in both tsA201 and rDAT-LLCPK cell lines. Differentially phosphorylated NDAT samples were used to test for specificity of Thr P53 in immunoblot assays. NDAT that was not phosphorylated or phosphorylated by PKC was not recognized by Thr 53 Ab. NDAT protein was present in all samples as it was demonstrated through the staining with mAb 16, and it showed an upward trend of NDAT induced by Thr phosphorylation Foster et al. (29711). The lysates from LLCPK cells were not immunostained by Thr(P) Ab but a strong reactivity was seen against an approximately 90 kDa in rDAT-LLCPK lysates showing specific identification of rDAT. We also noted that Thr (P) stained unglycosylated 60kDa form of DAT and also a 90kDa fully glycosylated form. Saturation analyses indicated that after stabilizing the transport of DA for comparative expression of the DAT plasma membrane, the two mutants had significant lower V values than the WT protein implicating changes in turnover rate rather than DA recognition as the mechanisms for reduced transport in mutants. T53 DAT was generated in order to determine if glutamic acid mutation of Thr would provide a strongest phosphomimetic substitution. ERK is the only proline kinase that has been recorded to have great effects on DAP. ERK inhibitors are responsible for the reduced DA transport activity. These findings show that DA transport functionality is positively controlled by tonic ERK signaling, and rapid control can be induced by upregulation of ERK receptor. These findings also make evident the role of AMPH-stimulated efflux, which is an essential factor in the amplification of neurotoxic traits of AMPH and other associated drugs Foster et al. (29712). A protein phosphatase activity was found to maintain a dephosphorylated state of DAT Thr in rat synaptosomes and cells treated with OA. Conclusion These results show the ability of phosphorylation of threonine can be controlled in two ways. One of the ways is through direct means via kinases and phosphatase enzymes that are guided by proline, whereas the other way is through PKC regulation of downstream paths. Consequently, the ability of threonine to act as a locus for the integration of DAT regulatory signs is demonstrated. Works Cited Foster, James D., Jae-Won Yang, Amy E. Moritz, Sathyavathi ChallaSivaKanaka, Margaret A. Smith, Marion Holy, Kyle Wilebski, Harald H. Sitte, and Roxanne A. Vaughan. “Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-stimulated Efflux.” Journal of Biological Chemistry 287.35(2012): 29702–29712. Read More