Abstract
Sulfotransferase (SULT) 4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to colocalize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and SULT1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, and this was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using small interference RNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases, suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells.
SIGNIFICANCE STATEMENT The catalytically inactive sulfotransferase (SULT) 4A1 may regulate the function of other SULTs by interacting with them via a conserved dimerization motif. In neuron-like cells, SULT4A1 is able to modulate dopamine toxicity by interacting with SULT1A3, potentially decreasing the metabolism of dopamine.
Introduction
The aryl sulfotransferases (SULTs) are cytosolic enzymes that catalyze the transfer of a sulfonate group from the common cofactor 3′-phosphoadenosine-5′-phosphosulfate to a wide range of endogenous and exogenous substrates. In humans, there are at least 13 different SULT genes, which have been grouped into three families based on amino acid sequence homology: SULT1, SULT2, and SULT4 (Blanchard et al., 2004; Freimuth et al., 2004). The SULT1A family consists of 1A1, 1A2, 1A3, and 1A4, in which the 1A3 and 1A4 genes appear to have arisen from a segmental duplication that encodes the same protein (Hildebrandt et al., 2004). The SULT1A sulfotransferases are expressed widely with high abundance in the liver, lung, brain, skin, platelets, gastrointestinal tissues, and kidney (Maus et al., 1982; Campbell et al., 1987; Heroux et al., 1989; Cappiello et al., 1990; Falany et al., 1990; Nakamura et al., 1990; Hume and Coughtrie, 1994; Kudlacek et al., 1995). In humans, the SULT1A subfamily members SULT1A1 and SULT1A2 primarily conjugate xenobiotics and have a role in the activation of carcinogens (Glatt, 2000), although they also sulfonate endogenous substrates (Falany, 1997; Strott, 2002). Human SULT1A3 has high catalytic activity toward catecholamines, such as dopamine, noradrenaline, and adrenaline (Veronese et al., 1994; Falany, 1997; Brix et al., 1999).
SULT4A1 is the only known member of the 4A family. It was originally cloned from human and rat brain (Falany et al., 2000), in which it is predominantly expressed. Although SULT4A1 shares low sequence homology with other human SULTs, it is highly conserved between species (Minchin et al., 2008). SULT4A1 may be catalytically inactive because the active site is reportedly too small to accommodate 3′-phosphoadenosine-5′-phosphosulfate (Allali-Hassani et al., 2007). SULT4A1 expression is regulated by the cAMP response element–binding protein and the activating transcription factor 2 (Butcher et al., 2010). Post-translationally, it is phosphorylated to generate a recognition site for the peptidyl-prolyl cis-trans isomerase Pin1 (Mitchell and Minchin, 2009; Mitchell et al., 2011). The SULT4A1 gene shows a very low rate of synonymous or nonsynonymous mutations within its exonic sequence (Lewis and Minchin, 2009) and has the lowest overall mutation rate among all of the known human sulfotransferases (Hildebrandt et al., 2007). Single nucleotide polymorphisms in the SULT4A1 gene have been associated with schizophrenia susceptibility (Brennan and Condra, 2005; Condra et al., 2007; Ramsey et al., 2011). More recently, deletion of Sult4a1 in a mouse model resulted in a severe phenotype that included absence seizures, tremors, and ataxia (Garcia et al., 2018). The SULT4A1 null animals had a lifespan of only 20–25 days. These experiments provide strong evidence that SULT4A1 may be an important protein in neurodevelopment. SULT4A1 has been identified in the mitochondrial fraction of neuronal cells, and it has been suggested that it may be involved in regulating redox homeostasis and provide protection against mitochondrial dysfunction and oxidative stress (Hossain et al., 2019). This study further highlights the potential physiologic importance of SULT4A1 expression.
Most mammalian cytosolic sulfotransferases are found as homodimers in solution, with the mouse estrogen sulfotransferase Sult1e1 being an exception. The physiologic consequence of dimer formation is still unclear, although studies have shown differences in function and stability between the monomeric and dimeric forms (Thomae et al., 2002; Cook et al., 2010; Sidharthan et al., 2014). Crystallographic and site-directed mutagenesis experiments have shown that a conserved sequence motif of 10 residues (KxxxTVxxxE) located toward the C terminus of the protein is responsible for the interaction between the two sulfotransferase monomers (Petrotchenko et al., 2001). The two residues (T and V) at the center of the dimerization motif make strong symmetric contacts between the monomers. Dimeric human SULT1E1 contains a dimerization motif, whereas monomeric mouse Sult1e1 contains the sequence PE in place of TV. Mutation of TV to PE in human SULT1E1 produced a monomer, whereas mutation of mouse PE to TV created a homodimer, establishing the validity of the motif as the dimerization interface (Petrotchenko et al., 2001). Interestingly, heterodimers can also form between different SULTs containing the dimerization motif (Petrotchenko et al., 2001). Enzyme kinetic studies indicated that the two monomers do not catalyze sulfonation independently when present as a dimer (Sun and Leyh, 2010; Leyh et al., 2013). Moreover, dimerization may allow for cofactor-dependent allosteric regulation of enzyme activity (Tibbs et al., 2015).
During a yeast two-hybrid screen of a human brain library with SULT4A1, interactions between SULT4A1 and a variety of cytosolic sulfotransferases were observed, including SULT1A1 and SULT1A3, which are also found in abundance in the brain (Salman et al., 2009; James and Ambadapadi, 2013). These observations prompted the current study into the interaction of SULT4A1 with SULT1A1 and SULT1A3 using human cell models to identify protein-protein binding and determine whether any observed interactions affected the toxicity of the SULT1A3 substrate dopamine.
Materials and Methods
Cell Lines, Transient Transfections, and Cell Differentiation.
HeLa, SK-N-MC, and SH-SY5Y cells were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in RPMI 1640 medium (Hela), Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (SH-SY5Y), or minimum essential medium (SK-N-MC) supplemented with 10% FBS and incubated at 37°C in 5% CO2. For transient transfections, cells were seeded in six-well plates at 0.6 × 106 cells per well. Cells were transfected for 24 hours, and total transfected DNA was held constant at 4 μg by addition of empty control plasmid. Plasmid DNA was transfected using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer’s instructions. SK-N-MC and SH-SY5Y were differentiated into neuron-like cells in normal medium supplemented with 10 μM trans–retinoic acid (RA) (Sigma-Aldrich) for 5 days and then in medium containing low serum (1% FBS) and RA (10 μM) for a further 5 days.
Construct Cloning.
The generation of the HA-tagged pHM6-SULT4A1 and FLAG-tagged pX3FLAG-CMV-7.1-SULT4A1 constructs have been described previously (Mitchell and Minchin, 2009). For SULT1A1 and SULT1A3, the following forward and reverse primers containing EcoRI/XbaI and HindIII/EcoRI restriction sites (underlined) were used for PCR and to clone each into pFLAG-CMV-7.1 or phosphate metabolism protein 6, respectively: FP 5′-GATCGAATTCGATGGAGCTGATCCAGGAC-3′ and RP 5′-TCTAGATCTAGATCACAGCTCAGAGCGGAAG-3′ and FP 5′-GATCAAGCTTGATGGAGCTGATCCAGGAGACC-3′ and RP 5′-GATCGAATTCTCACAGCTCAGAGCGGAAGC-3′. SULT1A1 and SULT1A3 dimerization mutants (V270E) were generated using the GeneTailor site-directed mutagenesis kit (Invitrogen) and the SULT constructs described above as templates with the following common primers: FP 5′-GACTGGAAGACCACCTTCACCGAGGCGCAGAATGAG-3′ and RP 5′-CGGTGAAGGTGGTCTTCCAGTCCCCAGCCATG-3′ (mutated bases underlined).
To generate p3XFLAG-CMV-10-SULT4A1 for stable transfection, SULT4A1 was excised from pFLAG-CMV-7.1 and ligated into the same sites of p3XFLAG-CMV-10 (Sigma-Aldrich), which contains a G418 selection cassette. This plasmid was then used as template to create the SULT4A1 dimerization mutant (KTV→EEE; K265E, T269E, V270E) in two steps using the GeneArt site-directed mutagenesis kit (Invitrogen). First, the T269E and V270E mutations were introduced with primers FP 5′- GGAAGGACATCTTCGAGGAGTCCATGAATGAGAAG-3′ and RP 5′-CTTCTCATTCATGGACTCCTCGAAGATGTCCTTCC-3′ (mutated bases underlined). Second, using the above construct as template, the K265E mutation was introduced with primers FP 5′-AGAGTTGGGCTGTGGGAGGACATCTTC-3′ and RP 5′-GAAGATGTCCTCCCACAGCCCAACTCT-3′ (mutated bases underlined). The lysine at position 265 is a site of polyubiquitination and was changed to stabilize the dimerization mutant SULT4A1 protein (Sidharthan et al., 2014). All clones were verified by DNA sequencing.
Stable Cell Lines.
To generate stable cell lines, SK-N-MC cells were transfected with p3XFLAG-CMV-10 plasmid containing either wild-type or dimerization mutant (KTV→EEE) SULT4A1 or empty vector using LipofectAMINE 2000 (Invitrogen). Transfected cells were then selected with 1 mg/ml Geneticin (Thermo Fisher Scientific), and single clones were isolated and expanded. Stable cell lines were maintained in medium supplemented with 0.3 mg/ml Geneticin. Expression of the SULT4A1 proteins was verified by Western blot with anti-FLAG antibody.
Immunocytochemistry.
Post-mortem human brain tissue from confirmed neurologically normal individuals was obtained from the Queensland Brain Bank in Brisbane, Australia under the University of Queensland Ethics Committee approval (2012001283). Frozen cryostat sections from the anterior cingulate cortex and formalin-fixed paraffin-embedded sections (7 μm–thick) from the substantia nigra were used. Antigen retrieval was performed using 0.05% citraconic anhydride (pH 7.4) at 90°C for 30 minutes, and this was followed by treatment with Historeveal solution (Abcam) according to the manufacturer’s instructions. Sections were then washed three times in PBS and incubated in blocking buffer (PBS containing 2% bovine serum albumin, 5% normal donkey serum, 0.1% Triton X-100, and 0.05% Tween-20) for 2 hours at room temperature. Sections were then washed once in PBS and incubated with primary antibodies for SULT4A1 (Proteintech, diluted 1:1000) and SULT1A1/1A3 (gift from Professor Mike Coughtrie, The University of British Columbia, Vancouver, Canada; diluted 1:1000) overnight at 4°C in antibody buffer (PBS with 2% bovine serum albumin and 2% normal donkey serum). Tyrosine hydroxylase (diluted 1:2000; Cell Signaling Technology) was used as a dopaminergic neuron marker and microtubule-associated protein 2 (MAP2) (diluted 1:2000; Cell Signaling Technology) was used as a pan-neuronal marker for colocalization studies. After five washes in PBS, sections were incubated with the respective Alexa dye–conjugated secondary antibodies (diluted 1:5000) for 2 hours at room temperature. Sections were then washed 5 times in PBS and treated using the Autofluorescence Eliminator Reagent (2160; Merck Millipore) for 5 minutes to eliminate lipofuscin-derived autofluorescence. Sections were then mounted using Prolong Gold antifade reagent (Molecular Probes) for confocal microscopy.
Coimmunoprecipitation Assays.
HeLa cells were transfected as described above with various combinations of plasmids that expressed FLAG-tagged and HA-tagged SULT proteins. Transiently transfected cells (1 × 106) were washed in PBS, and whole-cell extracts were prepared by incubation in 500 µl NP40 lysis buffer (1% NP40; 50 mM Tris, pH 8.0; 150 mM NaCl; 5 mM NaF; 1 mM Na3VO4; 10 mM β-glycerophosphate; 1 mM EDTA) and protease inhibitor cocktail (Sigma-Aldrich) for 15 minutes on ice. Lysates were cleared by centrifugation at 14,000g for 10 minutes at 4°C, and samples were incubated with or without rabbit anti-HA antibody (diluted 1:200; Sigma-Aldrich) for 2 hours at 4°C, after which 30 µl protein A-Sepharose beads (Sigma-Aldrich) were added, and the samples were incubated for a further 1 hour at 4°C. Beads were washed 3× with wash buffer [0.5% NP40, 50 mM Tris (pH 8.0), 150 mM NaCl] and then boiled for 5 minutes in Laemmli buffer. Samples (20 µl) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with mouse anti-FLAG M2 horseradish peroxidase–conjugated antibody (diluted 1:5000; Sigma-Aldrich) to detect FLAG-SULT binding. To validate HA-SULT pulldown, blots were incubated with mouse anti-HA antibody (diluted 1:1000; Sigma-Aldrich) followed by anti-mouse horseradish peroxidase–conjugated secondary IgG (diluted 1:30,000; Jackson ImmunoResearch Laboratories). Protein bands were visualized with Immu-Star ECL reagent (Bio-Rad Laboratories) as per the manufacturer’s instructions.
Western Blots.
At the times indicated, cells were washed twice with cold PBS, lysed directly in Laemmli buffer, and boiled for 5 minutes. Samples (10–20 µg protein) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for FLAG- and HA-tagged SULT proteins, endogenous SULT1A1/3 (anti-SULT1A1/3 antibody detects both isoforms and was a gift from Professor Mike Coughtrie, The University of British Columbia, Vancouver, Canada; diluted 1:1000), SULT4A1 (Proteintech, diluted 1:1000), and α-tubulin (diluted 1:2000; Cell Signaling Technology) as previously described (Sidharthan et al., 2013, 2014).
Small Interference RNA Studies.
SH-SY5Y cells were transiently transfected with small interference RNAs (siRNAs) directed against SULT4A1 (SR308535 A, B, and C; Origene Technologies) for a total of 72 hours using LipofectAMINE RNAiMAX (Invitrogen) according to manufacturer’s protocol. A universal scrambled siRNA (SR30004; Origene Technologies) was used as a negative control. All siRNAs were used at a final concentration of 20 nM. SULT4A1 gene knockdown was evaluated by Western blot.
Dopamine Toxicity.
SH-SY5Y cells were seeded in 96-well plates at a density of 2000 cells/well and allowed to adhere overnight. The cells were then treated with up to 1 mM dopamine (Sigma-Aldrich) in culture medium for 48 hours. Cell viability was then assessed using the CyQuant Cell Proliferation Assay kit (Invitrogen), according to the manufacturer’s protocol.
Statistical Analysis.
Data are presented as mean ± S.E.M. unless otherwise noted. Significant differences were determined by Student’s t test (P < 0.05) or ANOVA as indicated. IC50 values were estimated by fitting the dopamine toxicity data to a dose-response curve using nonlinear least-squares regression (GraphPad Prism).
Results
Colocalization of SULT4A1 and SULT1A1/3 in Human Brain.
The physiologic relevance of any interaction between the sulfotransferases is predicated on whether the proteins are expressed in the same cell type(s). Therefore, immunocytochemistry was performed with human brain tissue from two different regions to determine whether SULT4A1 colocalizes with SULT1A1/3 (Fig. 1). The SULT1A1/3 antibody recognizes both proteins (Riches et al., 2009), so it was not possible to determine whether the SULT1A1/3 staining represents either or both sulfotransferases. Nevertheless, in the anterior cingulate cortex, all cells that expressed SULT1A1/3 also expressed SULT4A1 (Fig. 1A). Moreover, these cells were of neuronal origin, as identified by costaining with anti-MAP2 antibody. There were some cells that expressed SULT4A1 but were negative for SULT1A1/3 (Fig. 1A, yellow arrows). These cells were also negative for MAP2 staining, suggesting they may be of non-neuronal origin. A similar colocalization was seen in the substantia nigra, where both of the sulfotransferases were expressed in dopaminergic neurons, which were identified by costaining with anti-tyrosine hydroxylase antibody (Fig. 1B). These results demonstrate colocalization of SULT4A1 with SULT1A1 and/or SULT1A3 in human brain and suggest that any interaction between these proteins may produce a physiologic effect.
SULT4A1, SULT1A1, and SULT1A3 Proteins Form Homodimers and Heterodimers that Require the Sulfotransferase Dimerization Motif.
Using HeLa cells that do not express SULT1A1/3 or SULT4A1, FLAG-tagged and HA-tagged SULTs were coexpressed and then coimmunoprecipitated with anti-HA antibody. Initially, each SULT was coexpressed with itself to determine homodimerization (Fig. 2A). Western blots for FLAG-tagged SULTs that coimmunoprecipitated with HA-tagged SULTs showed that all three SULTs formed homodimers in HeLa cells. Western blots of samples prior to immunoprecipitation (Input) confirmed expression of each FLAG-tagged SULT in the cells (Fig. 2A, lanes 2 to 3 in each blot). Furthermore, both expression (Input) and immunoprecipitation of the HA-tagged proteins were also confirmed (Fig. 2A, lower panels). Omitting the HA antibody showed there was no nonspecific binding to protein A beads (Fig. 2A, lanes 4–6 in each blot).
Coexpression of the different SULTs as FLAG-tagged and HA-tagged proteins was then performed to detect heterodimerization. All three proteins interacted with each other as detected by immunoprecipitation (Fig. 2B, last lanes). To determine whether the dimerization motif was required for protein-protein interactions, both SULT1A1 and SULT1A3 were mutated to the sequence found in mouse Sult1e1, which does not form homodimers (T269P, V270E) (Petrotchenko et al., 2001). Mutants bound neither to SULT4A1 nor to each other (Fig. 3, last lanes). Taken together, these results suggest that SULT4A1, SULT1A1, and SULT1A3 can interact with each other in the context of the whole cell. Moreover, this interaction appears to occur via the conserved dimerization motif in the C terminus of each protein.
A yeast-2–hybrid assay and an in vitro pulldown of glutathione S-transferase–tagged recombinant proteins were also investigated to confirm the immunoassay results. In both assays, SULT4A1 was shown to bind to SULT1A1/3 (Supplemental Fig. 1; Supplemental Table 1, respectively).
SULT4A1 Protein Expression Decreases SULT1A1/3 Protein Levels.
HeLa cells were transiently transfected with SULT4A1 and either SULT1A1 or SULT1A3, after which protein levels were determined by Western blotting. In the presence of SULT4A1, expression of SULT1A1 and SULT1A3 was significantly attenuated by 60% and 64%, respectively, compared with empty vector controls (Fig. 4A). To determine whether a similar effect is seen with endogenous SULTs, SK-N-MC cells were used because these cells constitutively express both SULT1A1 and SULT1A3 but very low levels of SULT4A1 (Fig. 4B). In cells stably transfected with SULT4A1, endogenous SULT1A1 and SULT1A3 proteins were decreased by 90% and 85%, respectively (Fig. 4B). However, when cells were stably transfected with a SULT4A1 mutant, there was no change in SULT1A1 or SULT1A3 protein (Fig. 4C). These experiments show that the presence of SULT4A1 leads to a decrease in SULT1A1/3 protein, most likely after protein-protein interactions.
It has been shown previously that SULT4A1 is induced after differentiation of SK-N-MC cells with retinoic acid (Sidharthan et al., 2013). Since SULT1A1 and SULT1A3 are constitutively expressed in these cells, this was a good model to determine whether upregulation of endogenous SULT4A1 affects endogenous SULT1A1/3 expression. The levels of SULT1A1/3 protein were quantified before and after treatment with 10 µM retinoic acid for 10 days (Fig. 5). There was an 11-fold increase in SULT4A1 protein after differentiation, which was accompanied by 20% and 50% decreases in SULT1A1 and SULT1A3 proteins, respectively (P < 0.05, Student’s t test). To determine whether this change was due to the increase in SULT4A1, cells were differentiated with retinoic acid and then treated with siRNA directed against SULT4A1 mRNA. Figure 6 (upper blot) shows efficient downregulation of SULT4A1 by the siRNA compared with a scrambled sequence. After treatment, the loss in SULT1A1/3 seen in Fig. 5 was reversed. These results show that endogenous SULT4A1 may regulate the level of SULT1A1/3 protein expression in cells.
SULT4A1 Upregulation During Neuronal Differentiation Enhances Dopamine Toxicity.
Dopamine is toxic to most cells, but this can be attenuated by SULT1A3, which metabolizes dopamine to dopamine sulfate (Sidharthan et al., 2013). Thus, a change in SULT1A3 expression leads to a change in cell survival. Unlike SK-N-MC cells, SH-SY5Y cells express low levels of SULT4A1 and SULT1A1 but high levels of SULT1A3. After 10 µM retinoic acid treatment of 10 days, the SH-SY5Y cells showed characteristic dendrite formation consistent with a neuronal-like phenotype (Fig. 7A). Moreover, the neuron-specific marker neuronal nuclei was up-regulated (Fig. 7B). Similar to that seen in the SK-N-MC cells, SULT4A1 protein increased, whereas SULT1A3 protein decreased upon differentiation. Expression of SULT1A1 was low both before and after treatment (Fig. 7B).
To determine whether the downregulation of SULT1A3 by endogenous SULT4A1 affects dopamine toxicity, SH-SY5Y cells were treated with increasing concentrations of dopamine before and after differentiation. The toxicity of dopamine appeared to be inversely related to SULT1A3 expression levels. This is seen in Fig. 8A, in which the IC50 of dopamine decreased from 137 ± 16 µM in undifferentiated cells (high SULT1A3) to 15 ± 1 µM after retinoic acid treatment (low SULT1A3). To determine whether this increase in toxicity was related to an increase in SULT4A1, the differentiated cells were treated with siRNA for SULT4A1 or a scrambled sequence as a control. Two independent siRNAs were used (B and C), both of which decreased SULT4A1 protein over 72 hours (Fig. 8B). Moreover, both siRNAs reversed the increase in dopamine toxicity to that seen in the undifferentiated cells (Fig. 8C). By contrast, the scrambled sequence had no effect on toxicity.
Discussion
Despite the discovery of SULT4A1 in the brain almost 20 years ago, there has been little work to uncover its role in mammalian cells. When its expression was inhibited in a zebrafish model, distinct phenotypic changes were seen, including sedentary behavior (Crittenden et al., 2015), and upregulation of genes associated with phototransduction (Crittenden et al., 2014). In a mouse gene knockout model, a severe phenotype was observed with animals surviving only 20–25 days postbirth (Garcia et al., 2018). A recent study showed that SULT4A1 expression in SH-SY5Y cells altered mitochondrial function and protected the cells from hydrogen peroxide–induced cell death (Hossain et al., 2019). Interestingly, it also reported that SULT4A1 was colocalized with the mitochondrial outer membrane translocase protein Translocase of outer membrane 20 kDa subunit, which forms part of a complex that imports proteins from the cytosol into mitochondria. The possible interaction of SULT4A1 with the translocase of outer membrane complex may be important for its role in altering mitochondrial function. In the present study, SULT4A1 colocalized with SULT1A1/3 in human neurons in at least two separate regions of the brain. SULT4A1 was also present in non-neuronal cells in the absence of the other sulfotransferases. SULT4A1 heterodimerized with both SULT1A1 and SULT1A3, and this interaction required an intact dimerization motif. This is the first study to identify heterodimerization between these proteins. Using ectopic expression of the sulfotransferases as well as changes in endogenous levels during cell differentiation, SULT4A1 was shown to decrease both SULT1A1 and SULT1A3 protein levels. For SULT1A3, this resulted in an increase in the toxicity of dopamine, a substrate for the enzyme. Collectively, these results show a reciprocal relationship between SULT4A1 and SULT1A1/3 in neuronal cells. The mechanism behind SULT4A1-induced SULT1A1/3 protein loss remains unclear and requires further study. One possible explanation may be that heterodimerization of the SULTs increases protein degradation, which is supported by the fact that mutation of the dimerization site prevents protein loss.
There is an emerging picture of SULT4A1 regulation as a complex cellular process involving mRNA splicing, post-translational modification, and protein-protein interactions. SULT4A1 mRNA is found in most tissues in the body but primarily as an aberrant transcript that does not translate a stable protein (Sidharthan et al., 2014). After correct splicing that removes a pseudoexon located between exons 6 and 7, the full-length stable protein is generated. Thus, SULT4A1 protein levels are regulated at a post-transcriptional level through alternative splicing. SULT4A1 expression is also regulated by both transcriptional and post-translational processes (Mitchell and Minchin, 2009; Butcher et al., 2010). In neuronal-like cells, SULT4A1 transcription is under the control of the cAMP response element–binding protein and the activating transcription factor-2 (Butcher et al., 2010). Post-translationally, the protein is phosphorylated in an ERK1/2-dependent manner, which promotes binding of the peptidyl-prolyl cis-trans isomerase Pin1 and subsequent SULT4A1 degradation. These pathways are summarized in Fig. 9.
In a previous study using SK-N-MC cells, low dopamine concentrations were shown to increase SULT1A1 and SULT1A3 transcription through activation of the D1 receptor (Sidharthan et al., 2013). Part of this pathway involved upregulation of mitogen-activated protein kinase kinase-ERK1/2 signaling. Thus, ERK1/2 is a point of convergence for the regulation of both SULT1A1/3 and SULT4A1 (Fig. 9). However, although ERK1/2 upregulates SULT1A1/3, it downregulates SULT4A1. The dopamine-ERK1/2-SULT1A3 axis may provide a feedback loop to regulate dopamine levels in the brain. Controlling SULT4A1 protein levels may be an additional pathway to ensure adequate SULT1A3 protein in the presence of excess dopamine. This may be an important observation that warrants further investigation. In addition, there are a number of drugs that can modulate ERK1/2 activity, including serotonin (Li et al., 2010), amphetamines (Choe and Wang, 2002), isoproterenol (Du et al., 2010), and cocaine (Hoffmann et al., 2012). Whether these treatments alter the levels of sulfotransferases in the brain remains to be determined.
Acknowledgments
The authors thank Professor Mike Coughtrie, The University of British Columbia, for the generous gift of SULT1A1/3 antibody.
Authorship Contributions
Participated in research design: Butcher, Minchin.
Conducted experiments: Idris, Mitchell, Gordon, Sidharthan, Butcher.
Performed data analysis: Idris, Minchin.
Wrote or contributed to the writing of the manuscript: Idris, Butcher, Minchin.
Conflict of interest. The authors declare that there are no competing interests associated with the manuscript.
Footnotes
- Received October 20, 2019.
- Accepted January 28, 2020.
This work was supported by the National Health and Medical Research Council of Australia [Grant NHMRC 511175 and 1005899].
↵This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- ERK
- extracellular signal-regulated kinase
- HA
- hemagglutinin
- MAP2
- microtubule-associated protein 2
- Pin1
- peptidyl-prolyl cis-trans isomerase never-in-mitosis-gene-A–interacting 1
- RA
- retinoic acid
- siRNA
- small interference RNA
- SULT
- sulfotransferase
- Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics