Role of SHP2 protein tyrosine phosphatase in SERT inhibition by enteropathogenic E. coli (EPEC)

The data presented in the current study reveal that intestinal serotonin transporter (SERT) is a target of the tyrosine phosphatase SHP2 and show a novel mechanism by which a common diarrheagenic pathogen, EPEC, activates cellular SHP2 to inhibit SERT function. These studies highlight host-pathogen interactions, which may be of therapeutic relevance in the management of diarrhea associated with enteric infections.

Abstract

Enteropathogenic Escherichia coli (EPEC), one of the diarrheagenic E. coli pathotypes, is among the most important food-borne pathogens infecting children worldwide. Inhibition of serotonin transporter (SERT), which regulates extracellular availability of serotonin (5-HT), has been implicated previously in EPEC-associated diarrhea. EPEC was shown to inhibit SERT via activation of protein tyrosine phosphatase (PTPase), albeit the specific PTPase involved is not known. Current studies aimed to identify EPEC-activated PTPase and its role in SERT inhibition. Infection of Caco-2 monolayers with EPEC strain E2348/69 for 30 min increased the activity of Src-homology-2 domain containing PTPase (SHP2) but not SHP1 or PTPase 1B. Similarly, Western blot studies showed increased tyrosine phosphorylation of (p-tyrosine) SHP2, indicative of its activation. Concomitantly, EPEC infection decreased SERT p-tyrosine levels. This was associated with increased interaction of SHP2 with SERT, as evidenced by coimmunoprecipitation studies. To examine whether SHP2 directly influences SERT phosphorylation status or function, SHP2 cDNA plasmid constructs (wild type, constitutively active, or dominant negative) were overexpressed in Caco-2 cells by Amaxa electroporation. In the cells overexpressing constitutively active SHP2, SERT polypeptide showed complete loss of p-tyrosine. In addition, there was a decrease in SERT function, as measured by Na⁺Cl⁻-sensitive [³H]5-HT uptake, and an increase in association of SERT with SHP2 in Caco-2 cells expressing constitutively active SHP2 compared with dominant-negative SHP2. Our data demonstrate that intestinal SERT is a target of SHP2 and reveal a novel mechanism by which a common food-borne pathogen uses cellular SHP2 to inhibit SERT.

NEW & NOTEWORTHY The data presented in the current study reveal that intestinal serotonin transporter (SERT) is a target of the tyrosine phosphatase SHP2 and show a novel mechanism by which a common diarrheagenic pathogen, EPEC, activates cellular SHP2 to inhibit SERT function. These studies highlight host-pathogen interactions, which may be of therapeutic relevance in the management of diarrhea associated with enteric infections.

despite major advances in the treatments, diarrheal diseases cause >1.2 million childhood deaths per year. Although oral rehydration therapy has significantly reduced mortality from infectious diarrhea (33), more efficacious drugs are needed to reduce morbidity and fluid loss associated with diarrhea caused by enteric infections. Thus agents that decrease intestinal fluid or ion secretion and/or increase absorption offer a number of potential drug targets to treat diarrhea. Among these, serotonin (5-HT) is an attractive candidate, because it is a well-known secretagogue, reduces Na⁺Cl⁻ absorption, and alters gastrointestinal motility (29, 31). In fact, elevated serotonin (5-HT) levels and a decrease in serotonin transporter (SERT) in the gut are known to underlie gastrointestinal disturbances, such as diarrheal and inflammatory disorders (2, 6, 39). Therefore, the regulation of the luminal availability of 5-HT may be beneficial to prevent diarrheal diseases.

5-HT availability is under the control of SERT, which limits the actions of 5-HT via its rapid uptake through an Na⁺Cl⁻-dependent process (5, 15, 36). In the absence of SERT, 5-HT remains in the extracellular space, allowing prolonged 5-HT receptor activation/desensitization. In fact, several lines of evidence indicate perturbation of SERT expression in gut disorders (11, 21, 29). For example, deletion of SERT in mice results in increased stool water content (indicative of a diarrheal phenotype), an abnormal pattern of motility, and increased susceptibility to colitis in IL-10-deficient mice, as well as in trinitrobenzene sulfate-treated mice (16, 25). However, little is known regarding modulation of intestinal SERT by enteric pathogens that cause diarrhea. Indeed, our recent studies demonstrated that SERT is a target of an important food-borne pathogen, enteropathogenic Escherichia coli (EPEC) (10).

EPEC is a major cause of infantile diarrhea, leading to dehydration and contributing to significant morbidity and mortality in infants worldwide (8, 34). EPEC does not express a toxin and is not invasive; therefore, the pathophysiology of EPEC-associated diarrhea has been more elusive. EPEC, however, induces changes in host epithelial cell functions via a type III secretion system (T3SS) that injects bacterial virulence factors directly into host cells (17, 32). Our previously published studies show that EPEC-induced diarrhea is a multifactorial process involving a decrease in Na⁺ and Cl⁻ transport, as well as an inhibition of SERT, leading to increased luminal 5-HT levels (10). The bacterium T3SS can affect a number of host cellular functions, such as microtubule network disruption by E. coli-secreted protein G (EspG), barrier disruption by EspF, alteration of pedestal or filopodia formation by EspH, and changes in mitochondrial membrane potential by mitochondrial-associated protein (8). However, our previous studies showed that deletion of these T3SS effector molecules did not influence SERT inhibition by EPEC, ruling out their involvement (10). Interestingly, EPEC-induced inhibition of SERT and activation of host protein tyrosine phosphatases (PTPases) are dependent on the T3SS, although the detailed mechanisms have not been defined.

Given that SERT inhibition by EPEC is PTPase dependent, the current study was undertaken to identify the specific PTPase involved and investigate the PTPase-dependent mechanism leading to EPEC inhibition of SERT. The results demonstrate that EPEC infection of intestinal epithelial cells induces the activity of the Src-homology-2 (SH2) domain containing PTPase (SHP2) but not SHP1 or PTPase 1B (PTP1B). Furthermore, EPEC infection increased the association of SHP2 with SERT, causing SERT dephosphorylation at tyrosine residues. Similar to EPEC, overexpression of constitutively active SHP2 decreased SERT phosphorylation and reduced its activity compared with cells expressing dominant-negative SHP2. These data indicate that EPEC-induced activation of SHP2 dephosphorylates SERT, inhibiting its function and providing the molecular basis for the role of intestinal SERT in the pathogenesis of EPEC-associated diarrhea. These studies define novel post-translational mechanisms of SERT regulation in intestinal epithelial cells and may be critical in exploiting SERT as an important pharmacological target.

MATERIALS AND METHODS

Cell culture.

Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in MEM containing 20% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For experiments, monolayers of confluent cells were used after 12 days of plating on 24-well plastic support.

Bacterial culture and cell infection.

Caco-2 monolayers were infected with wild-type EPEC strain E2348/69 at a multiplicity of infection of 100, as described previously (14, 20).

Transfection and [³H]serotonin uptake in Caco-2 monolayers.

Plasmid constructs of SHP2 (constitutively active, dominant negative, and wild-type) were a generous gift from Elizabeth A. Eklund (Northwestern University, Chicago, IL). Briefly, Caco-2 cells were transfected with wild-type, constitutively active, and dominant-negative SHP2 constructs using Amaxa electroporation, as described previously (10). Serotonin uptake studies were performed in transfected Caco-2 cells, as described previously (30). To determine the sodium dependency of serotonin uptake, Na⁺ in the buffer was isosmotically replaced with choline chloride. Uptake rates were measured as picomoles per milligram of protein per 5 min.

Western blotting.

Western blotting was carried out as described previously (10). After 60 min of EPEC infection, cells were washed with ice-cold PBS and lysed in cell lysis buffer (Cell Signaling Technology, Danvers, MA), freshly supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN). Following Bradford assays, proteins were separated on 7.5% SDS-PAGE gel and blotted onto a nitrocellulose membrane (EMD Millipore, Billerica, MA). The blotted membrane was blocked in PBS-Tween 20 containing 5% nonfat milk for 1 h at room temperature. Phosphorylated (p)-SHP2, p-SHP1, and p-PTP1B antibodies were used in Tris-buffered saline with Tween 20 containing 5% nonfat milk, and the blot was incubated at 4°C overnight in a rotary shaker. The bands were analyzed by enhanced chemiluminescence in an ECL kit, according to the manufacturer’s instructions (GE Healthcare Bio-Sciences, Pittsburgh, PA).

Immunoprecipitation.

Following EPEC infection, whole cell lysates and lysates of cells transfected with SHP2 constructs were incubated with anti-SERT antibody (Abcam, Cambridge, UK) at 4°C overnight in a rotary shaker. Lysates were incubated with protein A/G Plus-Agarose beads (Santa Cruz Biotechnology, Dallas, TX) for 2–3 h the following day. Agarose beads were collected by centrifugation, washed four times with PBS, supplemented with protease inhibitor cocktail, and heated to 95°C for 5 min after adding Laemmli buffer. The resulting immunoprecipitates were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with anti-SHP2 and p-tyrosine antibody (Cell Signaling Technology). Immunoblots were visualized using enhanced chemiluminescence (GE Healthcare Bio-Sciences).

In vitro phosphatase assay.

Phosphatase activity was assessed using the DuoSet IC Human/Mouse/Rat Active SHP2, according to the manufacturer’s instructions with some modifications. Briefly, immunoprecipitation capture assay was used to measure the activity of various phosphatases, including PTP1B, SHP1, and SHP2, in Caco-2 cell lysates. Protein A/G Plus-Agarose beads (Santa Cruz Biotechnology) were used instead of beads conjugated to SHP2 to measure the activity of various phosphatases. After washing away unbound material, a synthetic phosphopeptide substrate was added that is dephosphorylated by active phosphatase to generate free phosphate and unphosphorylated peptide. The beads were pelleted by centrifugation, and the supernatant was transferred to a microplate. The amount of free phosphate in the supernatant was determined by a sensitive dye-binding assay using malachite green and molybdic acid. The activity of phosphatase was determined by calculating the rate of phosphate release.

Statistics.

Results were expressed as means ± SE of three separate experiments performed on separate occasions with control taken as one. One-way ANOVA (Tukey) or Student’s t-test was used for statistical analysis. P < 0.05 or less was considered statistically significant.

RESULTS

Activation of SHP2 by EPEC.

Our previous studies demonstrated that EPEC-mediated inhibition of SERT in Caco-2 monolayers was dependent on the activation of PTPases (10). There are ~17 nonreceptor PTPases; of these, PTPN1 (PTP1B), PTPN2 (PTPT), PTPN6 (SHP1), PTPN11 (SHP2), and PTPN22 (PTPN8) are present in the intestine. Several enteric pathogens, including EPEC, are known to recruit SH2-containing PTPases to establish infection (3). To identify the specific host PTPase activated by EPEC, we examined in vitro PTP activity in Caco-2 cells using a commercially available kit. Nonreceptor PTPs (SHP2, SHP1, PTP1B) were immunoprecipitated from lysates of uninfected or EPEC-infected Caco-2 cells and incubated with phosphopeptides (26). Phosphate release into the supernatant was detected by addition of malachite green using the commercially available kit. SHP2 activity levels were increased significantly in response to EPEC infection (Fig. 1). However, there was no alteration in the activity of SHP1 or PTP1B (Fig. 1).

Fig. 1.

EPEC infection increases the activity of Src-homology-2 domain containing PTPase (SHP2) in Caco-2 cells. Fully differentiated Caco-2 cells (12 days postplating) were infected with EPEC for 60 min. Immunoprecipitation was carried out with SHP1, SHP2, and PTP1B antibodies from total cell lysates from control and EPEC-infected Caco-2 cells. Phosphate released in response to EPEC infection was measured using the phosphatase activity assay (DuoSet IC kit). Results are depicted as means ± SE of 3 separate experiments performed on separate occasions, with control taken as 1. *P < 0.05 vs. control by one-way ANOVA.

Increased phosphorylation of SHP2 by EPEC.

To examine further whether EPEC infection activates SHP2, Western blotting was performed. SHP2 is phosphorylated at Tyr 542 and Tyr 580 in its carboxy terminus in response to growth factor receptor activation. Phosphorylation alleviates basal inhibition and stimulates SHP2 tyrosine phosphatase activity (27). With the use of a specific antibody that detects phosphorylation at Tyr 542 of SHP2, our results demonstrated that EPEC infection increased phosphorylation of SHP2, indicating its activation (Fig. 2B). The total levels of SHP2 remained unaltered in control and EPEC-infected cells (Fig. 2B). Densitometric analysis showed a significant increase in p-SHP2 levels in EPEC-infected cells vs. uninfected cells. The phosphorylation levels of SHP1 and PTP1B were also examined using phospho-specific antibodies, anti-SHP1 (S591) and anti-PTP1B (Y152), respectively, following EPEC infection. Infection with EPEC did not increase the phosphorylation of either SHP1 or PTP1B (not shown). These data confirm that EPEC specifically activates SHP2 PTPase in host intestinal epithelial cells.

Fig. 2.

EPEC infection activates SHP2 PTPase in Caco-2 cells. Fully differentiated Caco-2 monolayers (12 days postplating) were either left uninfected or infected with EPEC for 60 min. Total cell lysates were analyzed by 7.5% SDS-PAGE, followed by transfer of proteins to nitrocellulose, and probed with phosphorylated (p)-SHP2 antibody. The blots were stripped and reprobed with the SHP2 antibody to normalize for equal loading of protein or equal input in each lane. A: Western blot for phosphorylation levels of SHP2 PTPase in response to EPEC infection. B: densitometric analysis of the phosphorylated levels of SHP2 PTPase. Results are expressed as arbitrary units of p-SHP2/total SHP2. Results are depicted as means ± SE of 3 separate experiments performed on separate occasions, with control (C) taken as 1. *P < 0.05 vs. control with Student’s t-test.

EPEC induces SERT tyrosine dephosphorylation.

The data shown above demonstrate that EPEC induces SHP2 activity. To confirm the involvement of SHP2 in EPEC-mediated inhibition on SERT, the effect of EPEC infection on SERT phosphorylation was examined. SERT was immunoprecipitated from control and EPEC-infected (60 min) lysates with anti-SERT antibodies and immunoblotted with p-tyrosine MAb. Compared with control cells, infection with EPEC decreases p-tyrosine of the SERT polypeptide (Fig. 3, A and B). This decrease can be attributed to increased SHP2 activity following EPEC infection.

Fig. 3.

EPEC infection induces dephosphorylation of SERT at tyrosine residues. SERT was immunoprecipitated (IP) from whole cell lysates prepared from control or EPEC-infected cells. The samples were analyzed by 7.5% SDS-PAGE, followed by transfer of proteins to nitrocellulose and probed with p-tyrosine antibody. The blots [immunoblots (IB)] were stripped and reprobed with the SERT antibody to normalize for input. A: representative Western blot showing SERT dephosphorylation at tyrosine residue and total levels of SERT. B: densitometric analysis showing phosphorylated SERT levels. Results are expressed as p-tyrosine/total SERT. Results are depicted as means ± SE of 3 separate experiments performed on separate occasions, with control taken as 1. *P < 0.05 vs. control with Student’s t-test.

Involvement of SHP2 in SERT dephosphorylation.

To confirm directly the involvement of SHP2 in SERT dephosphorylation, cDNA plasmid constructs of SHP2 (constitutively active, dominant negative, and wild type) were overexpressed in Caco-2 cells. Caco-2 cells were transfected with a vector to overexpress wild-type SHP2, a dominant-negative form of SHP2 (C463S-SHP2), or a constitutively active form of SHP2 (E76K-SHP2). Vector overexpressing wild-type SHP2 was used as a control. Transfectants were analyzed for endogenous SERT p-tyrosine levels. For these studies, lysates from different transfectants were immunoprecipitated using SERT antibody and then probed with p-tyrosine antibody. Similar to EPEC infection, expression of constitutive SHP2 in Caco-2 reduced SERT phosphorylation (Fig. 4A). Densitometric analysis of phosphorylation levels normalized to cellular levels of SERT (Fig. 4B) showed a complete loss of SERT phosphorylation in cells overexpressing the constitutively active form of SHP2.

Fig. 4.

Role of SHP2 PTPase in modulating SERT phosphorylation. SHP2 PTPase constructs [wild-type (Wt), constitutively active, and dominant negative (-ve)] were overexpressed in Caco-2 cells by Amaxa electroporation. SERT was immunoprecipitated from whole cell lysates and probed with p-tyrosine antibody to check the phosphorylation levels of SERT at tyrosine residues. The blot was also probed with SHP2 PTPase antibody to show that the total levels of the phosphatase remained unchanged. A: representative Western blot for phosphorylation levels of SERT at tyrosine residues and SHP2 PTPase levels in total lysates. B: densitometric analysis for phosphorylated SERT levels. Results are expressed as p-tyrosine/total SERT. Values represent means ± SE of 3 different experiments. ***P < 0.005 vs. control with one-way ANOVA.

Activation of SHP2 inhibits SERT function.

The previous studies show an inhibition of SERT in response to EPEC infection of Caco-2 cells (10). As demonstrated above, EPEC activates SHP2 and dephosphorylates SERT. Thus we next examined whether activation of SHP2 directly inhibits SERT activity. For these studies, Na⁺Cl⁻-sensitive [³H]5-HT uptake was measured in cells transfected with different SHP2 constructs. Interestingly, compared with wild-type SHP2-transfected cells, overexpression of constitutively active SHP2 inhibited SERT function to a similar degree as EPEC. In contrast, overexpression of dominant-negative SHP2 increased SERT function (Fig. 5). These data provide direct evidence for the reduction in SERT activity in intestinal epithelial cells via SHP2-induced tyrosine dephosphorylation.

Fig. 5.

Constitutively active construct of SHP2 PTPase inhibits SERT function. Caco-2 cells were transiently transfected with the SHP2 PTPase constructs (wild-type, constitutively active, and dominant negative) by Amaxa electroporation. SERT function was measured 24 h post-transfection as [³H]5-HT uptake (control values in picomoles per milligram of protein per 5 min). *P < 0.05 vs. control, n = 3, by one-way ANOVA.

SHP2 directly binds to SERT.

SHP2 is preferentially localized to the cytoplasm; however, SHP2 binding to a p-tyrosine-containing ligand promotes relocalization of SHP2 to the cell membrane (12). In this regard, our previous studies show that EPEC infection inhibits SERT function at the membrane level, independent of membrane trafficking events. Thus it was critical to examine whether EPEC induces SHP2 activation and interaction with SERT to inhibit its function. For these studies, SERT was immunoprecipitated from control and EPEC-infected cells and then probed for SHP2. The data showed that infection with EPEC increases the association of SERT with SHP2 (Fig. 6). Overexpression of constitutively active SHP2 mimicked the effects of EPEC infection in increasing association of SERT with SHP2 (Fig. 7). These data suggest that SERT and SHP2 are present in the same complexes on the membrane in response to EPEC infection or constitutive activation of SHP2 in Caco-2 cells.

Fig. 6.

Interaction of SHP2 PTPase and SERT enhances in response to EPEC infection. SERT was immunoprecipitated from the control and EPEC-infected whole cell lysates. The samples were analyzed by 7.5% SDS-PAGE, followed by transfer of proteins to nitrocellulose membrane, and probed with SHP2 antibody. The blots were stripped and reprobed with the SERT antibody to normalize for equal loading of protein or equal input in each lane. A: representative Western blot showing SHP2 PTPase levels in SERT-immunoprecipitated samples post-EPEC infection. B: densitometric analysis for SHP2 PTPase with total levels of SERT. Values represent means ± SE of 3 different experiments. *P < 0.05 vs. control with Student’s t-test.

Fig. 7.

Interaction of SERT with SHP2 PTPase in Caco-2 cells. Caco-2 cells were transiently transfected with wild-type (w+), constitutively active (c+), and dominant-negative (e−) constructs of SHP2 PTPase by Amaxa electroporation. SERT was immunoprecipitated from the whole cell lysates. The samples were analyzed by 7.5% SDS-PAGE, followed by transfer of proteins to nitrocellulose, and probed with SHP2 antibody. The blots were stripped and reprobed with the SERT antibody to show equal loading. A: representative Western blot showing the association of SHP2 with SERT in different constructs of SHP2 PTPase. B: densitometric analysis for SHP2 PTPase with total levels of SERT. Values represent means ± SE of 3 different experiments. **P < 0.05 vs. control by one-way ANOVA test.

DISCUSSION

Our previous studies using Caco-2 cells as an in vitro cell culture model and C57BL/6J as an in vivo mouse model demonstrated a two-phase process of SERT inhibition by EPEC. In the first phase, SERT function is reduced in a bacterial T3SS-dependent manner and via involvement of cellular PTPases, as evidenced in the in vitro model of acute infection. In the second phase, a decrease in SERT mRNA and protein expression occurs, as evidenced in the in vivo mouse model (10). The current data extend our previous studies to demonstrate that EPEC infection specifically activates SHP2, and not SHP1 or PTP1B, causing tyrosine dephosphorylation of the SERT polypeptide, thus inhibiting its function in Caco-2 cells, a model intestinal epithelial cell line. Although derived from the large intestine, Caco-2 cells manifest many features of enterocytes upon differentiation, including expression of small intestinal transporters, tight junctions, and microvilli. Over the years, they have proven to be a great in vitro model of human small intestinal epithelial cells to study regulation of intestinal transporters, including SERT (10, 13, 28).

PTPases belong to a structurally diverse family of tightly regulated enzymes, characterized by a conserved catalytic domain with an active-site cysteine residue essential for catalysis (35, 37). PTPases act in a coordinated fashion with PTKs to control numerous signaling pathways that regulate fundamental physiological processes (42). The PTPase family is broadly divided into transmembrane receptor-like proteins and nontransmembrane or nonreceptor cytoplasmic PTPs. Nonreceptor PTPases contain one PTP domain, which is mostly linked to domains mediating protein–protein interactions, such as the SH2 domain. Previous studies using RAW264.7 and human embryonic kidney 293 T cells demonstrated that the EPEC-secreted effector molecule, translocated intimin receptor, interacts with nonreceptor tyrosine phosphatases, SHP1 and SHP2, to suppress host immune response and establish infection (41). Similar mechanisms of interactions between SHP2 and bacterial proteins were reported for enterohemorrhagic E. coli and Epstein-Barr virus (18, 23). Interestingly, recent studies showed that the parasite Cryptosporidium parvum also uses SHP2 to increase infectivity in human intestinal epithelial cells (38).

Besides acting as negative regulators to oppose the effects of PTKs, PTPases, such as SHP2, have many biological cellular functions important for development and embryogenesis. Several lines of evidence indicate that SHP2 helps promote cell proliferation, differentiation, or survival via activation of the Ras–MAPK signaling pathway, functioning downstream of growth factor receptors and cytokines (7). SHP2 is also implicated in modulating the activity of the small GTP-binding protein Rho, which may play a role in cell adhesion or migration (24). Recent studies have shown that SHP2/MAPK signaling controls goblet and Paneth cell fate in the intestine (19) and may control epithelial homeostasis (40).

Interestingly, our studies demonstrated that EPEC infection specifically activates SHP2, but not SHP1 or PTP1B, in intestinal epithelial cells to inhibit SERT, which may contribute to the associated diarrhea. This is supported by the data demonstrating SHP2 activity and increased phosphorylation of SHP2 at Tyr 542 in response to EPEC infection. In general, SHP2 exhibits a low basal catalytic activity due to autoinhibition caused by close interactions between the SH2 and PTP domains (22). Activation of SHP2 requires releasing these interactions to open the molecule. Activating mutations in SHP2 (E76K- SHP2) have been identified in samples from human subjects with myelodysplastic syndromes, acute myeloid leukemia, or juvenile chronic myelomonocytic leukemia (4).

For establishing a direct functional link between SHP2 and SERT inhibition, we overexpressed different SHP2 mutants, representing constitutively active SHP2 (E76K- SHP2), and compared them with dominant-negative SHP2 (C463S-SHP2) or wild-type SHP2. Immunoprecipitation studies showed increased association between SERT and SHP2 in cells overexpressing constitutively active SHP2. In parallel, the cells overexpressing active SHP2 displayed loss of SERT p-tyrosine and an inhibition in SERT-mediated Na⁺Cl⁻-sensitive 5-HT uptake in Caco-2 cells. These studies indicate that SHP2-mediated dephosphorylation of SERT at tyrosine residues decreases its function. These studies show for the first time that SERT is a direct substrate for SHP2 in intestinal epithelial cells.

SERT is a 630-amino acid protein with 12 transmembrane domains. Our in silico analysis and previous reports have shown that SERT has multiple p-tyrosine sites (1). The study mentions that the SERT sequence contains four tyrosine residues (Tyr 47, Tyr 142, Tyr 350, and Tyr 358), which face the inside of the cell, and could be potential phosphorylation targets for tyrosine kinase(s)/phosphatases (1). These previous studies, using heterologous expression of SERT in human placental trophoblasts, demonstrated a role of p-tyrosine at residues Tyr 47 and Tyr 142 in enhancing SERT protein stability and promoting its activity (1). Further site-directed mutagenesis studies, aimed at investigating the role of potential tyrosine residues responsible for SHP2-mediated dephosphorylation of SERT, are warranted in intestinal epithelial cells. It is interesting to note that our previous studies showed that EPEC infection of Caco-2 cells decreased SERT via alteration in V_max (but not K_m) of the transporter and independent of any changes in the plasma membrane levels of SERT (10). Coimmunoprecipitation studies show that SERT and SHP2 are present in the same complex. Since SHP2 and SERT protein are of almost similar sizes (~72–75 kDa), it is possible that coimmunoprecipitation with SERT antibody and probing with p-tyrosine antibody detect phosphorylation levels of not only SERT but also other associated proteins, such as SHP2. In this regard, immunoprecipitation with SERT antibody showed a decrease in p-tyrosine of a 72-kDa band in cells infected with EPEC. A similar pattern in p-tyrosine levels was observed in Caco-2 cells with constitutive activation of SHP2 when immunoprecipitated with the SERT antibody. Furthermore, it remains to be determined what other chaperone proteins or downstream signaling events are involved in the recruitment of SHP2 to the plasma membrane, where SERT is primarily localized. We speculate that EPEC-induced activation of SHP2 dephosphorylates SERT, thereby altering its turnover rate at the membrane level (reflected in altered V_max) in response to EPEC infection. In conclusion, the studies presented here provide novel post-translational mechanisms of regulation of SERT in intestinal epithelial cells, which may be of therapeutic significance in diarrheal and inflammatory disorders.

GRANTS

Support for these studies was provided by the National Institute of Diabetes and Digestive and Kidney Diseases [Grants R01 DK098170 and R03 DK096258 (to R. K. Gill); R01 DK54016, R01 DK81858, and R01DK92441 (to P. K. Dudeja); and R01 DK71596 (to W. A. Alrefai)] and the U.S. Department of Veterans Affairs (VA; BX 002011, to P. K. Dudeja; BX002867-01A1, to S. Saksena; and BX000152, to W. A. Alrefai), VA Research Career Scientist Award (to W. A. Alrefai), and VA Senior Research Career Scientist Award (to P. K. Dudeja).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

R.K.G. conceived and designed research; M.S., C.M., V.S., and R.K.G. performed experiments; M.S., W.A.A., and R.K.G. analyzed data; R.K.G. interpreted results of experiments; M.S. and R.K.G. prepared figures; M.S. and R.K.G. drafted manuscript; M.S., C.M., W.A.A., S.S., G.A.H., P.K.D., and R.K.G. edited and revised manuscript; M.S., C.M., V.S., W.A.A., S.S., G.A.H., P.K.D., and R.K.G. approved final version of manuscript.