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

Abstract

Enteropathogenic (EPEC), one of the diarrheagenic 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 NaCl-sensitive [H]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. 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.

Serotonin is an endogenous regulator of intestinal CYP1A1 via AhR

“…For SERT overexpression experiments, Caco-2 cells were transiently transfected by electroporation utilizing Amaxa Nucleofector T (Lonza) as described 61 . For transient transfection experiments without SERT overexpression, Caco-2 cells were transiently co-transfected with pGudLuc7.5 XRE luciferase reporter plasmid (kind gift from Dr. Michael Denison, University of California, Davis) and CMV- β , a β -gal mammalian expression plasmid (BD Biosciences) using lipofectamine 2000 transfection reagent (Invitrogen) as recommended by the manufacturer.…”

Post-translational modifications of transporters

“…Additionally, the serotonin transporter (SERT; SLC6A4 ) (25, 26) and apical sodium-dependent bile acid transporter (ASBT; SLC10A2 ) (27) are tyrosine phosphorylated and regulated by Src family kinases, suggesting that TKIs may have some off-target risk (22-24, 28-30). Both ASBT and SERT have reduced pTyr status and reduced function during enteropathogenic E. coli (EPEC) infections (25-27).…”

Environment Controls LEE Regulation in Enteropathogenic Escherichia coli

“…In fact, EPEC infection causes an increase in epithelial short circuit current that is partially dependent on an induction of chloride secretion ( Collington et al, 1998 ). EPEC infection causes an increase in protein tyrosine phosphatase (PTPase), specifically by activation of Src-homology-2 domain containing PTPase, which decreases tyrosine phosphorylation of the serotonin transporter, decreasing transporter function and thus deregulation of serotonin uptake ( Singhal et al, 2017 ). The EPEC T3SS is necessary for many instances of host homeostasis disruption.…”