Abstract
The intestinal trefoil factor (ITF/TFF3) protects intestinal epithelia from a range of insults and contributes to mucosal repair. However, the signaling events that mediate healing responses are only partially understood. To identify ITF signaling pathways, proteins that were Ser/Thr phosphorylated in response to ITF stimulation were immunoprecipitated from human colon carcinoma cell lines and identified by mass spectrometry. We demonstrated that Van Gogh-like protein 1 (also designated Vang-like 1 or Vangl1), a protein with four transmembrane domains, was Ser/Thr phosphorylated in response to ITF stimulation. Vangl1 was present in normal human colon and all intestinal epithelial cell lines (IEC) tested. In transfected IEC, FLAG-Vangl1 was mostly present in the Nonidet P-40 soluble fraction as detected by Western blotting, corresponding to the localization of endogenous protein in cytoplasmic vesicular structures by confocal microscopy with rabbit polyclonal anti-human Vangl1 antibody (alpha-Vangl1). Vangl1 cell membrane association increased with differentiation, as demonstrated by co-localization with E-cadherin in differentiated IEC. Increased Vangl1 phosphorylation after stimulation with ITF corresponded to decreased cell membrane association with E-cadherin. Functionally, Vangl1 overexpression enhanced ITF unstimulated and stimulated wound closure of IEC, whereas siRNA directed against Vangl1 inhibited the migratory response to ITF. Vangl1 protein may serve as an effector mediating the ITF healing response of the intestinal mucosa.
Highlights
MATERIALS AND METHODSAntibodies and Reagents—Phospho-Ser/Thr antibodies were as follows: anti-phospho-Ser/Thr (Phe) rabbit polyclonal antibody (␣-Ser/ Thr, clone number 9631, Cell Signaling, Beverly, MA) detects phosphoserine/threonine in the context of tyrosine, tryptophan, or phenylalanine at the Ϫ1 position or phenylalanine at the ϩ1 position
ITF is a 6-kDa polypeptide [6], naturally occurring as a 12-kDa protease-resistant homo-dimer (6 –9), that is abundantly secreted onto the mucosal surface by the goblet cells of the distal gastrointestinal tract [6]
ITF is associated with intestinal restitution, a process necessary for healing of wounded intestinal mucosa [5]
Summary
Antibodies and Reagents—Phospho-Ser/Thr antibodies were as follows: anti-phospho-Ser/Thr (Phe) rabbit polyclonal antibody (␣-Ser/ Thr, clone number 9631, Cell Signaling, Beverly, MA) detects phosphoserine/threonine in the context of tyrosine, tryptophan, or phenylalanine at the Ϫ1 position or phenylalanine at the ϩ1 position. 2 mg of protein in cell lysates from transfected cells (HCT116 after starvation and stimulation) were incubated overnight with 2 g of either ␣-FLAG M2 or ␣-Ser/Thr antibodies together with 30 l of Hitrap protein A/G-Sepharose beads (Amersham Biosciences) for 12 h of rocking at 4 °C. HCT116 cells were cultured on 6-well plates and transfected with plasmids containing FLAG-Vangl or GFP control. 24 h after transfection, cells were starved for 24 h in serum-free Dulbecco’s modified Eagle’s medium, washed with phosphate-free Dulbecco’s modified Eagle’s medium (Invitrogen), and incubated for 10 min, 1, or 6 h with 37 °C warmed phosphate-free Dulbecco’s modified Eagle’s medium containing 0.15 mCi/ml 32Pi (H332P-O4, PerkinElmer Life Sciences). Cells were blocked for 30 min with TBST (Tris-buffered saline with 0.1% Tween 20) containing 5% goat serum and incubated 2 h with primary antibody as indicated. A p value of 0.05 was considered to be statistically significant
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