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The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

Yang J, Sarker R, Singh V, Sarker P, Yin J, Chen TE, Chaerkady R, Li X, Tse CM, Donowitz M - Biochem. J. (2015)

Bottom Line: The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin.Furthermore, phosphorylation of Ser(303) located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate.Moreover, increased phosphorylation of Ser(303) was functionally significant preventing acute stimulation of NHE3 (Na(+)-H(+) exchanger 3) activity by dexamethasone.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.

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Identification of NHERF2 phosphorylation sites Ser303 and Thr305 by LC–MS/MS analysis of FLAG–NHERF2 IP sampleNHERF2 sample was immunoprecipitated from Caco-2/Bbe cells and proteolysed for LC–MS/MS analysis. Two phosphorylation sites were identified within the EBRS of NHERF2. MS/MS spectrum (A) shows the phosphorylation at Ser303. Presence of b12 and y8 ions with neutral loss phosphate indicate phosphorylation at Ser303 and in (B) presence of b14, y6 ions with the neutral loss phosphate indicate phosphorylation at Thr305 respectively. Phosphorylation analysis using MS was repeated in four separate experiments. Maximum Mascot score for Ser303 was 128 (range 42–128) and for Thr305 was 73 (range 43–73).
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Figure 7: Identification of NHERF2 phosphorylation sites Ser303 and Thr305 by LC–MS/MS analysis of FLAG–NHERF2 IP sampleNHERF2 sample was immunoprecipitated from Caco-2/Bbe cells and proteolysed for LC–MS/MS analysis. Two phosphorylation sites were identified within the EBRS of NHERF2. MS/MS spectrum (A) shows the phosphorylation at Ser303. Presence of b12 and y8 ions with neutral loss phosphate indicate phosphorylation at Ser303 and in (B) presence of b14, y6 ions with the neutral loss phosphate indicate phosphorylation at Thr305 respectively. Phosphorylation analysis using MS was repeated in four separate experiments. Maximum Mascot score for Ser303 was 128 (range 42–128) and for Thr305 was 73 (range 43–73).

Mentions: Cell lysate was prepared with lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 0.5% Triton X-100 and protease inhibitors). For experiments in Figure 2, 1 mg of cell lysate was mixed with 10 μl of pre-washed anti-FLAG M2 magnetic beads and incubated at 4°C for 3 h on a rotating shaker. For experiments in Figures 7 and 9, 1.5 mg of cell lysate and 20 μl of anti-FLAG M2 magnetic beads were used and overnight incubations were performed. Beads were washed with the same lysis buffer four times and eluted with 1.5× Laemmli sample buffer without 2-mercaptoethanol. Samples were analysed by Western blot. For MS studies in Figure 7, FLAG–NHERF2 was eluted from beads with 100 mM glycine/HCl, pH 3.0, and adjusted to pH 7.0 with 0.5 M HEPES buffer pH 7.4.


The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

Yang J, Sarker R, Singh V, Sarker P, Yin J, Chen TE, Chaerkady R, Li X, Tse CM, Donowitz M - Biochem. J. (2015)

Identification of NHERF2 phosphorylation sites Ser303 and Thr305 by LC–MS/MS analysis of FLAG–NHERF2 IP sampleNHERF2 sample was immunoprecipitated from Caco-2/Bbe cells and proteolysed for LC–MS/MS analysis. Two phosphorylation sites were identified within the EBRS of NHERF2. MS/MS spectrum (A) shows the phosphorylation at Ser303. Presence of b12 and y8 ions with neutral loss phosphate indicate phosphorylation at Ser303 and in (B) presence of b14, y6 ions with the neutral loss phosphate indicate phosphorylation at Thr305 respectively. Phosphorylation analysis using MS was repeated in four separate experiments. Maximum Mascot score for Ser303 was 128 (range 42–128) and for Thr305 was 73 (range 43–73).
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Related In: Results  -  Collection

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Figure 7: Identification of NHERF2 phosphorylation sites Ser303 and Thr305 by LC–MS/MS analysis of FLAG–NHERF2 IP sampleNHERF2 sample was immunoprecipitated from Caco-2/Bbe cells and proteolysed for LC–MS/MS analysis. Two phosphorylation sites were identified within the EBRS of NHERF2. MS/MS spectrum (A) shows the phosphorylation at Ser303. Presence of b12 and y8 ions with neutral loss phosphate indicate phosphorylation at Ser303 and in (B) presence of b14, y6 ions with the neutral loss phosphate indicate phosphorylation at Thr305 respectively. Phosphorylation analysis using MS was repeated in four separate experiments. Maximum Mascot score for Ser303 was 128 (range 42–128) and for Thr305 was 73 (range 43–73).
Mentions: Cell lysate was prepared with lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 0.5% Triton X-100 and protease inhibitors). For experiments in Figure 2, 1 mg of cell lysate was mixed with 10 μl of pre-washed anti-FLAG M2 magnetic beads and incubated at 4°C for 3 h on a rotating shaker. For experiments in Figures 7 and 9, 1.5 mg of cell lysate and 20 μl of anti-FLAG M2 magnetic beads were used and overnight incubations were performed. Beads were washed with the same lysis buffer four times and eluted with 1.5× Laemmli sample buffer without 2-mercaptoethanol. Samples were analysed by Western blot. For MS studies in Figure 7, FLAG–NHERF2 was eluted from beads with 100 mM glycine/HCl, pH 3.0, and adjusted to pH 7.0 with 0.5 M HEPES buffer pH 7.4.

Bottom Line: The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin.Furthermore, phosphorylation of Ser(303) located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate.Moreover, increased phosphorylation of Ser(303) was functionally significant preventing acute stimulation of NHE3 (Na(+)-H(+) exchanger 3) activity by dexamethasone.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.

Show MeSH