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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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Binding of NHERF2 to ezrin is modulated by phosphorylation at Ser303(A) Pull down was performed by mixing 3 μg of purified GST-N–ezrin and 1.5 mg cell lysate prepared from HEK-293A cells transiently transfected with FLAG–NHERF2–WT or mutants (S303A, S303D, T305A, T305D, S303A/T305A, S303D/T305D). The non-transfected HEK293 cell was used as a negative control (Ctrl). Samples were analysed by Western blot with antibodies against FLAG and GST. (B) HEK-293A cells transiently transfected with FLAG–NHERF2–WT or S303A or S303D mutant, were immunoprecipitated with anti-FLAG M2 magnetic beads. IP/co-IP samples were analysed by Western blot with antibodies against FLAG, ezrin and P-ezrin.
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Figure 9: Binding of NHERF2 to ezrin is modulated by phosphorylation at Ser303(A) Pull down was performed by mixing 3 μg of purified GST-N–ezrin and 1.5 mg cell lysate prepared from HEK-293A cells transiently transfected with FLAG–NHERF2–WT or mutants (S303A, S303D, T305A, T305D, S303A/T305A, S303D/T305D). The non-transfected HEK293 cell was used as a negative control (Ctrl). Samples were analysed by Western blot with antibodies against FLAG and GST. (B) HEK-293A cells transiently transfected with FLAG–NHERF2–WT or S303A or S303D mutant, were immunoprecipitated with anti-FLAG M2 magnetic beads. IP/co-IP samples were analysed by Western blot with antibodies against FLAG, ezrin and P-ezrin.

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)

Binding of NHERF2 to ezrin is modulated by phosphorylation at Ser303(A) Pull down was performed by mixing 3 μg of purified GST-N–ezrin and 1.5 mg cell lysate prepared from HEK-293A cells transiently transfected with FLAG–NHERF2–WT or mutants (S303A, S303D, T305A, T305D, S303A/T305A, S303D/T305D). The non-transfected HEK293 cell was used as a negative control (Ctrl). Samples were analysed by Western blot with antibodies against FLAG and GST. (B) HEK-293A cells transiently transfected with FLAG–NHERF2–WT or S303A or S303D mutant, were immunoprecipitated with anti-FLAG M2 magnetic beads. IP/co-IP samples were analysed by Western blot with antibodies against FLAG, ezrin and P-ezrin.
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Related In: Results  -  Collection

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Figure 9: Binding of NHERF2 to ezrin is modulated by phosphorylation at Ser303(A) Pull down was performed by mixing 3 μg of purified GST-N–ezrin and 1.5 mg cell lysate prepared from HEK-293A cells transiently transfected with FLAG–NHERF2–WT or mutants (S303A, S303D, T305A, T305D, S303A/T305A, S303D/T305D). The non-transfected HEK293 cell was used as a negative control (Ctrl). Samples were analysed by Western blot with antibodies against FLAG and GST. (B) HEK-293A cells transiently transfected with FLAG–NHERF2–WT or S303A or S303D mutant, were immunoprecipitated with anti-FLAG M2 magnetic beads. IP/co-IP samples were analysed by Western blot with antibodies against FLAG, ezrin and P-ezrin.
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