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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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NHERF2–ezrin interaction is stronger than NHERF1–ezrin interaction(A) One nanomole of GST-N–ezrin was mixed with 3 nmol of NHERF2–C109 and varied amounts of NHERF1–C127 as indicated for pull-down. Samples were analysed by Coomassie Blue staining. (B) Indicated amounts of GST-N–ezrin protein were pre-incubated with His6–thioredoxin or His6–thioredoxin fused NHERF1 or NHERF2 C-termini and then mixed with Caco-2 lysates for pull-down as described in ‘Experimental’. Samples were analysed by Western blot. GST was used as negative control and did not pull down NHERF1 or NHERF2. NHERF1–C127 and NHERF2–C109 efficiently competed with full-length NHERF1 and NHERF2, whereas NHERF1–C30 and NHERF2–C30 did not. Experiments were repeated three times and one representative result is shown.
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Figure 4: NHERF2–ezrin interaction is stronger than NHERF1–ezrin interaction(A) One nanomole of GST-N–ezrin was mixed with 3 nmol of NHERF2–C109 and varied amounts of NHERF1–C127 as indicated for pull-down. Samples were analysed by Coomassie Blue staining. (B) Indicated amounts of GST-N–ezrin protein were pre-incubated with His6–thioredoxin or His6–thioredoxin fused NHERF1 or NHERF2 C-termini and then mixed with Caco-2 lysates for pull-down as described in ‘Experimental’. Samples were analysed by Western blot. GST was used as negative control and did not pull down NHERF1 or NHERF2. NHERF1–C127 and NHERF2–C109 efficiently competed with full-length NHERF1 and NHERF2, whereas NHERF1–C30 and NHERF2–C30 did not. Experiments were repeated three times and one representative result is shown.

Mentions: For Figures 2–5 and 8, 1 nmol of recombinant GST-N–ezrin was used as bait for pull-downs. For binary interaction studies, 3 nmol of purified proteins were used as prey. For competition experiments between two NHERF fragments or full-length proteins, 3 nmol of each purified protein were used. For competition experiments described in Figure 4, 1 nmol of GST-N–ezrin was first pre-incubated with 3 nmol of His6–thioredoxin or His6–thioredoxin fused NHERF C-terminus fragments for 30 min, then 1 mg of Caco-2 cell lysate was added. The volume of final mixture was adjusted to 500 μl. Each bait–prey mixture was mixed with 10 μl of pre-washed GSH-resin and incubated at 4°C for 3 h on a rotating shaker. Resin was washed four times and eluted with lysis buffer plus 10 mM glutathione. For Figure 9, 3 μg of purified GST-N–ezrin was mixed with 1.5 mg of HEK-293A cell lysate expressing FLAG–NHERF2 or mutants and 10 μl of GSH–resin for overnight incubation at 4°C. Resin was washed four times and finally eluted with 2× Laemmli buffer at 80°C.


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)

NHERF2–ezrin interaction is stronger than NHERF1–ezrin interaction(A) One nanomole of GST-N–ezrin was mixed with 3 nmol of NHERF2–C109 and varied amounts of NHERF1–C127 as indicated for pull-down. Samples were analysed by Coomassie Blue staining. (B) Indicated amounts of GST-N–ezrin protein were pre-incubated with His6–thioredoxin or His6–thioredoxin fused NHERF1 or NHERF2 C-termini and then mixed with Caco-2 lysates for pull-down as described in ‘Experimental’. Samples were analysed by Western blot. GST was used as negative control and did not pull down NHERF1 or NHERF2. NHERF1–C127 and NHERF2–C109 efficiently competed with full-length NHERF1 and NHERF2, whereas NHERF1–C30 and NHERF2–C30 did not. Experiments were repeated three times and one representative result is shown.
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Related In: Results  -  Collection

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Figure 4: NHERF2–ezrin interaction is stronger than NHERF1–ezrin interaction(A) One nanomole of GST-N–ezrin was mixed with 3 nmol of NHERF2–C109 and varied amounts of NHERF1–C127 as indicated for pull-down. Samples were analysed by Coomassie Blue staining. (B) Indicated amounts of GST-N–ezrin protein were pre-incubated with His6–thioredoxin or His6–thioredoxin fused NHERF1 or NHERF2 C-termini and then mixed with Caco-2 lysates for pull-down as described in ‘Experimental’. Samples were analysed by Western blot. GST was used as negative control and did not pull down NHERF1 or NHERF2. NHERF1–C127 and NHERF2–C109 efficiently competed with full-length NHERF1 and NHERF2, whereas NHERF1–C30 and NHERF2–C30 did not. Experiments were repeated three times and one representative result is shown.
Mentions: For Figures 2–5 and 8, 1 nmol of recombinant GST-N–ezrin was used as bait for pull-downs. For binary interaction studies, 3 nmol of purified proteins were used as prey. For competition experiments between two NHERF fragments or full-length proteins, 3 nmol of each purified protein were used. For competition experiments described in Figure 4, 1 nmol of GST-N–ezrin was first pre-incubated with 3 nmol of His6–thioredoxin or His6–thioredoxin fused NHERF C-terminus fragments for 30 min, then 1 mg of Caco-2 cell lysate was added. The volume of final mixture was adjusted to 500 μl. Each bait–prey mixture was mixed with 10 μl of pre-washed GSH-resin and incubated at 4°C for 3 h on a rotating shaker. Resin was washed four times and eluted with lysis buffer plus 10 mM glutathione. For Figure 9, 3 μg of purified GST-N–ezrin was mixed with 1.5 mg of HEK-293A cell lysate expressing FLAG–NHERF2 or mutants and 10 μl of GSH–resin for overnight incubation at 4°C. Resin was washed four times and finally eluted with 2× Laemmli buffer at 80°C.

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