What question did this study set out to answer?

To identify the specific site of constitutive NHERF phosphorylation and determine the kinase responsible for this modification.

August 1, 1999Journal of Biological ChemistryOpen Access

G Protein-coupled Receptor Kinase 6A Phosphorylates the Na+/H+ Exchanger Regulatory Factor via a PDZ Domain-mediated Interaction

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Population

Human embryonic kidney 293 (HEK-293) cells and in vitro models

Comparison

G protein-coupled receptor kinase 6A (GRK6A) vs GRK6B, GRK6C, and GRK2

Design

Preclinical

Authors

RHRandy A. HallEmory University RSRobert F. SpurneyDuke University RPRichard T. PremontUniversity Hospitals of Cleveland

Discussion

Member takes

Implication

GRK6A emerges as regulator of NHERF; leaves open its in vivo roles and therapeutic relevance.

Key Points

To identify the specific site of constitutive NHERF phosphorylation and determine the kinase responsible for this modification.
Mapped constitutive NHERF phosphorylation sites and measured phosphorylation stoichiometry in HEK-293 cells.
Conducted protein overlay and co-immunoprecipitation assays to test binding between NHERF PDZ domains and GRK6 splice variants (GRK6A, GRK6B, GRK6C).
Assayed in vitro phosphorylation of NHERF by GRK variants and evaluated endogenous NHERF kinase activity in HEK-293 cell lysates.
Identified Ser(289) as the primary constitutive phosphorylation site on NHERF with a stoichiometry near 1 mol/mol.
Demonstrated that NHERF selectively binds with high affinity to GRK6A possessing a carboxyl-terminal T-R-L motif, but not to GRK6B or GRK6C.
Showed that GRK6A efficiently phosphorylates NHERF at Ser(289) in vitro (unlike GRK6B, GRK6C, or GRK2) and accounts for endogenous NHERF kinase activity in HEK-293 lysates.

Structured PICO

Population

Human embryonic kidney 293 (HEK-293) cells and in vitro models

Intervention

G protein-coupled receptor kinase 6A (GRK6A)

Comparator

GRK6B, GRK6C, and GRK2

Outcome

Phosphorylation of NHERF on Ser(289) and binding affinity

GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction, identifying it as the kinase responsible for constitutive NHERF phosphorylation in HEK-293 cells.

Cite This Study

Hall et al. (1999) studied this question.

synapsesocial.com/papers/6a7d5116eecc944e79aa0567 https://doi.org/10.1074/jbc.274.34.24328

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G Protein-coupled Receptor Kinase 6A Phosphorylates the Na+/H+ Exchanger Regulatory Factor via a PDZ Domain-mediated Interaction

Discussion

Member takes

Implication

Key Points

Structured PICO

Cite This Study

G Protein-coupled Receptor Kinase 6A Phosphorylates the Na+/H+ Exchanger Regulatory Factor via a PDZ Domain-mediated Interaction

Discussion

Member takes

Implication

Key Points

Structured PICO

Cite This Study

Also Consider

Also Consider