G protein-coupled receptor kinase 6A phosphorylates the Na+/H+ exchanger regulatory factor via a PDZ domain-mediated interaction

被引:121
作者
Hall, RA
Spurney, RF
Premont, RT
Rahman, N
Blitzer, JT
Pitcher, JA
Lefkowitz, RJ
机构
[1] Duke Univ, Med Ctr, Howard Hughes Med Inst, Dept Med, Durham, NC 27710 USA
[2] Duke Univ, Med Ctr, Dept Biochem, Durham, NC 27710 USA
关键词
D O I
10.1074/jbc.274.34.24328
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Na+/H+ exchanger regulatory factor (NHERF) is constitutively phosphorylated in cells, but the site(s) of this phosphorylation and the kinase(s) responsible for it have not been identified. We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK-293) cells is Ser(289), and that the stoichiometry of phosphorylation is near 1 mol/mol, NHERF contains two PDZ domains that recognize the sequence S/T-X-L at the carboxyl terminus of target proteins, and thus we examined the possibility that kinases containing this motif might associate with and phosphorylate NHERF, Overlay experiments and co-immunoprecipitation studies revealed that NRERF binds with high affinity to a splice variant of the G protein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R-L, NHERF does not associate with GRK6B or GRK6C, alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini, GRK6A phosphorylates NHERF efficiently on Ser(289) in vitro, whereas GRK6B, GRK6C, and GRK2 do not, Furthermore, the endogenous "NHERF kinase" activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction and that GRK6A is the kinase in HER-293 cells responsible for the constitutive phosphorylation of NHERF.
引用
收藏
页码:24328 / 24334
页数:7
相关论文
共 31 条
  • [1] BENOVIC JL, 1993, J BIOL CHEM, V268, P19521
  • [2] Binding and phosphorylation of tubulin by G protein-coupled receptor kinases
    Carman, CV
    Som, T
    Kim, CM
    Benovic, JL
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (32) : 20308 - 20316
  • [3] RABBIT ILEAL VILLUS CELL BRUSH-BORDER NA+/H+ EXCHANGE IS REGULATED BY CA-2+/CALMODULIN-DEPENDENT PROTEIN KINASE-II, A BRUSH-BORDER MEMBRANE-PROTEIN
    COHEN, ME
    REINLIB, L
    WATSON, AJM
    GORELICK, F
    RYSSIKORA, K
    TSE, M
    ROOD, RP
    CZERNIK, AJ
    SHARP, GWG
    DONOWITZ, M
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (22) : 8990 - 8994
  • [4] Crystal structures of a complexed and peptide-free membrane protein-binding domain: Molecular basis of peptide recognition by PDZ
    Doyle, DA
    Lee, A
    Lewis, J
    Kim, E
    Sheng, M
    MacKinnon, R
    [J]. CELL, 1996, 85 (07) : 1067 - 1076
  • [5] Molecular cloning of two rat GRK6 splice variants
    Firsov, D
    Elalouf, JM
    [J]. AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 1997, 273 (03): : C953 - C961
  • [6] PHOSPHORYLATION AND DESENSITIZATION OF THE HUMAN BETA(1)-ADRENERGIC RECEPTOR - INVOLVEMENT OF G-PROTEIN-COUPLED RECEPTOR KINASES AND CAMP-DEPENDENT PROTEIN-KINASE
    FREEDMAN, NJ
    LIGGETT, SB
    DRACHMAN, DE
    PEI, G
    CARON, MG
    LEFKOWITZ, RJ
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (30) : 17953 - 17961
  • [7] GTP-binding-protein-coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin
    Haga, K
    Ogawa, H
    Haga, T
    Murofushi, H
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1998, 255 (02): : 363 - 368
  • [8] The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange
    Hall, RA
    Premont, RT
    Chow, CW
    Blitzer, JT
    Pitcher, JA
    Claing, A
    Stoffel, RH
    Barak, LS
    Shenolikar, S
    Weinman, EJ
    Grinstein, S
    Lefkowitz, RJ
    [J]. NATURE, 1998, 392 (6676) : 626 - 630
  • [9] A C-terminal motif found in the β2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins
    Hall, RA
    Ostedgaard, LS
    Premont, RT
    Blitzer, JT
    Rahman, N
    Welsh, MJ
    Lefkowitz, RJ
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1998, 95 (15) : 8496 - 8501
  • [10] Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase - Phosphorylation-dependent and -independent mechanisms
    Kurashima, K
    Yu, FH
    Cabado, AG
    Szabo, EZ
    Grinstein, S
    Orlowski, J
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (45) : 28672 - 28679