Nucleoside diphosphate kinase and Cl--sensitive protein phosphorylation in apical membranes from ovine airway epithelium

Richmond Muimo, Stephen J. Banner, Lindsay J. Marshall, Anil Mehta

Research output: Contribution to journalArticle

Abstract

We have previously shown that nucleotide species (adenosine triphosphate [ATP] or guanosine triphosphate [GTP]), [Cl-], and anion species determine the steady-state phosphorylation of apical membrane proteins within human airway epithelium in vitro. We found that a C1-regulated 37-kD protein (p37) principally phosphorylated with GTP but not ATP as substrate. Here we show that apical membranes from sheep tracheal epithelium also contain a Cl(-)-regulated 37-kD ϒphosphoprotein (p37s) and characterize one of the kinases involved in the regulation of p37s. Analysis of phosphorylation of apical membrane proteins with ϒ[32P]GTP in the presence of MgCl2 showed that two proteins circa 19 and 21 kD (p19s and p21s) were transiently phosphorylated before p37s. Renaturation of apical membrane proteins within polyacrylamide gels showed that p19s and p21s autophosphorylated with either ϒ[32P]GTP or ϒ[32P]ATP as substrates, suggesting that the two proteins were kinases. Immunoblotting and immunoprecipitation with a specific polyclonal antibody showed that p21s was a membrane-bound isoform of nucleoside diphosphate kinase (NDPK, EC 2.7.4.6), a protein kinase which catalyzes transfer of terminal phosphate from ATP to diphosphate nucleotides and is, among other functions, essential for cell secretion. Incubation of apical membrane proteins in the presence of ϒ[32P]ATP and guanosine diphosphate (GDP) (but not GDPβS) resulted in enhancement of phosphorylation of p37s. Dephosphorylation of NDPK was stimulated by the addition of Mg2+, Mn2+, and Co2+ (but not Zn2+ or Ca2+). Our data show that ovine trachea is a good model for further characterization of the chloride-dependent cascade in airway epithelium.
Original languageEnglish
Pages (from-to)270-8
Number of pages9
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume18
Issue number2
DOIs
Publication statusPublished - Feb 1998

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Nucleoside-Diphosphate Kinase
Phosphorylation
Sheep
Guanosine Triphosphate
Epithelium
Adenosine Triphosphate
Membranes
Membrane Proteins
Diphosphates
Proteins
Protein Kinases
Phosphotransferases
Nucleotides
Magnesium Chloride
Guanosine
Phosphoproteins
Substrates
Trachea
Immunoprecipitation
Immunoblotting

Keywords

  • Adult
  • Animals
  • Anions
  • Cations, Divalent
  • Cell Membrane
  • Cell Polarity
  • Cells, Cultured
  • Chlorides
  • Epithelial Cells
  • Guanosine Diphosphate
  • Guanosine Triphosphate
  • Humans
  • Magnesium Chloride
  • Meglumine
  • Membrane Proteins
  • Molecular Weight
  • Nucleoside-Diphosphate Kinase
  • Phosphorylation
  • Sheep
  • Temperature
  • Trachea

Cite this

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abstract = "We have previously shown that nucleotide species (adenosine triphosphate [ATP] or guanosine triphosphate [GTP]), [Cl-], and anion species determine the steady-state phosphorylation of apical membrane proteins within human airway epithelium in vitro. We found that a C1-regulated 37-kD protein (p37) principally phosphorylated with GTP but not ATP as substrate. Here we show that apical membranes from sheep tracheal epithelium also contain a Cl(-)-regulated 37-kD ϒphosphoprotein (p37s) and characterize one of the kinases involved in the regulation of p37s. Analysis of phosphorylation of apical membrane proteins with ϒ[32P]GTP in the presence of MgCl2 showed that two proteins circa 19 and 21 kD (p19s and p21s) were transiently phosphorylated before p37s. Renaturation of apical membrane proteins within polyacrylamide gels showed that p19s and p21s autophosphorylated with either ϒ[32P]GTP or ϒ[32P]ATP as substrates, suggesting that the two proteins were kinases. Immunoblotting and immunoprecipitation with a specific polyclonal antibody showed that p21s was a membrane-bound isoform of nucleoside diphosphate kinase (NDPK, EC 2.7.4.6), a protein kinase which catalyzes transfer of terminal phosphate from ATP to diphosphate nucleotides and is, among other functions, essential for cell secretion. Incubation of apical membrane proteins in the presence of ϒ[32P]ATP and guanosine diphosphate (GDP) (but not GDPβS) resulted in enhancement of phosphorylation of p37s. Dephosphorylation of NDPK was stimulated by the addition of Mg2+, Mn2+, and Co2+ (but not Zn2+ or Ca2+). Our data show that ovine trachea is a good model for further characterization of the chloride-dependent cascade in airway epithelium.",
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author = "Richmond Muimo and Banner, {Stephen J.} and Marshall, {Lindsay J.} and Anil Mehta",
year = "1998",
month = "2",
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Nucleoside diphosphate kinase and Cl--sensitive protein phosphorylation in apical membranes from ovine airway epithelium. / Muimo, Richmond; Banner, Stephen J.; Marshall, Lindsay J.; Mehta, Anil.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 18, No. 2, 02.1998, p. 270-8.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Nucleoside diphosphate kinase and Cl--sensitive protein phosphorylation in apical membranes from ovine airway epithelium

AU - Muimo, Richmond

AU - Banner, Stephen J.

AU - Marshall, Lindsay J.

AU - Mehta, Anil

PY - 1998/2

Y1 - 1998/2

N2 - We have previously shown that nucleotide species (adenosine triphosphate [ATP] or guanosine triphosphate [GTP]), [Cl-], and anion species determine the steady-state phosphorylation of apical membrane proteins within human airway epithelium in vitro. We found that a C1-regulated 37-kD protein (p37) principally phosphorylated with GTP but not ATP as substrate. Here we show that apical membranes from sheep tracheal epithelium also contain a Cl(-)-regulated 37-kD ϒphosphoprotein (p37s) and characterize one of the kinases involved in the regulation of p37s. Analysis of phosphorylation of apical membrane proteins with ϒ[32P]GTP in the presence of MgCl2 showed that two proteins circa 19 and 21 kD (p19s and p21s) were transiently phosphorylated before p37s. Renaturation of apical membrane proteins within polyacrylamide gels showed that p19s and p21s autophosphorylated with either ϒ[32P]GTP or ϒ[32P]ATP as substrates, suggesting that the two proteins were kinases. Immunoblotting and immunoprecipitation with a specific polyclonal antibody showed that p21s was a membrane-bound isoform of nucleoside diphosphate kinase (NDPK, EC 2.7.4.6), a protein kinase which catalyzes transfer of terminal phosphate from ATP to diphosphate nucleotides and is, among other functions, essential for cell secretion. Incubation of apical membrane proteins in the presence of ϒ[32P]ATP and guanosine diphosphate (GDP) (but not GDPβS) resulted in enhancement of phosphorylation of p37s. Dephosphorylation of NDPK was stimulated by the addition of Mg2+, Mn2+, and Co2+ (but not Zn2+ or Ca2+). Our data show that ovine trachea is a good model for further characterization of the chloride-dependent cascade in airway epithelium.

AB - We have previously shown that nucleotide species (adenosine triphosphate [ATP] or guanosine triphosphate [GTP]), [Cl-], and anion species determine the steady-state phosphorylation of apical membrane proteins within human airway epithelium in vitro. We found that a C1-regulated 37-kD protein (p37) principally phosphorylated with GTP but not ATP as substrate. Here we show that apical membranes from sheep tracheal epithelium also contain a Cl(-)-regulated 37-kD ϒphosphoprotein (p37s) and characterize one of the kinases involved in the regulation of p37s. Analysis of phosphorylation of apical membrane proteins with ϒ[32P]GTP in the presence of MgCl2 showed that two proteins circa 19 and 21 kD (p19s and p21s) were transiently phosphorylated before p37s. Renaturation of apical membrane proteins within polyacrylamide gels showed that p19s and p21s autophosphorylated with either ϒ[32P]GTP or ϒ[32P]ATP as substrates, suggesting that the two proteins were kinases. Immunoblotting and immunoprecipitation with a specific polyclonal antibody showed that p21s was a membrane-bound isoform of nucleoside diphosphate kinase (NDPK, EC 2.7.4.6), a protein kinase which catalyzes transfer of terminal phosphate from ATP to diphosphate nucleotides and is, among other functions, essential for cell secretion. Incubation of apical membrane proteins in the presence of ϒ[32P]ATP and guanosine diphosphate (GDP) (but not GDPβS) resulted in enhancement of phosphorylation of p37s. Dephosphorylation of NDPK was stimulated by the addition of Mg2+, Mn2+, and Co2+ (but not Zn2+ or Ca2+). Our data show that ovine trachea is a good model for further characterization of the chloride-dependent cascade in airway epithelium.

KW - Adult

KW - Animals

KW - Anions

KW - Cations, Divalent

KW - Cell Membrane

KW - Cell Polarity

KW - Cells, Cultured

KW - Chlorides

KW - Epithelial Cells

KW - Guanosine Diphosphate

KW - Guanosine Triphosphate

KW - Humans

KW - Magnesium Chloride

KW - Meglumine

KW - Membrane Proteins

KW - Molecular Weight

KW - Nucleoside-Diphosphate Kinase

KW - Phosphorylation

KW - Sheep

KW - Temperature

KW - Trachea

UR - http://www.atsjournals.org/doi/abs/10.1165/ajrcmb.18.2.2850

U2 - 10.1165/ajrcmb.18.2.2850

DO - 10.1165/ajrcmb.18.2.2850

M3 - Article

VL - 18

SP - 270

EP - 278

JO - American Journal of Respiratory Cell and Molecular Biology

JF - American Journal of Respiratory Cell and Molecular Biology

SN - 1044-1549

IS - 2

ER -