Purification and characterization of a protein tyrosine phosphatase which dephosphorylates the nicotinic acetylcholine receptor

Lin Mei, Richard L. Huganir

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The nicotinic acetylcholine receptor (nAChR) is phosphorylated to a high stoichiometry on tyrosine residues both in vitro and in vivo. Moreover, tyrosine phosphorylation has been shown to regulate the functional properties of the receptor. We report here the purification and characterization of a protein tyrosine phosphatase that dephosphorylates tyrosine-phosphorylated nAChR from Torpedo electroplax, a tissue highly enriched in the nAChR. The 32P-labeled tyrosine phosphorylated nAChR was used as a substrate to monitor the enzyme activity during purification. The protein tyrosine phosphatase activity was purified using three consecutive cation-exchange columns (phosphocellulose, S Sepharose Fast Flow, Bio-Rex 70), followed by two affinity matrices (p-aminobenzylphosphonic acid-agarose and thiophosphotyrosyl nAChR-Sepharose 4B). The enzyme activity was purified to homogeneity, with an overall purification of 25,000-fold and a yield of 20%. The purified enzyme had an apparent molecular mass of 43 kDa on sodium dodecyl sulfate-polyacrylamide gels and migrated as a monomer during Superose 12 chromatography. It had a neutral pH optimum and a specific activity of 18 μmol/mg of protein/min, with a Km of 4.7 μM for tyrosine-phosphorylated nAChR. The phosphatase was specific for tyrosine phosphorylated nAChR; it showed no activity towards the nAChR phosphorylated on serine residues by cAMP-dependent protein kinase. The enzyme also dephosphorylated 32P-labeled poly(Glu-Tyr) (4:1). However, it did not dephosphorylate p-nitrophenylphosphate. The tyrosine phosphatase was inhibited by ammonium molybdate (IC50 of 2 μM), sodium vanadate (IC50 of 150 μM) and the divalent cations Mg2+, Mn2+, and Ca2+ at millimolar concentrations, but not by 100 μM ZnCl or 10 mM NaF. Poly(Glu, Tyr) (4:1) and heparin inhibited the enzyme activity at micromolar concentrations. These unique properties of the purified enzyme suggest that it may be a novel protein tyrosine phosphatase that specifically dephosphorylates the nAChR.

Original languageEnglish (US)
Pages (from-to)16063-16072
Number of pages10
JournalJournal of Biological Chemistry
Volume266
Issue number24
StatePublished - Dec 1 1991

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Protein Tyrosine Phosphatases
Nicotinic Receptors
Purification
Tyrosine
Enzyme activity
Enzymes
Sepharose
Phosphoric Monoester Hydrolases
Inhibitory Concentration 50
Torpedo
Phosphorylation
Vanadates
Divalent Cations
Molecular mass
Cyclic AMP-Dependent Protein Kinases
Chromatography
Sodium Dodecyl Sulfate
Stoichiometry
Serine
Heparin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of a protein tyrosine phosphatase which dephosphorylates the nicotinic acetylcholine receptor. / Mei, Lin; Huganir, Richard L.

In: Journal of Biological Chemistry, Vol. 266, No. 24, 01.12.1991, p. 16063-16072.

Research output: Contribution to journalArticle

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N2 - The nicotinic acetylcholine receptor (nAChR) is phosphorylated to a high stoichiometry on tyrosine residues both in vitro and in vivo. Moreover, tyrosine phosphorylation has been shown to regulate the functional properties of the receptor. We report here the purification and characterization of a protein tyrosine phosphatase that dephosphorylates tyrosine-phosphorylated nAChR from Torpedo electroplax, a tissue highly enriched in the nAChR. The 32P-labeled tyrosine phosphorylated nAChR was used as a substrate to monitor the enzyme activity during purification. The protein tyrosine phosphatase activity was purified using three consecutive cation-exchange columns (phosphocellulose, S Sepharose Fast Flow, Bio-Rex 70), followed by two affinity matrices (p-aminobenzylphosphonic acid-agarose and thiophosphotyrosyl nAChR-Sepharose 4B). The enzyme activity was purified to homogeneity, with an overall purification of 25,000-fold and a yield of 20%. The purified enzyme had an apparent molecular mass of 43 kDa on sodium dodecyl sulfate-polyacrylamide gels and migrated as a monomer during Superose 12 chromatography. It had a neutral pH optimum and a specific activity of 18 μmol/mg of protein/min, with a Km of 4.7 μM for tyrosine-phosphorylated nAChR. The phosphatase was specific for tyrosine phosphorylated nAChR; it showed no activity towards the nAChR phosphorylated on serine residues by cAMP-dependent protein kinase. The enzyme also dephosphorylated 32P-labeled poly(Glu-Tyr) (4:1). However, it did not dephosphorylate p-nitrophenylphosphate. The tyrosine phosphatase was inhibited by ammonium molybdate (IC50 of 2 μM), sodium vanadate (IC50 of 150 μM) and the divalent cations Mg2+, Mn2+, and Ca2+ at millimolar concentrations, but not by 100 μM ZnCl or 10 mM NaF. Poly(Glu, Tyr) (4:1) and heparin inhibited the enzyme activity at micromolar concentrations. These unique properties of the purified enzyme suggest that it may be a novel protein tyrosine phosphatase that specifically dephosphorylates the nAChR.

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