Tyrosine-specific dephosphorylation-phosphorylation with alkaline phosphatases and epidermal growth factor receptor kinase as evidenced by 31P NMR spectroscopy.
The dephosphorylation of phospho-amino acids with alkaline phosphatase (AlPase) from calf intestine or Escherichia coli and the phosphorylation of bovine serum albumin (BSA) with epidermal growth factor (EGF) receptor kinase from human A431 epidermoid carcinoma cells were investigated by 31P NMR spectroscopy. The initial rates of the dephosphorylation of phospho-tyrosine (P-Tyr) and phosphoserine (P-Ser) with AlPase were essentially the same in the one-substrate system. In the two-substrate system (P-Tyr plus P-Ser), however, the ratio of the initial rate for P-Tyr vs. P-Ser was 2.4 to 4.5 depending on the buffer and pH conditions employed. This substantiates for the first time the specificity of AlPases to P-Tyr over P-Ser at the free amino acid level. In the stationary phase of the overall process, the dephosphorylation of P-Ser became slow compared to that of P-Tyr in the one-substrate system. The decrease in the rate for P-Ser was further pronounced in the two-substrate system. For this remarkable effect, the rephosphorylation of serine was responsible, as demonstrated in the reaction mixture containing serine, Pi, and AlPase. BSA phosphorylated by EGF receptor kinase exhibited sharp 31P resonances around 0 ppm at neutral pH, far distant from the peak positions (4.9 ppm) of histone H1 phosphorylated by cAMP-dependent protein kinase. These NMR data are directed evidence that BSA was phosphorylated exclusively at the tyrosyl residues, whereas the phosphorylation of histone H1 was at the seryl residues.
Takahashi
K
K
Department of Biochemistry, Okayama University Dental School.
Shimidzu
M
M
Shindo
H
H
Kawamoto
T
T
Nishi
M
M
Matsumoto
U
U
Taniguchi
S
S
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D066246:ErbB Receptors; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007422:Intestines / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010758:Phosphorus; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014443:Tyrosine
D012710:Serum Albumin, Bovine; D010758:Phosphorus; D014443:Tyrosine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D000469:Alkaline Phosphatase