Properties of a purified estradiol-dependent calf uterus tyrosine kinase.
A uterus tyrosine kinase has been purified to a single 67-kDa protein when analyzed by SDS-PAGE. Under nondenaturing conditions the molecular weight of the enzyme ranges from 114 to 136 kDa, depending on the procedure employed. The kinase binds calmodulin in a Ca(2+)-dependent manner and the ATP analog [(fluorosulfonyl)benzoyl]adenosine. The purified enzyme phosphorylates the phosphatase-treated uterus estradiol receptor on tyrosine and activates its hormone binding. The kinase phosphorylates actin, calmodulin, and histone H2B. Whatever the substrate, the enzymic activity is dependent on purified estradiol-receptor complex and is activated by Ca(2+)-calmodulin. The kinase activates and phosphorylates the human estradiol receptor (HEO) within the hormone binding domain (HBD) [Migliaccio, et al. (1989) Mol Endocrinol. 3, 1061-1069] as well as four of the five mutants of the HEO obtained by substituting each of the five tyrosine residues present in the HBD of the receptor with phenylalanine by site-directed mutagenesis. The mutant substituted at tyrosine 537 is the only one that is neither phosphorylated nor activated by the kinase. This proves a causal relationship between the phosphorylation of estradiol receptor on tyrosine 537 and its hormone binding activity. A synthetic peptide corresponding to 11 out of 13 amino acids surrounding tyrosine at position 537 of the human estrogen receptor can be phosphorylated by the kinase. This and other findings indicate that this kinase, unlike other tyrosine kinases, phosphorylates tyrosyl residues with acidic amino acids close to the carboxyl side.
Castoria
G
G
Istituto di Patologia Generale ed Oncologia, Facoltà di Medicine e Chirurgia, II Università di Napoli, Italia.
Migliaccio
A
A
Green
S
S
Di Domenico
M
M
Chambon
P
P
Auricchio
F
F
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011489:Protein Denaturation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011959:Receptors, Estradiol / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014599:Uterus / Q000201:enzymology*
D000199:Actins; D002147:Calmodulin; D006657:Histones; D011959:Receptors, Estradiol; D019000:Phosphotyrosine; D014443:Tyrosine; D010649:Phenylalanine; D004958:Estradiol; D011505:Protein-Tyrosine Kinases; D002118:Calcium