Prostatic epithelial cells in culture: phosphorylation of protein tyrosyl residues and tyrosine protein kinase activity.
The ability of dividing canine prostatic epithelial cells in primary monolayers to phosphorylate protein tyrosyl residues was evaluated by metabolic studies performed through incorporation of [32P]-phosphate into alkali-resistant phosphoproteins and by the assay of their tyrosine protein kinase activity. The presence of sodium orthovanadate during cell incubation with [32P]-phosphate greatly enhanced the relative labelling intensity of a 44 kDa alkali-resistant phosphoprotein and the total cellular content of phosphotyrosine in proteins; in this respect, growth factors such as epidermal growth factor, insulin, and insulin-like growth factor I, and the steroids dihydrotestosterone and estradiol were inactive. When the cells were solubilized, sodium orthovanadate stimulated their tyrosine protein kinase activity and inhibited their phosphotyrosine phosphatase activity. To characterize the tyrosine protein kinase of these cultured cells, conditions for optimal activity were established using the substrate poly [Glu80Na, Tyr20]. The subcellular localization of the enzyme was determined upon cell fractionation: 88% of the kinase activity was associated with the particulate fraction and 30% of this activity was partially solubilized with 0.5% Triton X-100; this solubilization was improved to 83% in the presence of 0.25 M KCI. The enzyme directly solubilized from prostatic cells with Triton X-100 (38% of activity) mainly catalyzed the alkali-resistant phosphorylation of pp63, pp59, and pp44, which contained phosphotyrosine. These proteins were also phosphorylated by the major peak of kinase activity which was eluted at an apparent molecular weight of 300-350 kDa upon gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)
Bourassa
C
C
Research Center, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada.
Nguyen
L T
LT
Durocher
Y
Y
Roberts
K D
KD
Chevalier
S
S
D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D006133:Growth Substances / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D019000:Phosphotyrosine; D011467:Prostate / Q000166:cytology / Q000201:enzymology*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013256:Steroids / Q000494:pharmacology; D013347:Subcellular Fractions; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D006133:Growth Substances; D013256:Steroids; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D008345:Manganese; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D008274:Magnesium