Zn²⁺-dependent tyrosine phosphorylation of a 68-kDa protein and its differentiation from Mg²⁺-dependent tyrosine phosphorylation in sheep platelets

Abstract

A 68-kDa protein that was tyrosine phosphorylated in the presence of Zn²⁺ and two proteins of 52 and 46 kDa that were tyrosine phosphorylated in the presence of Mg²⁺ were separated by column chromatography of a sheep platelet high speed supernatant on poly(Glu,Tyr)4: 1 copolymer-Sepharose or tyrosine-Sepharose. Phosphorylation of the 68-kDa protein occurred maximally in the presence of Zn²⁺ while Mg²⁺ was ineffective. The kinases responsible for the Zn²⁺- and Mg²⁺-dependent tyrosine phosphorylation could also tyrosine phosphorylate poly (Glu,Tyr)4:1, histone, and angiotensin II with the same metal ion specificity. The two tyrosine kinase activities could be also distinguished by their differential response to polyamines and quercetin. Zn²⁺ stimulation did not appear to be due to the inhibition of a protein tyrosine phosphatase. Sephadex G-100 gel filtration of the fraction showing Zn²⁺-dependent tyrosine phosphorylation of the 68-kDa protein showed that the tyrosine kinase activity corresponded to a molecular mass of 68,000 and it showed a protein band of 68 kDa as detected by silver staining on sodium dodecyl sulfate-polyacrylamide gel.