Kinetic properties of the C-terminal Src kinase, p50 csk

Abstract

Csk is an important regulator of tyrosine kinases of the Src family. In this paper, we have characterised the kinetics and catalytic properties of a highly active and stable enzyme obtained in milligram amounts by expressing the enzyme as a fusion protein with glutathione- S-transferase (GST) in Escherichia coli. Using the synthetic polyamino acid poly(Glu,Tyr) as substrate, phosphotransferase activity was linear for 7–8 min with Mg 2+ and 5 min with Mn 2+. With Mg 2+ and Mn 2+, respectively, K m(ATP) was 56.9±6.2 and 5.4±0.6 μM and V max was 293±52 and 217±38 pmol phosphate transferred ( μg Csk) −1 min −1. Optimal concentrations of Mg 2+ and Mn 2+ were 4–10 mM and 2–3 mM, respectively, and higher concentrations of both cations were inhibitory. The Csk activity was highly sensitive to monovalent (Na +, K +) and divalent (Ca 2+) cations, the sensitivity being 2–5-fold higher with Mg 2+ than Mn 2+. Physiological concentrations of Ca 2+ (less than 10 μM) were without effect. Autophosphorylation of Csk was demonstrated in vitro, but did not influence the catalytic activity. Addition of inorganic phosphate above 100 μM strongly inhibited Csk catalytic activity towards poly(Glu,Tyr) in the presence of Mn 2+, but not in the presence of Mg 2+. Phosphorylation of a physiological substrate (Lck) and autophosphorylation of Csk was not inhibited by phosphate, indicating that the phosphate-dependent inhibition of Csk activity was substrate specific.