Kinetic properties of the C-terminal Src kinase, p50csk.
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 Mg2+ and 5 min with Mn2+. With Mg2+ and Mn2+, respectively, K(m) (ATP) was 56.9 +/- 6.2 and 5.4 +/- 0.6 microM and Vmax was 293 +/- 52 and 217 +/- 38 pmol phosphate transferred (microgram Csk)-1 min-1. Optimal concentrations of Mg2+ and Mn2+ 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 (Ca2+) cations, the sensitivity being 2-5-fold higher with Mg2+ than Mn2+. Physiological concentrations of Ca2+ (less than 10 microM) were without effect. Autophosphorylation of Csk was demonstrated in vitro, but did not influence the catalytic activity. Addition of inorganic phosphate above 100 microM strongly inhibited Csk catalytic activity towards poly(Glu, Tyr) in the presence of Mn2+, but not in the presence of Mg2+. 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.
Vang
T
T
Institute of Medical Biochemistry, University of Oslo, Norway. torkel.vang@basalmed.uio.no
Taskén
K
K
Skålhegg
B S
BS
Hansson
V
V
Levy
F O
FO
D001667:Binding, Competitive; D000081247:CSK Tyrosine-Protein Kinase; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D008345:Manganese; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D008274:Magnesium