Requirement for an Additional Divalent Metal Cation To Activate Protein Tyrosine Kinases

Abstract

In addition to the magnesium ion needed to form the true phosphate-donating substrate (ATP-Mg complex), we have determined that at least one additional Mg2+ ion is essential for the activation of protein tyrosine kinases. This activation was investigated in detail using purified Csk, Src, and the fibroblast growth factor receptor kinase, which led to the following conclusions. (1) The catalytic activity of these kinases is dependent on the Mg2+ concentration present in the assay, approaching saturation at 5-8 mM MgCl2, while ATP was saturated at approximately 1 mM MgCl2. (2) Extrapolation to zero free Mg2+ at a constant ATP-Mg concentration predicts zero activity, suggesting that free magnesium ion in excess of that needed to bind to ATP is essential for the activation of these enzymes. (3) The free magnesium ion activates Csk and Src kinase activity by increasing the Vmax but does not change their apparent Km(ATP-Mg). In contrast, the free magnesium ion activates the fibroblast growth factor receptor kinase activity by increasing its Vmax and decreasing its apparent Km(ATP-Mg). These and previous studies with the insulin receptor tyrosine kinase suggest that receptor-type protein tyrosine kinases respond to the concentration of free Mg2+ differently than soluble protein tyrosine kinases. (4) With the phosphate-accepting substrate as the variable ligand, increases in the concentration of free Mg2+ resulted in increases in the apparent Vmax for all tyrosine kinases examined, but the apparent Km response is dependent on the enzyme and the substrate used. While these studies do not pinpoint a single kinetic mechanism, they do suggest that additional magnesium ion(s) is(are) an essential activator for protein tyrosine kinases in addition to being a part of the ATP-Mg complex. The difference among protein tyrosine kinases in their kinetic response to the additional divalent metal cation and the potential biological significance of such are discussed.

Biochemical Characterization of the Arabidopsis Protein Kinase SOS2 That Functions in Salt Tolerance

“…These enzymes can also be activated or inactivated by binding of a cation to an additional site of interaction (Armstrong et al, 1979;Sun and Budde, 1997). To determine the divalent cation preferences in vitro of these mutants, we measured peptide substrate phosphorylation in the presence of various concentrations of either Mg 2ϩ or Mn 2ϩ (Fig.…”

Effect of Mg2+ on the thermal inactivation and unfolding of creatine kinase

“…The modi®cation of protein structure and activity by millimolar amounts of divalent cations was reported for high molecular weight oligomeric hemoglobins [25], hemocyanins [26], Groel oligomer [27], coagulation factor IX [28] and protein tyrosine kinases [6]. Some factors a ecting cell surface receptors were found to signi®cantly increase the Mg 2+ in¯ux and intracellular concentration of free Mg 2+ in a variety of cells [6].…”

“…Some factors a ecting cell surface receptors were found to signi®cantly increase the Mg 2+ in¯ux and intracellular concentration of free Mg 2+ in a variety of cells [6]. This raises the possibility that Mg 2+ plays an essential role in the activity and the stability of the structures of many oligomer proteins in the cells.…”

“…The excitation wavelength was 295 nm [6]. The course of the conformational changes was followed by rapidly mixing a small volume of the enzyme with a solution of 50 mM Glycine± NaOH bu er of pH 9.0 with Mg(Ac) 2 at the desired concentration and preheated to the required temperature.…”

“…In the second, magnesium binds to a ligand, thereby making a suitable substrate (e.g. ATP±Mg) [6,7]. The third mechanism is through allosteric activation in which binding to one or www.elsevier.com/locate/ijbcb more forms of the enzyme in the reaction course induces a change in its conformation [8,9].…”