Physiological concentrations of divalent magnesium ion activate the serine/threonine specific protein kinase ERK2

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by phorbol esters, serum, and growth factors, and displays enhanced activity in several human tumors. Despite its important biological function, its mechanism of catalysis and mode of regulation are poorly understood. Recently, we showed that in the presence of 10 mM magnesium chloride, ERK2 phosphorylates the transcription factor Ets-1 through a random-ordered ternary-complex mechanism [Waas, W. F., and Dalby, K. N. (2002) J. Biol. Chem. 277, 12532]. Now we provide kinetic evidence that ERK2 must bind two divalent magnesium ions to facilitate catalysis at a physiologically relevant rate, because a second magnesium ion promotes both MgATP(2-) binding and phosphoryl transfer. The velocity dependence on magnesium at saturating concentrations of the protein substrate, EtsDelta138, over a range of ATP(4-) and Mg2+ ion concentrations, supports the notion that magnesium is an essential activator of ERK2. At high (greater than or equal to 1 mM) concentrations of ATP(4-), the velocity dependence on total Mg2+ is sigmoidal, but plateaus at high concentrations of free Mg2+, where the enzyme is fully activated. At concentrations of Mg2+ of less than or equal to4 mM, the velocity dependence on ATP(4-) displays a peak when the concentration of ATP(4-) approaches that of total Mg2+ and tends to zero at high concentrations of ATP(4-), where the enzyme is predominantly unactivated. The observed velocity dependencies are consistent with the notion that ERK2.EtsDelta138 complexes and ATP(4-) compete for the same pool of Mg2+ ions in solution. No binding of ATP(4-) (0-2.5 mM) by ERK2 (65 muM) can be detected using isothermal titration calorimetry at 27 degreesC, pH 8.0, and an ionic strength of 0.15 M (KCl), suggesting that the complex, MgATP(2-), is the true substrate for ERK2. In contrast, 5-iodotubericidin binds ERK2 tightly (K-d = 1 muM) and displays a competitive inhibition pattern toward MgATP(2-) and a mixed pattern toward free Mg2+, suggesting that the binding of Mg2+ before MgATP(2-) is not compulsory.