VANADIUM OXOANIONS AND CAMP-DEPENDENT PROTEIN-KINASE - AN ANTI-SUBSTRATE INHIBITOR

Vanadium oxoions have been shown to elicit a wide range of effects in biological systems, including an increase in the quantity of phosphory lated proteins. This response has been attributed to the inhibition of protein phosphatases, the indirect activation of protein kinases via stimulation of enzymes at early steps in signal transduction pathways and/or the direct activation of protein kinases. We have evaluated the latter possibility by exploring the effects of vanadate, decavanadate and vanadyl cation species on the activity of the cAMP-dependent prot ein kinase (PKA), a serine/threonine kinase. Vanadate, in the form of monomer, dimer, tetramer and pentamer species, neither inhibits nor ac tivates PKA. In marked contrast, decavanadate is a competitive inhibit or (K-i = 1.8+/-0.1 mM) of kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), a p eptide-based substrate. This inhibition pattern is especially surprisi ng, since the negatively charged decavanadate would not be predicted t o bind to the region of the active site of the enzyme that accommodate s the positively charged kemptide substrate. Our studies suggest that decavanadate can associate with kemptide in solution, which would prev ent kemptide from interacting with the enzyme. Vanadium(IV) also inhib its the PKA-catalysed phosphorylation of kemptide, but with an IC50 of 366 +/- 10 mu M. However, in this case V4+ appears to bind to the Mg2 +-binding site, since it can substitute for Mg2+. In the absence of Mg 2+, the optimal concentration of vanadium(IV) for the PKA-catalysed ph osphorylation of kemptide is 100 mu M, with concentrations above 100 m u M being markedly inhibitory. However, even at the optimal 100 mu M V 4+ concentration, the V-max and K-m values (for kemptide) are signific antly less favourable than those obtained in the presence of 100 mu M Mg2+. In summary, we have found that oxovanadium ions can directly alt er the activity of the serine/threonine-specific PKA.