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 phosphorylated 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 protein kinase (PKA), a serine/threonine kinase. Vanadate, in the form of monomer, dimer, tetramer and pentamer species, neither inhibits nor activates PKA. In marked contrast, decavandate is a competitive inhibitor (Ki = 1.8 +/- 0.1 mM) of kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), a peptide-based substrate. This inhibition pattern is especially surprising, since the negatively charged decavanadate would not be predicted to bind to the region of the active site of the enzyme that accommodates the positively charged kemptide substrate. Our studies suggest that decavanadate can associate with kemptide in solution, which would prevent kemptide from interacting with the enzyme. Vanadium(IV) also inhibits the PKA-catalysed phosphorylation of kemptide, but with an IC50 of 366 +/- 10 microM. However, in this case V4+ appears to bind to the Mg(2+)-binding site, since it can substitute for Mg2+. In the absence of Mg2+, the optimal concentration of vanadium(IV) for the PKA-catalysed phosphorylation of kemptide is 100 microM, with concentrations above 100 microM being markedly inhibitory. However, even at the optimal 100 microM V4+ concentration, the Vmax and K(m) values (for kemptide) are significantly less favourable than those obtained in the presence of 100 microM Mg2+. In summary, we have found that oxovanadium ions can directly alter the activity of the serine/threonine-specific PKA.