The vast majority of serine/threonine protein kinases have a strong preference for ATP over GTP as a phosphate donor. CK2 (Casein kinase 2) is an exception to this rule and in this study we investigate whether calcium/calmodulin-dependent protein kinase II (CaMKII) has the same extended nucleotide range. Using the Drosophila enzyme, we have shown that CaMKII uses Mg(2+)GTP with a higher K-m and V-max compared to Mg(2+)ATP. Substitution of Mn2+ for Mg2+ resulted in a much lower K-m for GTP, while nearly abolishing the ability of CaMKII to use ATP. These similar results were obtained with rat alpha CaMKII, showing the ability to use GTP to be a general property of CaMKII. The V-max difference between Mg(2+)ATP and Mg(2+)GTP was found to be due to the fact that ADP is a potent inhibitor of phosphorylation, while GDP has modest effects. There were no differences found between sites autophosphorylated by ATP and GTP, either by partial proteolysis or mass spectrometry. Phosphorylation of fly head extract revealed that similar proteins are substrates for CaMKII whether using Mg(2+)ATP or Mg(2+)GTP. This new information confirms that CaMKII can use both ATP and GTP, and opens new avenues for the study of regulation of this kinase. (C) 2009 Elsevier Inc. All rights reserved.