Modification of calmodulin on Lys-75 by carbamoylating nitrosoureas

This paper describes characterization of the reaction of calmodulin with a series of nitrosoureas which are capable of releasing amine-reactive isocyanates of varying hydrophobic character. The site of calcium-dependent carbamoylation on calmodulin by the antineoplastic agent 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (methyl CCNU) was determined to be Lys-75 as demonstrated using [ring-14C]methyl CCNU and sequence analysis of the sole labeled peptide obtained from tryptic digestion of reversed-phase high pressure liquid chromatography (HPLC)-purified radiolabeled calmodulin. CCNU, the 4-desmethylcyclohexyl derivative of methyl CCNU, and its reactive hydrolysis product, cyclohexyl isocyanate, were also determined to modify calmodulin in a similar manner and at the same site, as demonstrated by specific blockade of modification by the calmodulin antagonist calmidazolium. Nitrosoureas which release the less hydrophobic 4-hydroxy- and 4-carboxycyclohexyl isocyanates are unable to modify calmodulin at 25-fold higher concentrations than those required for modification with methyl CCNU, CCNU, or cyclohexyl isocyanate. With this monomodified Lys-75 derivative, purified to homogeneity by HPLC, differential effects of modification on the activation of bovine brain 3',5'-cyclic nucleotide phosphodiesterase (phosphodiesterase) and human erythrocyte Ca2+,Mg2+-ATPase were observed. Compared to the amounts of native calmodulin needed, phosphodiesterase required 7-fold higher amounts of this derivative to reach maximal activation, whereas the activation of the ATPase was unaffected. Clearly, different regions of calmodulin are responsible for the activation of phosphodiesterase and the ATPase. We conclude that Lys-75 is not essential for the function of calmodulin but is in a region of the molecule involved in interaction with phosphodiesterase as well as the binding of certain hydrophobic calmodulin antagonists.