Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases

Abstract

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.