1.Phosrestide-1, a peptide derived from the Drosophila photoreceptor protein phosrestin I, is a potent substrate for Ca2+/calmodulin-dependent protein kinase II from rat brain.
Kahn ES;Kinumi T;Tobin SL;Matsumoto H Comp Biochem Physiol B Biochem Mol Biol. 1998 Apr;119(4):739-46.
Multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMK II) plays a crucial role in mediation of cellular responses to rising cytosolic Ca2+ levels. We find that the novel peptide substrate PGTIEKKRSNAMKKMKSIEQHR serves as a highly potent substrate for CaMK II enzymes purified from both Drosophila and rat. The peptide is derived from a photoreceptor-specific protein, phosrestin I, of the Drosophila compound eye and is designated as phosrestide-1. Using saturating substrate concentrations, the enzymes from both species transfer the gamma-phosphoryl group of ATP to phosrestide-1 at a level three to ten times greater than to the commercially available mammalian-derived CaMK II substrates, autocamtide-3 and syntide-2. This indicates a conservation of substrate preferences for CaMK II derived from distantly related species, a dipteran fly and a mammal. Although phosrestide-1 contains two potential serine residues for CaMK II phosphorylation, we find that only the C-terminal serine is phosphorylated by rat CaMK II. However, removal of the upstream sequence containing the N-terminal serine substantially reduced the potency of phosrestide-1 as a CaMK II substrate to a level comparable to that of syntide-2 or autocamtide-3.
2.The effect of KN-62, Ca2+/calmodulin dependent protein kinase II inhibitor on cell cycle.
Minami H;Inoue S;Hidaka H Biochem Biophys Res Commun. 1994 Feb 28;199(1):241-8.
The isoquinolinesulfonamide derivative, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin dependent protein kinase II (CaM kinase II) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M., and Hidaka, H.(1990) J. Biol. Chem. 265, 4315-4320). KN-62 inhibits growth of K562 cells, in a dose-dependent manner. Flow cytometric analysis demonstrates that the treatment of K562 cells with 10 microM KN-62 causes an accumulation of cells in S phase. Immunoblotting studies showed that specific antibodies against CaM kinase II recognized the 65 kDa of protein in K562 cells. This protein showed protein kinase activity as examined by the activity gel method. The inhibition of this enzyme activity by KN-62 was dose-dependent. The immunoprecipitates with the antibodies from K562 cells phosphorylates the synthetic peptide substrates, syntide-2. These results suggest that CaM kinase II plays an important role in the mechanisms for the cell growth in K562 cells.
3.Identification, characterization, immunocytochemical localization, and developmental changes in the activity of calcium/calmodulin-dependent protein kinase II in the CNS of Bombyx mori during postembryonic development.
Shanavas A;Dutta-Gupta A;Murthy CR J Neurochem. 1998 Apr;70(4):1644-51.
In the present investigation, in vitro phosphorylation of CNS proteins of the silkworm Bombyx mori during the postembryonic development have been studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of phosphorylated proteins revealed the presence of major phosphoproteins of 59/60 kDa. Based on molecular mass, calcium/calmodulin-dependent autophosphorylation, substrate specificity, KN-62 inhibition, apparent Km for ATP and syntide-2, these proteins were identified as calcium/calmodulin-dependent protein kinase II (CaM kinase II). Anti-rat CaM kinase II monoclonal antibody showed immunoreactivity with Bombyx CaM kinase II isoforms. This kinase showed a high degree of autophosphorylation in neural tissue. During postembryonic development of Bombyx, two distinct peaks of enzyme activity could be noticed, one at the late-larval and another at the late-pupal stage, which were associated with an increase in amount of the enzyme. These results suggested that the expression of CaM kinase II in the CNS of Bombyx was developmentally regulated.
