Phosrestide-1, a peptide derived from the Drosophila photoreceptor protein phosrestin I, is a potent substrate for Ca/calmodulin-dependent protein kinase II from rat brain |
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| Authors: | Esther S. Kahn Tomoya Kinumi Sara L. Tobin Hiroyuki Matsumoto |
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| Affiliation: | aDepartment of Biochemistry and Molecular Biology and The NSF EPSCoR Oklahoma Laser Mass Spectrometry Facility, The University of Oklahoma, Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190, USA;bDepartment of Physiology, University of Arizona, 1609 North Warren Avenue, Biomedical Research Laboratories, A-114, Tucson, AZ 85724, USA;cDepartment of Applied Physics and Chemistry, The University of Electro-Communications, 1-5-1 Chofugaoka, Chofu, Tokyo 182, Japan;dCenter for Biomedical Ethics, Program for Genomics, Ethics, and Society, Stanford University, 701A Welch Road, Suite 1105, Palo Alto, CA 94304, USA |
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| Abstract: | 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 γ-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. We also find that a peptide representing the N-terminal segment of phosrestide-1 does not inhibit either CaMK II. Therefore, the enhanced potency of phosrestide-1 as a CaMK II substrate is likely to be due to a preferred conformation of the peptide induced by the N-terminal segment rather than to a specific binding of the enzymes to the N-terminus of the peptide. To the best of our knowledge, phosrestide-1 is the first CaMK II substrate which is designed based on an invertebrate sequence. The high phosphorylation level of phosrestide-1 by CaMK II of mammalian origin may reflect highly conserved CaMK II signaling cascades between vertebrates and invertebrates. |
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| Keywords: | Drosophila melanogaster CaM kinase II Peptide substrate Syntide-2 Autocamtide-3 MALDI-TOFMS |
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