Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src

Nobuhiro Hayashi, Chisako Nakagawa, Yutaka Ito, Akihiko Takasaki, Yuji Jinbo, Yoshinori Yamakawa, Koiti Titani, Keiichiro Hashimoto, Yoshinobu Izumi, Norio Matsushima

Research output: Contribution to journalArticle

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Abstract

pp60v-src tyrosine protein kinase was suggested to interact with Ca2+-bound calmodulin (Ca2+/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca2+/CaM; (1) the N terminus of pp60 v-src is myristoylated like CAP-23/NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60v-src. To verify this possibility, we investigated the direct interaction between pp60 v-src and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60v-src. The binding assay indicated that only the myristoylated peptide binds to Ca 2+/CaM, and the non-myristoylated peptide is not able to bind to Ca2+/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (KD) of 2.07×10 -9M (KD1) and 3.93×10-6M (K D2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca2+/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca2+/CaM complex is 2-3Å smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60v-src and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions.

Original languageEnglish
Pages (from-to)169-180
Number of pages12
JournalJournal of Molecular Biology
Volume338
Issue number1
DOIs
Publication statusPublished - 16-04-2004

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Calmodulin
Calcium
Peptides
Oncogene Protein pp60(v-src)
Serine
Phosphorylation
src-Family Kinases
Protein Kinase C
Lysine
X-Rays
Neurons

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Cite this

Hayashi, N., Nakagawa, C., Ito, Y., Takasaki, A., Jinbo, Y., Yamakawa, Y., ... Matsushima, N. (2004). Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src. Journal of Molecular Biology, 338(1), 169-180. https://doi.org/10.1016/j.jmb.2004.02.041
Hayashi, Nobuhiro ; Nakagawa, Chisako ; Ito, Yutaka ; Takasaki, Akihiko ; Jinbo, Yuji ; Yamakawa, Yoshinori ; Titani, Koiti ; Hashimoto, Keiichiro ; Izumi, Yoshinobu ; Matsushima, Norio. / Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src. In: Journal of Molecular Biology. 2004 ; Vol. 338, No. 1. pp. 169-180.
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title = "Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src",
abstract = "pp60v-src tyrosine protein kinase was suggested to interact with Ca2+-bound calmodulin (Ca2+/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca2+/CaM; (1) the N terminus of pp60 v-src is myristoylated like CAP-23/NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60v-src. To verify this possibility, we investigated the direct interaction between pp60 v-src and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60v-src. The binding assay indicated that only the myristoylated peptide binds to Ca 2+/CaM, and the non-myristoylated peptide is not able to bind to Ca2+/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (KD) of 2.07×10 -9M (KD1) and 3.93×10-6M (K D2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca2+/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca2+/CaM complex is 2-3{\AA} smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60v-src and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions.",
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Hayashi, N, Nakagawa, C, Ito, Y, Takasaki, A, Jinbo, Y, Yamakawa, Y, Titani, K, Hashimoto, K, Izumi, Y & Matsushima, N 2004, 'Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src', Journal of Molecular Biology, vol. 338, no. 1, pp. 169-180. https://doi.org/10.1016/j.jmb.2004.02.041

Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src. / Hayashi, Nobuhiro; Nakagawa, Chisako; Ito, Yutaka; Takasaki, Akihiko; Jinbo, Yuji; Yamakawa, Yoshinori; Titani, Koiti; Hashimoto, Keiichiro; Izumi, Yoshinobu; Matsushima, Norio.

In: Journal of Molecular Biology, Vol. 338, No. 1, 16.04.2004, p. 169-180.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src

AU - Hayashi, Nobuhiro

AU - Nakagawa, Chisako

AU - Ito, Yutaka

AU - Takasaki, Akihiko

AU - Jinbo, Yuji

AU - Yamakawa, Yoshinori

AU - Titani, Koiti

AU - Hashimoto, Keiichiro

AU - Izumi, Yoshinobu

AU - Matsushima, Norio

PY - 2004/4/16

Y1 - 2004/4/16

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