Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate

Ariko Fujise, Keiko Mizuno, Yoshihiko Ueda, Shin Ichi Osada, Syu Ichi Hirai, Atushi Takayanagi, Nobuyoshi Shimizu, M. Koji Owada, Hiroshi Nakajima, Shigeo Ohno

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Abstract

Although myristoylated alanine-rich C kinase substrate (MARCKS), has been employed as an indicator for the activation of protein kinase C (PKC) in intact cells, little is known about its specificity for PKC family members. To address this question, we partially purified human MARCKS from baculovirus-infected cells and compared the kinetic parameters for phosphorylation by PKC isozymes, conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ), all of which are distributed in a wide variety of cells. cPKCα, nPKCδ, and nPKCε efficiently phosphorylated intact MARCKS protein in vitro. The affinity of MARCKS for cPKCα, nPKCδ, and nPKCε was extremely high and decreased in the order α > δ > ε with K(m) values of 10.7, 20.7, and 29.8 nM, respectively. The rate of phosphorylation also decreased in the same order. In contrast, aPKCζ did not phosphorylate MARCKS efficiently, and we were unable to estimate the kinetic parameters. These results suggest that cPKCα, nPKCδ, and nPKCε but not aPKCζ are enzymes that phosphorylate MARCKS in response to PKC activators in intact cells. The structural requirements of MARCKS for efficient phosphorylation by these PKC members were then examined using a peptide that surrounds the phosphorylation site of MARCKS (peptide MARCKS). Interestingly, intact MARCKS showed a 90-150 times lower rate of phosphorylation by PKCs compared with peptide MARCKS, whereas the former showed a 40-180 times higher affinity for these PKC members. This implies that intact MARCKS protein retains a very high affinity for PKC with the sacrifice of its phospho-accepting activity. The structural requirements of PKC were then examined using a calpain-cleaved active fragment of nPKCδ. MARCKS was phosphorylated by the active catalytic fragment as efficiently as by intact nPKCδ, indicating that the kinase domain is sufficient for the high affinity interaction with intact MARCKS. However, gel overlay assay revealed that both intact nPKCδ and its regulatory domain bind to MARCKS, suggesting that both the kinase and regulatory domains of nPKCδ are involved in the high affinity interaction with intact MARCKS protein.

Original languageEnglish
Pages (from-to)31642-31648
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number50
Publication statusPublished - 1994 Dec 16

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Alanine
Protein Kinase C
Substrates
Phosphorylation
myristoylated alanine-rich C kinase substrate
Kinetic parameters
Peptides
Phosphotransferases
Proteins
Calpain
Baculoviridae
Isoenzymes
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Fujise, A., Mizuno, K., Ueda, Y., Osada, S. I., Hirai, S. I., Takayanagi, A., ... Ohno, S. (1994). Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate. Journal of Biological Chemistry, 269(50), 31642-31648.

Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate. / Fujise, Ariko; Mizuno, Keiko; Ueda, Yoshihiko; Osada, Shin Ichi; Hirai, Syu Ichi; Takayanagi, Atushi; Shimizu, Nobuyoshi; Owada, M. Koji; Nakajima, Hiroshi; Ohno, Shigeo.

In: Journal of Biological Chemistry, Vol. 269, No. 50, 16.12.1994, p. 31642-31648.

Research output: Contribution to journalArticle

Fujise, A, Mizuno, K, Ueda, Y, Osada, SI, Hirai, SI, Takayanagi, A, Shimizu, N, Owada, MK, Nakajima, H & Ohno, S 1994, 'Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate', Journal of Biological Chemistry, vol. 269, no. 50, pp. 31642-31648.
Fujise, Ariko ; Mizuno, Keiko ; Ueda, Yoshihiko ; Osada, Shin Ichi ; Hirai, Syu Ichi ; Takayanagi, Atushi ; Shimizu, Nobuyoshi ; Owada, M. Koji ; Nakajima, Hiroshi ; Ohno, Shigeo. / Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 50. pp. 31642-31648.
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abstract = "Although myristoylated alanine-rich C kinase substrate (MARCKS), has been employed as an indicator for the activation of protein kinase C (PKC) in intact cells, little is known about its specificity for PKC family members. To address this question, we partially purified human MARCKS from baculovirus-infected cells and compared the kinetic parameters for phosphorylation by PKC isozymes, conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ), all of which are distributed in a wide variety of cells. cPKCα, nPKCδ, and nPKCε efficiently phosphorylated intact MARCKS protein in vitro. The affinity of MARCKS for cPKCα, nPKCδ, and nPKCε was extremely high and decreased in the order α > δ > ε with K(m) values of 10.7, 20.7, and 29.8 nM, respectively. The rate of phosphorylation also decreased in the same order. In contrast, aPKCζ did not phosphorylate MARCKS efficiently, and we were unable to estimate the kinetic parameters. These results suggest that cPKCα, nPKCδ, and nPKCε but not aPKCζ are enzymes that phosphorylate MARCKS in response to PKC activators in intact cells. The structural requirements of MARCKS for efficient phosphorylation by these PKC members were then examined using a peptide that surrounds the phosphorylation site of MARCKS (peptide MARCKS). Interestingly, intact MARCKS showed a 90-150 times lower rate of phosphorylation by PKCs compared with peptide MARCKS, whereas the former showed a 40-180 times higher affinity for these PKC members. This implies that intact MARCKS protein retains a very high affinity for PKC with the sacrifice of its phospho-accepting activity. The structural requirements of PKC were then examined using a calpain-cleaved active fragment of nPKCδ. MARCKS was phosphorylated by the active catalytic fragment as efficiently as by intact nPKCδ, indicating that the kinase domain is sufficient for the high affinity interaction with intact MARCKS. However, gel overlay assay revealed that both intact nPKCδ and its regulatory domain bind to MARCKS, suggesting that both the kinase and regulatory domains of nPKCδ are involved in the high affinity interaction with intact MARCKS protein.",
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AU - Fujise, Ariko

AU - Mizuno, Keiko

AU - Ueda, Yoshihiko

AU - Osada, Shin Ichi

AU - Hirai, Syu Ichi

AU - Takayanagi, Atushi

AU - Shimizu, Nobuyoshi

AU - Owada, M. Koji

AU - Nakajima, Hiroshi

AU - Ohno, Shigeo

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N2 - Although myristoylated alanine-rich C kinase substrate (MARCKS), has been employed as an indicator for the activation of protein kinase C (PKC) in intact cells, little is known about its specificity for PKC family members. To address this question, we partially purified human MARCKS from baculovirus-infected cells and compared the kinetic parameters for phosphorylation by PKC isozymes, conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ), all of which are distributed in a wide variety of cells. cPKCα, nPKCδ, and nPKCε efficiently phosphorylated intact MARCKS protein in vitro. The affinity of MARCKS for cPKCα, nPKCδ, and nPKCε was extremely high and decreased in the order α > δ > ε with K(m) values of 10.7, 20.7, and 29.8 nM, respectively. The rate of phosphorylation also decreased in the same order. In contrast, aPKCζ did not phosphorylate MARCKS efficiently, and we were unable to estimate the kinetic parameters. These results suggest that cPKCα, nPKCδ, and nPKCε but not aPKCζ are enzymes that phosphorylate MARCKS in response to PKC activators in intact cells. The structural requirements of MARCKS for efficient phosphorylation by these PKC members were then examined using a peptide that surrounds the phosphorylation site of MARCKS (peptide MARCKS). Interestingly, intact MARCKS showed a 90-150 times lower rate of phosphorylation by PKCs compared with peptide MARCKS, whereas the former showed a 40-180 times higher affinity for these PKC members. This implies that intact MARCKS protein retains a very high affinity for PKC with the sacrifice of its phospho-accepting activity. The structural requirements of PKC were then examined using a calpain-cleaved active fragment of nPKCδ. MARCKS was phosphorylated by the active catalytic fragment as efficiently as by intact nPKCδ, indicating that the kinase domain is sufficient for the high affinity interaction with intact MARCKS. However, gel overlay assay revealed that both intact nPKCδ and its regulatory domain bind to MARCKS, suggesting that both the kinase and regulatory domains of nPKCδ are involved in the high affinity interaction with intact MARCKS protein.

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