Studies with synthetic peptide substrates derived from the neuronal protein neurogranin reveal structural determinants of potency and selectivity for protein kinase C

Shu Jen Chen, Eric Klann, Morganne C. Gower, Craig M. Powell, J. Suzanne Sessoms, J. David Sweatt

Research output: Contribution to journalArticle

Abstract

The neuronal protein neurogranin, also known as RC3, is a selective substrate for protein kinase C (PKC). We synthesized a peptide corresponding to the phosphorylation domain of neurogranin (amino acids 28-43) and characterized its properties as a PKC substrate. Neurogranin(28-43) was phosphorylated by purified PKC with a Km of 150 nM. No significant phosphorylation of the peptide by either cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase II could be detected. Thus, neurogranin(28-43) is a potent and selective substrate for PKC. We tested several peptide analogues of neurogranin(28-43) for their substrate potency and specificity as kinase substrates, in order to help elucidate the structural determinants involved in the phosphorylation of substrates by PKC. Substituting Arg36 with He caused a significant reduction in the affinity for PKC. Replacing Lys30 with Arg enhanced the catalytic efficiency ( Vmax/Km) for PKC but diminished the selectivity of the substrate for PKC. These results support the generally held model that basic amino acids on both sides of the phosphorylated Ser are important structural determinants in PKC substrates. However, the data also suggest that the presence of particular basic amino acids (Arg vs Lys) can contribute to the degree of selectivity of a substrate for PKC. Replacement with Ala of Phe35, the amino acid adjacent to the Ser34 phosphorylation site, resulted in a peptide with greatly diminished potency as a PKC substrate. This finding indicates a critical role of Phe35 in modulating binding and phosphorylation of neurogranin-derived peptides by PKC. Substituting He in this same position caused only a slight decrease in potency as a PKC substrate, suggesting that the hydrophobic character of the amino acid in this position was an important potency determinant.

Original languageEnglish (US)
Pages (from-to)1032-1039
Number of pages8
JournalBiochemistry
Volume32
Issue number4
StatePublished - 1993

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Neurogranin
Protein Kinase C
Peptides
Substrates
Phosphorylation
Proteins
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Basic Amino Acids
Amino Acids
Calcium-Calmodulin-Dependent Protein Kinase Kinase
Calcium-Calmodulin-Dependent Protein Kinases

ASJC Scopus subject areas

  • Biochemistry

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Studies with synthetic peptide substrates derived from the neuronal protein neurogranin reveal structural determinants of potency and selectivity for protein kinase C. / Chen, Shu Jen; Klann, Eric; Gower, Morganne C.; Powell, Craig M.; Suzanne Sessoms, J.; David Sweatt, J.

In: Biochemistry, Vol. 32, No. 4, 1993, p. 1032-1039.

Research output: Contribution to journalArticle

Chen, Shu Jen ; Klann, Eric ; Gower, Morganne C. ; Powell, Craig M. ; Suzanne Sessoms, J. ; David Sweatt, J. / Studies with synthetic peptide substrates derived from the neuronal protein neurogranin reveal structural determinants of potency and selectivity for protein kinase C. In: Biochemistry. 1993 ; Vol. 32, No. 4. pp. 1032-1039.
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abstract = "The neuronal protein neurogranin, also known as RC3, is a selective substrate for protein kinase C (PKC). We synthesized a peptide corresponding to the phosphorylation domain of neurogranin (amino acids 28-43) and characterized its properties as a PKC substrate. Neurogranin(28-43) was phosphorylated by purified PKC with a Km of 150 nM. No significant phosphorylation of the peptide by either cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase II could be detected. Thus, neurogranin(28-43) is a potent and selective substrate for PKC. We tested several peptide analogues of neurogranin(28-43) for their substrate potency and specificity as kinase substrates, in order to help elucidate the structural determinants involved in the phosphorylation of substrates by PKC. Substituting Arg36 with He caused a significant reduction in the affinity for PKC. Replacing Lys30 with Arg enhanced the catalytic efficiency ( Vmax/Km) for PKC but diminished the selectivity of the substrate for PKC. These results support the generally held model that basic amino acids on both sides of the phosphorylated Ser are important structural determinants in PKC substrates. However, the data also suggest that the presence of particular basic amino acids (Arg vs Lys) can contribute to the degree of selectivity of a substrate for PKC. Replacement with Ala of Phe35, the amino acid adjacent to the Ser34 phosphorylation site, resulted in a peptide with greatly diminished potency as a PKC substrate. This finding indicates a critical role of Phe35 in modulating binding and phosphorylation of neurogranin-derived peptides by PKC. Substituting He in this same position caused only a slight decrease in potency as a PKC substrate, suggesting that the hydrophobic character of the amino acid in this position was an important potency determinant.",
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N2 - The neuronal protein neurogranin, also known as RC3, is a selective substrate for protein kinase C (PKC). We synthesized a peptide corresponding to the phosphorylation domain of neurogranin (amino acids 28-43) and characterized its properties as a PKC substrate. Neurogranin(28-43) was phosphorylated by purified PKC with a Km of 150 nM. No significant phosphorylation of the peptide by either cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase II could be detected. Thus, neurogranin(28-43) is a potent and selective substrate for PKC. We tested several peptide analogues of neurogranin(28-43) for their substrate potency and specificity as kinase substrates, in order to help elucidate the structural determinants involved in the phosphorylation of substrates by PKC. Substituting Arg36 with He caused a significant reduction in the affinity for PKC. Replacing Lys30 with Arg enhanced the catalytic efficiency ( Vmax/Km) for PKC but diminished the selectivity of the substrate for PKC. These results support the generally held model that basic amino acids on both sides of the phosphorylated Ser are important structural determinants in PKC substrates. However, the data also suggest that the presence of particular basic amino acids (Arg vs Lys) can contribute to the degree of selectivity of a substrate for PKC. Replacement with Ala of Phe35, the amino acid adjacent to the Ser34 phosphorylation site, resulted in a peptide with greatly diminished potency as a PKC substrate. This finding indicates a critical role of Phe35 in modulating binding and phosphorylation of neurogranin-derived peptides by PKC. Substituting He in this same position caused only a slight decrease in potency as a PKC substrate, suggesting that the hydrophobic character of the amino acid in this position was an important potency determinant.

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