Molecular Basis for the Substrate Specificity of a Serine/Threonine-Specific Protein Kinase

Tae Ryong Lee, Marianne Mendelow, Jaya Srinivasan, Young Guen Kwon, David S. Lawrence

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Protein kinases typically phosphorylate the aliphatic alcohols of serine/threonine residues or the aromatic alcohol of tyrosine residues but not both. We report herein the first example of aromatic alcohol phosphorylation by a serine/threonine-specific protein kinase. The cAMP-dependent protein kinase phosphorylates the C-terminal aromatic alcohol in the active site-directed peptide Gly-Arg-Thr-Gly-Arg-Arg-Asn-(o-aminophenol). In contrast, corresponding peptides containing m- and p-aminophenols failed to serve as protein kinase substrates. These results indicate that the orientation of the aromatic hydroxyl group relative to the adjacent peptide backbone bond is a critical structural motif employed in substrate recognition by the enzyme. This observation also provides an explanation for the demonstrated inability of the cAMP-dependent protein kinase to catalyze the phosphorylation of tyrosine residues in proteins. The observed Km (793 ± 39 µM) for the o-aminophenol-containing substrate is consistent with values previously reported for other alcohol-bearing residues containing β-substituents. In contrast, the kcat (2.77 ±0.16 min−1) is significantly lower than those generally observed for the cAMP-dependent protein-kinase-catalyzed phosphorylation of aliphatic alcohols. However, this kcat does compare favorably with values reported for tyrosine-kinase catalyzed phosphorylations. The corresponding phosphopeptide of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(o-aminophenol) was isolated and subsequently characterized by fast atom bombardment mass spectrometry and 1H-coupled 31P nuclear magnetic resonance spectroscopy. In the latter case, a singlet was observed at −1.87 ppm upfield from H3PO4, which is the expected coupling pattern and chemical shift for a phosphorylated aromatic alcohol. These observations indicate that a serine/threonine-specific protein kinase possesses the inherent ability to catalyze phosphoryl transfer to an acceptor moiety other than an aliphatic alcohol and consequently expands the realm of possibilities with respect to inhibitor design.

Original languageEnglish
Pages (from-to)9888-9891
Number of pages4
JournalJournal of the American Chemical Society
Volume115
Issue number22
DOIs
Publication statusPublished - 1993 Nov 1

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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