Analysis of protein kinase autophosphorylation using expressed protein ligation and computational modeling

Kerry A. Pickin, Sidhartha Chaudhury, Blair C.R. Dancy, Jeffrey J. Gray, Philip A. Cole

Research output: Contribution to journalArticlepeer-review

Abstract

Protein kinases represent a family of enzymes that are critical in cell signaling. One mechanism by which protein kinases are regulated is via autophosphorylation. In the studies described here, we have examined the mechanism of autophosphosphorylation at serine 338 in the regulation of protein kinase A (PKA). Expressed protein ligation allowed for the covalent linkage of an ATP moiety to a Ser mimic at this phosphorylation site. Using a combination of size exclusion chromatography, fluorescence nucleotide binding, kinase measurements, and limited proteolysis assays on this semisynthetic ATP-linked protein, we have obtained unique evidence for an intramolecular autophosphorylation mechanism in PKA regulation. Computational analysis provided a plausible model for a PKA conformation consistent with intramolecular phosphoryl transfer. This approach could be applied to other autoprocessing enzymes by exploiting appropriate transition state analogue motifs in the context of protein semisynthesis.

Original languageEnglish (US)
Pages (from-to)5667-5669
Number of pages3
JournalJournal of the American Chemical Society
Volume130
Issue number17
DOIs
StatePublished - Apr 30 2008

ASJC Scopus subject areas

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

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