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

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 consiste

SUBMITTER: Pickin KA 

PROVIDER: S-EPMC2654778 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature