Analysis of Protein Kinase Autophosphorylation Using Expressed Protein Ligation and Computational Modeling

Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, United States
Journal of the American Chemical Society (Impact Factor: 12.11). 05/2008; 130(17):5667-9. DOI: 10.1021/ja711244h
Source: PubMed


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.

Download full-text


Available from: Sidhartha Chaudhury, Jan 10, 2014
  • Source
    • "The EPL technique (Muir et al., 1998;Schwarzer and Cole, 2005) involves coupling intein technology to cysteine ligation methodology inherent in the parent native chemical ligation procedure (Dawson et al., 1994). EPL may be regarded as the state of the art for chemical biological manipulation of side chain functionality, and is especially useful for studying PTM's and their relation to signal transduction (Flavell and Muir, 2009;Pickin et al., 2008;Rauh and Waldmann, 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: This overview focuses on the (alpha,alpha-difluoromethylene)phosphonate mimic of phosphoserine (pCF(2)Ser) and its application to the study of kinase-mediated signal transduction-pathways of great interest to drug development. The most versatile modes of access to these chemical biological tools are discussed, organized by method of PCF(2)-C bond formation. The pCF(2)-Ser mimic may be site-specifically incorporated into peptides (SPPS) and proteins (expressed protein ligation). This isopolar, dianionic pSer mimic results in a "constitutive phosphorylation" phenotype and is seen to support native protein-protein interactions that depend on serine phosphorylation. Signal transduction pathways studied with this chemical biological approach include the regulation of p53 tumor suppressor protein activity and of melatonin production. Given these successes, the future is bright for the use of such "teflon phospho-amino acid mimics" to map kinase-based signaling pathways.
    Full-text · Article · Sep 2009 · Chemistry & biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Introduction and objectives The aim of this study was to obtain an estimate of the incidence of cerebrovascular disease (CVD) in the Spanish population in 2002. Methods The study involved data on patients aged over 24 years for the year 2002 contained in both the death register and the Minimum Basic Data Set from 65 of the 84 Catalan general hospitals (i.e., 90.7% of all acute hospital beds in Catalonia). Total and age-adjusted mortality rates, cumulative incidence, and hospitalization rates, and the 28-day case fatality rate for CVD in the Catalan population were calculated after cases of traumatic and transient disease had been excluded. Results The unadjusted CVD mortality rate per 100,000 population aged over 24 years in Catalonia was 92 in men and 119 in women. The age-adjusted rates were 58 (95% confidence interval or CI, 56-61) and 43 (95% CI, 41-44), respectively. The cumulative incidence of CVD per 100,000 population was 218 (95% CI, 214-221) in men and 127 (95% CI, 125-128) in women. The unadjusted 28-day case fatality rate in the population was 36.2%: 30.3% in men and 42.0% in women. Some 62.5% of patients (57.2% of men and 66.4% of women) died from CVD outside hospital. Conclusions These findings indicate that CVD mortality and incidence rates in Catalonia are among the lowest in developed countries. More than half of the deaths that took place within 28 days after the onset of symptoms occurred outside hospital.
    Full-text · Article · Jun 2007 · Revista Espa de Cardiologia
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Selective protein cleavage at methionine residues is a useful method for the production of bacterially derived protein fragments containing an N-terminal cysteine residue required for native chemical ligation. Here we describe an optimised procedure for cyanogen bromide-mediated protein cleavage, and ligation of the resulting fragments to afford biologically active proteins.
    Full-text · Article · Dec 2008 · Organic & Biomolecular Chemistry
Show more