Linked Life Data

15450128

Source:http://linkedlifedata.com/resource/pubmed/id/15450128

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
9
pubmed:dateCreated
2004-9-28
pubmed:abstractText
PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
1079-9907
pubmed:author
pubmed:copyrightInfo
Copyright Mary Ann Liebert, Inc.
pubmed:issnType
Print
pubmed:volume
24
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
522-35
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
Biochemical analyses of multiple fractions of PKR purified from Escherichia coli.
pubmed:affiliation
Department of Cancer Biology, NB40, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.