Functional expression and characterization of the interferon-induced double-stranded RNA activated P68 protein kinase from Escherichia coli

Journal Article · Tue Oct 22 04:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00106a038· OSTI ID:5617834

Barber, G N; Tomita, J; Katze, M G ^[1]; Hovanessian, A G; Meurs, E ^[2]

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1. Univ. of Washington, Seattle (United States)
2. Institut Pasteur, Paris (France)

The P68 protein (referred to as P68 on the basis of its molecular weight of 68000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by double-stranded (ds) RNAs. Although extensively studied, little is currently known about the regulation of kinase function at the molecular level. What is known is that activation of this enzyme triggers as series of events which lead to an inhibition of protein synthesis initiation and may, in turn, play an integral role in the antiviral response to interferon. To begin to understand P68 and its biological functions in the eukaryotic cell, the authors have expressed the protein kinase in Escherichia coli under control of the bacteriophage T7 promoter. In rifampicin-treated cells, metabolically labeled with ({sup 35}S)methionine and induced by IPTG, the P68 kinase was the predominant labeled product. Further, P68 was recovered from extracts as a fully functional enzyme, shown by its ability to become activated and phosphorylate its natural substrate, the {alpha} subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). Moreover, P68 was phosphorylated in vivo in E. coli, providing conclusive evidence that the kinase has the capacity to phosphorylate and activate itself in the absence of other eukaryotic proteins. In contrast, a mutant P68 protein, containing a single amino acid substitution in the invariant lysine in catalytic domain II, was completely inactive. Finally, the expressed kinase could be isolated from contaminating E. coli proteins in an active form by immunoaffinity chromatography with a monoclonal antibody specific for P68.

• OSTI ID: 5617834
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:42; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DRUGS
ENZYMES
ESCHERICHIA COLI
EVEN-ODD NUCLEI
GROWTH FACTORS
HYDROXY ACIDS
INHIBITION
INTERFERON
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
LYMPHOKINES
LYSINE
METHIONINE
MICROORGANISMS
MITOGENS
MUTANTS
NUCLEI
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
RADIOISOTOPES
RNA
SERINE
SULFUR 35
SULFUR ISOTOPES
THREONINE
TRANSFERASES