Identification of the double-stranded RNA-binding domain of the human interferon-inducible protein kinase.

R.C Patel,G.C Sen

doi:10.1016/s0021-9258(18)42567-7

Abstract

The interferon-inducible double-stranded (ds) RNA-activated protein kinase (p68 kinase) is a physiologically important enzyme that regulates the rate of cellular and viral protein synthesis by phosphorylating and thereby inactivating the peptide chain initiation factor 2. We have generated a cDNA clone of the human p68 kinase by polymerase chain reaction cloning using the recently published sequence of this enzyme. Active enzyme was synthesized by in vitro transcription-translation of the cDNA clone. This system was used for mapping the dsRNA-binding domain of the enzyme. Progressive deletions from the carboxyl terminus were introduced by digesting the cDNA with suitable restriction enzymes. Expression of proteins harboring deletions from the amino terminus was achieved by cloning DNA fragments into appropriately constructed expression vectors. Affinity of the truncated proteins for dsRNA was examined by testing their capacity to bind to dsRNA-agarose beads. Our results demonstrated that the dsRNA-binding domain lies at the amino terminus of the protein. A truncated protein containing the first 170 amino acid residues from the amino terminus could bind to dsRNA. However, deletion of 34 residues from the amino terminus or 41 residues from the carboxyl terminus of this truncated protein eliminated its dsRNA-binding activity. Comparison of the primary structure and the secondary structure of this region of p68 kinase and the corresponding region of 2'-5'-oligoadenylate synthetase revealed no apparent similarity.

Highlights

DsRNA is needed for the activation of the kinase that is accompanied by its autophosphorylation on several serine and threonine residues [10]
Synthetase re- role of this enzyme in cell growth and differentiation has been suggested [18], and this enzyme has been implicated in the host cell shut-off of protein synthesis upon infection with a number of viruses [14, 19]
About 4 pg ofRNAwas synthesized in a standard reaction

Summary

Introduction

DsRNA is needed for the activation of the kinase that is accompanied by its autophosphorylation on several serine and threonine residues [10]. Among the many proteins whose synthesis is induced in anisms to evade the deleterious action of this enzyme on viral cells upon treatment with interferons (IFNs),’one of the early protein synthesis Some viruses, such as encephalomyocardiones to be characterized is a proteinkinase that needs double- tis virus, cause sequestration of this enzyme [20].Others, such stranded (ds) RNA as a co-factor [1,2,3]. Human immunodeficiency reticulocyte [4] and human peripheral mononuclear cells [5] virus 1 encodes gene products that can modulate the action that have not been deliberately treated with IFN The human of this enzyme [23, 24]. Re- encode inhibitory proteins [25, 26], and influenza virus acticently, the protein has been purified to homogenicity [6, 7], vates a cellular inhibitor of p68 kinase [27]

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Conclusion