Molecular cloning of two new interferon-induced, highly related nuclear phosphoproteins.
During the molecular cloning of the human dsRNA activated-p68 kinase (PKR), polyclonal antibodies against PKR selected, in addition to cDNAs corresponding to PKR, another cDNA presenting only slight homology with PKR cDNA. This cDNA recognized an mRNA species of 2 kilobases induced by both alpha- and gamma-interferons. Its transcription did not require protein synthesis. On further library screening, it selected two highly related cDNAs, referred to as 75 and 41, displaying perfect homology over 612 base pairs and divergent at both ends. In addition, cDNA 75 presents an insertion of 150 base pairs highly homologous to a region common to both sequences. The 75 and 41 peptidic sequences are very hydrophilic, rich in basic amino acid residues, and contain several potential phosphorylation sites for different serine/threonine kinases. Furthermore, they present two protamine- and histone-like nuclear targeting sequences as well as some homology with helix-loop-helix motifs of some DNA-binding proteins. The 75-encoded product, which resolved as a 52-kDa protein after in vitro expression in rabbit reticulocyte lysates, was found to migrate as a 65-67-kDa protein after in vivo expression in insect cells. In accord with sequence data, this 65-67-kDa protein was found to be phosphorylated in vivo in the insect cells and was recovered from the membrane/nuclear pellet. In contrast, the 41-encoded product (30-kDa protein in reticulocyte lysates) could not be expressed in vivo, as it provoked a rapid and severe shut-off of protein synthesis in insect cells. The function of the 75 and 41 proteins and their relation to PKR remains to be determined. However, the presence of nuclear targeting sequences, phosphorylation sites, and helix-loop-helix motif is consistent with a role of these proteins in the mechanism of transduction of the interferon action.
Kadereit
S
S
Unit of Virology and Cellular Immunology, Institut Pasteur, Paris, France.
Gewert
D R
DR
Galabru
J
J
Hovanessian
A G
AG
Meurs
E F
EF
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000502:physiology*; D008969:Molecular Sequence Data; D009036:Moths; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics*; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics*; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic; D019892:eIF-2 Kinase
D000906:Antibodies; D018076:DNA, Complementary; D009687:Nuclear Proteins; D010750:Phosphoproteins; C084090:nuclear phosphoprotein 41; C084089:nuclear phosphoprotein 75; D007372:Interferons; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase