MOLECULAR-CLONING OF 2 NEW INTERFERON-INDUCED, HIGHLY RELATED NUCLEARPHOSPHOPROTEINS

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 wi th PKR cDNA. This cDNA recognized an mRNA species of 2 kilobases induc ed by both alpha- and gamma-interferons. Its transcription did not req uire protein synthesis. On further library screening, it selected two highly related cDNAs, referred to as 75 and 41, displaying perfect hom ology over 612 base pairs and divergent at both ends. In addition, cDN A 75 presents an insertion of 150 base pairs highly homologous to a re gion common to both sequences. The 75 and 41 peptidic sequences are ve ry hydrophilic, rich in basic amino acid residues, and contain several potential phosphorylation sites for different serine/threonine kinase s. Furthermore, they present two protamine- and histone-like nuclear t argeting sequences as well as some homology with helix-loop-helix moti fs of some DNA-binding proteins. The 75-encoded product, which resolve d 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 ex pression in insect cells. In accord with sequence data, this 65-67-kDa protein was found to be phosphorylated in vivo in the insect cells an d 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 prote in synthesis in insect cells. The function of the 75 and 41 proteins a nd their relation to PKR remains to be determined. However, the presen ce of nuclear targeting sequences, phosphorylation sites, and helix-lo op-helix motif is consistent with a role of these proteins in the mech anism of transduction of the interferon action.