STRUCTURAL REQUIREMENTS FOR DOUBLE-STRANDED-RNA BINDING, DIMERIZATION, AND ACTIVATION OF THE HUMAN EIF-2-ALPHA KINASE DAI IN SACCHAROMYCES-CEREVISIAE

The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha s ubunit of translation initiation factor 2 (eIF-2 alpha). DAI is activa ted in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N termi nal to the kinase domain in DAI. High-level expression of DAI in Sacch aromyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protei n kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenot ypes were used to characterize structural requirements for DAI functio n in vivo, by examining the effects of amino acid substitutions at mat ching positions in the two dsRNA-binding motifs and of replacing one c opy of the motif with the other. We found that both copies of the dsRN A-binding motif are required for high-level kinase function and that t he N-terminal copy is more important than the C-terminal copy for acti vation of DAI in S. cerevisiae. On the basis of these findings,,ve con clude that the requirements for dsRNA binding in vitro and for activat ion of DAI kinase function in vivo closely coincide. Two mutant allele s containing deletions of the first or second binding motif functional ly complemented when coexpressed in yeast cells, strongly suggesting t hat the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing diff erent deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating poi nt mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We s uggest that large structural alterations in the kinase domain impair a n interaction between the two protomers in a DAT, dimer that is necess ary for activation by dsRNA or for catalysis of eIF-2 alpha phosphoryl ation.