SERINE PHOSPHORYLATION WITHIN A CONCISE AMINO-TERMINAL DOMAIN IN NUCLEAR RESPIRATORY FACTOR-1 ENHANCES DNA-BINDING

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator th at acts on a diverse set of nuclear genes required for mitochondrial r espiratory function in mammalian cells, These genes encode respiratory proteins as well as components of the mitochondrial transcription, re plication, and heme biosynthetic machinery, Here, we establish that NR F-1 is a phosphoprotein in vivo. Phosphorylation occurs on serine resi dues within a concise NH2-terminal domain with the major sites of phos phate incorporation at serines 39, 44, 46, 47, and 52. The in vivo pho sphorylation pattern can be approximated in vitro by phosphorylating r ecombinant NRF-1 with purified casein kinase II, Phosphate incorporati on at the sites utilized in vivo results in a marked stimulation of DN A binding activity which is not observed in mutated proteins lacking t hese sites, Pairwise expression of the wild-type protein with each of a series of truncated derivatives in transfected cells results in the formation of a dimer between wild-type and mutant forms demonstrating that a homodimer is the active binding species. Although NRF-1 can dim erize in the absence of DNA, phosphorylation does not enhance the form ation of these dimers, These findings suggest that phosphorylation res ults in an intrinsic change in the NRF-1 dimer enhancing its ability t o bind DNA.