Serine Phosphorylation within a Concise Amino-terminal Domain in Nuclear Respiratory Factor 1 Enhances DNA Binding*
- From the Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611
Abstract
Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator that acts on a diverse set of nuclear genes required for mitochondrial respiratory function in mammalian cells. These genes encode respiratory proteins as well as components of the mitochondrial transcription, replication, and heme biosynthetic machinery. Here, we establish that NRF-1 is a phosphoprotein in vivo. Phosphorylation occurs on serine residues within a concise NH2-terminal domain with the major sites of phosphate incorporation at serines 39, 44, 46, 47, and 52. The in vivo phosphorylation pattern can be approximated in vitro by phosphorylating recombinant NRF-1 with purified casein kinase II. Phosphate incorporation at the sites utilized in vivo results in a marked stimulation of DNA binding activity which is not observed in mutated proteins lacking these 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 dimerize in the absence of DNA, phosphorylation does not enhance the formation of these dimers. These findings suggest that phosphorylation results in an intrinsic change in the NRF-1 dimer enhancing its ability to bind DNA.
Footnotes
-
↵* This work was supported by United States Public Health Service Grant GM32525-14 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‡ To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611.
-
↵1 The abbreviations used are: NRF-1, nuclear respiratory factor 1; HA, hemagglutinin; CKII, casein kinase II; SRF, serum response factor; MEF2C, myocyte enhancer factor 2C; PAGE, polyacrylamide gel electrophoresis.
-
↵2 S. Gugneja and R. C. Scarpulla, unpublished observations.
-
- Received March 17, 1997.
- Revision received May 14, 1997.
