J. Biol. Chem., Vol. 266, Issue 19, 12234-12241, 07, 1991
Purification of PO-B, a protein that has increased affinity for the pro- opiomelanocortin gene promoter after dephosphorylation
A Wellstein, AF Dobrenski, MN Radonovich, JF Brady and AT Riegel
Department of Pharmacology, Vincent T Lombardi Cancer Center, Georgetown University School of Medicine, Washington, D.C. 20007.
The region -15 to -3 of the pro-opiomelanocortin (POMC) gene promoter
specifically binds a transcription factor previously designated PO-B. This
region of the POMC gene is involved in the control of constitutive POMC
gene expression since mutation of the PO-B DNA-binding site severely
reduces transcription from the POMC promoter both in vivo and in vitro
(Riegel, A. T., Remenick, J., Wolford, R., Berard, D., and Hager, G. (1990)
Nucleic Acids Res. 18, 4513-4521). We have now purified PO-B from HeLa
cells approximately 25,000-fold to greater than 90% homogeneity by a
combination of ion exchange and reversed phase chromatography. In addition
we have studied post-translational modifications that alter the affinity of
purified PO-B for its cognate DNA binding site. In Southwestern analysis of
column fractions, two bands of apparent molecular masses of 54 and 56 kDa
bound specifically to the PO-B recognition sequence. The two copurified
components have indistinguishable amino acid composition, are highly
hydrophobic, and are heat and acid stable. DNA-binding specificity studies
suggest that PO-B does not represent any previously described transcription
factor. In addition, dephosphorylation of both species with acid
phosphatase induced an about 30-fold increase in DNA binding but failed to
produce any significant change in electrophoretic mobility. We conclude
that the purified PO-B species represent products of the same gene and
suggest that the in vivo function of PO-B may be regulated by its
phosphorylation status.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
