Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein

Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein

Departments of ^*Molecular Physiology and Biological Physics and ^‡Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908-0011; ^†Department of Pharmacology, Merck Research Laboratories, West Point, PA 19486; and ^§Cardiovascular Drug Discovery, Bristol–Myers Squibb, Princeton, NJ 08543

Communicated by Oscar L. Miller, Jr., University of Virginia, Charlottesville, VA (received for review June 2, 1998)

Abstract

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF in vivo in response to stimulation with different growth factors or in vitro with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF–TBP binding depended on the C-terminal ‘acidic tail’ of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.

Footnotes

↵ ¶ To whom reprint requests should be addressed at: Department of Molecular Physiology and Biological Physics, Jordan Hall, Box 10011, Charlottesville, VA 22908-0011. e-mail: gko{at}virginia.edu.
ABBREVIATIONS:

SL1,

selectivity factor 1;

UBF,

upstream binding factor;

TBP,

TATA-binding protein;

A-II,

angiotensin-II;

SMC,

smooth muscle cells;

SFM,

serum-free medium;

FBS,

fetal bovine serum;

PDGF-BB,

platelet-derived growth factor BB;

rUBF,

recombinant UBF;

SAP,

shrimp alkaline phosphatase;

CKII,

casein kinase II