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

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.

Figure 1

Western blot analysis showing increased levels of UBF in TBP immunoprecipitates from growth-stimulated vs. quiescent cells. (A) Western blot analysis of UBF levels in TBP immunoprecipitates from SMC growth arrested in a defined SFM and stimulated with A-II, PDGF-BB, and FBS, as described in Methods. Cells were harvested and immunoprecipitations were performed as described in Methods. The blots were probed with a UBF antibody (a generous gift from L. Rothblum, Weis Research Institute, Danville, PA). Blots were detected with enhanced chemiluminescence (ECL, Amersham) according to manufacturer’s directions. (B) The same TBP immunoprecipitates assayed in A were assayed for TBP by Western blotting with a TBP antibody (Santa Cruz Biotechnology) as described in Methods. Because of a greater recovery of TBP, the loadings in B were proportionally reduced for all samples by a factor of eight, as compared with the corresponding lane in A.

Figure 2

TBP Far Western blot analysis of SMC nuclear extracts showing enhanced TBP–UBF binding in growth-stimulated SMC that was reduced by treatment with SAP. (A) Far Western blot of TBP binding proteins in SMC nuclear extracts. Cells were growth-arrested in SFM and then stimulated with A-II or FBS as described in Methods. Nuclear extracts were prepared as described (23). Four μg of each extract were treated with either SAP (10 units/ml, United States Biochemical) or glycerol (vehicle) and were incubated for 1 h at 37°C before electrophoresis and Far Western blotting. Lanes 1–3 show blots of nuclear extracts from SMC treated with 10% FBS, A-II, or SFM vehicle, respectively. Lanes 4–6 show blots of nuclear extracts treated with SAP. The band corresponding to the UBF1/UBF2 doublet is indicated, although the two UBF isoforms are not well resolved under the conditions of these assays. (B) Control Western blot analyses with a UBF antibody confirmed the position of UBF and demonstrated that treatment with SAP had no effect on UBF protein content. Similar results to these were obtained when TBP Far Western analyses were performed by using immunoprecipitates of UBF derived from vehicle and growth factor-stimulated SMC (data not shown), as compared with the whole nuclear extracts as shown here.

Figure 3

Far Western analysis of rUBF1 phosphorylated in vitro showed phosphorylation-dependent UBF–TBP binding. (A) ³²P-autoradiograph analysis (Top) of rUBF1 phosphorylated in vitro by SMC nuclear extract. The phosphorylation reaction was performed as described in Methods. SAP was added for the last 30 min of one of the 60-min time point samples. The membrane was dried and exposed to film for 18 h to detect ³²P incorporation into rUBF1. Far Western analysis (Middle) of TBP binding to rUBF1 was performed as described in Methods. Control UBF Western blot (Bottom) showing equal amounts of rUBF in each of the experimental groups. (B) Autoradiographic analysis of ³²P incorporation (Top) or Far Western blot analysis of TBP binding (Middle) by using truncated rUBF lacking the C-terminal acidic tail (amino acid residues 1–656, a generous gift from L. Rothblum, Weis Research Institute, Danville, PA) or full-length rUBF. (Bottom) A UBF Western blot showing equal amounts of rUBF in each of the experimental groups. Phosphorylation reactions and Far Western analyses were performed as described in Methods, with incubation times as indicated. Western blot analyses with UBF antibody showed that equivalent amounts of truncated and full-length rUBF1 were present in each lane (data not shown).

Figure 4

Two-dimensional phosphotryptic peptide maps of UBF phosphorylated in vivo showed that growth stimulation did not induce the phosphorylation of new sites. (A) Two-dimensional phosphotryptic peptide maps of phosphorylated UBF from quiescent and growth-stimulated SMC. Postconfluent growth-arrested SMC were switched to a low-phosphate SFM and labeled in vivo with ³²P-orthophosphoric acid (0.5 mCi/ml, 6,000 Ci/mmol) for 8 h before being treated with A-II (10⁻⁶M), SFM, or 10% FBS for 1 h. UBF was immunoprecipitated from SMC nuclear extracts as described previously (13). The maps were generated as described in Methods. (B) Two-dimensional phosphotryptic peptide maps of full-length and truncated rUBF1 that was phosphorylated in vitro by SMC nuclear extract. One μg of either full-length rUBF1 or truncated rUBF1 was phosphorylated in vitro by A-II-treated SMC crude nuclear extract (0.2 μg) and 300 μM [γ ⁻³²P]ATP (250 cpm/pmol), 7.5 μM MgCl₂. The reactions were terminated by the addition of SDS sample buffer. The maps were generated as described in Methods.

Figure 5

Far Western analysis showed that CKII phosphorylated rUBF1 in vitro but did not increase UBF–TBP binding. Autoradiographic analysis of ³²P incorporation (Upper) or Far Western blot rUBF1 (Lower) phosphorylated in vitro by CKII. Phosphorylation reactions and Far Western analysis were performed as described in Methods but with substitution of 20 ng of CKII (750 units/mg activity, Upstate Biotechnology, Lake Placid, NY) in place of the SMC nuclear extract.