Regulation of TFII-I activity by phosphorylation.

The transcription factor TFII-I binds to distinct promoter sequences including an initiator element in several eukaryotic genes. Here we demonstrate that TFII-I is phosphorylated in vivo at serine/threonine and tyrosine residues in the absence of any apparent extracellular signals. This "basal" phosphorylation of TFII-I is not required and does not affect its specific DNA binding, but is critical for its in vitro transcriptional properties via the Vbeta promoter. To better assess the functional role of phosphorylation in regulating TFII-I activity, we focused on tyrosine phosphorylation of TFII-I. Ectopically expressed recombinant TFII-I, like its native counterpart, exhibits tyrosine phosphorylation in the absence of distinct extracellular signals. More important, mutation of a potential consensus tyrosine phosphorylation site in TFII-I leads to severe reduction in its basal transcriptional activation of the Vbeta promoter in vivo. Taken together, these data suggest that tyrosine phosphorylation of TFII-I is important for its initiator-dependent transcriptional activity.

The transcription factor TFII-I binds to distinct promoter sequences including an initiator element in several eukaryotic genes. Here we demonstrate that TFII-I is phosphorylated in vivo at serine/threonine and tyrosine residues in the absence of any apparent extracellular signals. This "basal" phosphorylation of TFII-I is not required and does not affect its specific DNA binding, but is critical for its in vitro transcriptional properties via the V␤ promoter. To better assess the functional role of phosphorylation in regulating TFII-I activity, we focused on tyrosine phosphorylation of TFII-I. Ectopically expressed recombinant TFII-I, like its native counterpart, exhibits tyrosine phosphorylation in the absence of distinct extracellular signals. More important, mutation of a potential consensus tyrosine phosphorylation site in TFII-I leads to severe reduction in its basal transcriptional activation of the V␤ promoter in vivo. Taken together, these data suggest that tyrosine phosphorylation of TFII-I is important for its initiator-dependent transcriptional activity.
TFII-I is a multifunctional transcription factor with broad biochemical and biological activities and may be involved in several genetic disorders (1)(2)(3)(4)(5)(6). Therefore, it is important to undertake biochemical dissection of TFII-I to gain functional insights into this novel protein. We have previously shown that TFII-I can bind to the Inr 1 element in the natural TATA-less but Inr-containing T-cell receptor V␤ promoter and is required for its Inr-dependent transcription in vitro (4) and in vivo (7). TFII-I can also bind specifically to the Inr element in the TATA-and Inr-containing adenovirus major late promoter and, together with the upstream stimulatory factor, can markedly enhance adenovirus major late promoter transcription via the Inr element in vivo (6). However, the function of TFII-I is not necessarily restricted to the Inr element, as TFII-I can bind to an upstream regulatory element (E-box) both independently and synergistically with the upstream stimulatory factor and stimulate transcription in vivo through E-box elements in the absence of an Inr (6). Thus, TFII-I may serve as a transcriptional cofactor that potentially integrates signals from the regulatory components to the basal machinery. Consistent with its multifunctional potentials, TFII-I promotes formation of serum response factor and homeodomain protein Phox1 complexes on the c-fos promoter at the upstream serum response element (8). TFII-I functions through the upstream c-sis-inducible factor element and serum response elements, and ectopic expression of TFII-I leads to enhanced transcriptional activation of the c-fos promoter to a variety of stimuli (e.g. epidermal growth factor, platelet-derived growth factor, and 12-O-tetradecanoylphorbol-13-acetate) (9). Furthermore, growth factor stimulation leads to increased tyrosine phosphorylation of TFII-I (9). Finally, TFII-I is identical to the recently discovered protein BAP-135, which is associated in vivo with Btk (Bruton's tyrosine kinase), a mutation that leads to X-linked immune deficiency in humans and mice (10). 2 TFII-I is tyrosine-phosphorylated by Btk in vitro and upon immunoglobulin receptor cross-linking in B-cells (10). These observations suggest that TFII-I is a novel factor that can link signal-responsive activator complexes to the general transcription machinery perhaps in a cell type-dependent fashion. Interestingly, TFII-I has also been identified in the breakpoint regions of the 7q11.23 Williams-Beuren syndrome deletion (11).
To begin to understand the specific role that phosphorylation plays in regulating the functional activity of TFII-I, we have undertaken biochemical analysis of this multifunctional protein. Here we show that TFII-I is phosphorylated basally (i.e. in the absence of apparent extracellular signals) at serine/threonine and tyrosine residues. In vitro dephosphorylation of native TFII-I suggests that phosphorylation is dispensable for its specific DNA binding activity, but is required for its transcriptional activity. Extending these studies in vivo, we show that compared with wild-type TFII-I, a mutant TFII-I (in which the consensus tyrosine phosphorylation site is changed to phenylalanine) failed to significantly activate the V␤ promoter. Thus, tyrosine phosphorylation of TFII-I is critical for its basal transcriptional properties.

EXPERIMENTAL PROCEDURES
In Vivo Labeling-In vivo labeling of cultured cells with inorganic phosphate and subsequent processing were done essentially as described (12). HeLa cells (3 ϫ 10 5 /ml ϫ 66 ml) were grown in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum and harvested. Cell pellets were washed and resuspended in serum-free and phosphate-free Dulbecco's modified Eagle's medium. The resuspended cells were supplemented with 1 mCi of [ 32 P]orthophosphate (3000 Ci/mmol). Labeling proceeded for 4 h at 37°C Immunoprecipitation and Phosphoamino Acid Analysis (PAA)-Phosphate-labeled cells were harvested and washed in ice-cold Trisbuffered saline (3 ϫ 10 ml), and nuclear extracts were prepared (13). Anti-TFII-I antiserum (5 l) was coupled to protein A-agarose (15 l, 1:1 slurry) in buffer A (20 mM Tris (pH 7.9 at 4°C), 0.2 mM EDTA, 5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol, and 100 mM KCl) containing 0.05% Nonidet P-40, incubated for 40 min at 4°C, washed in buffer A with 0.05% Nonidet P-40 (6 ϫ 1 ml), mixed with labeled nuclear extract, and incubated for 1 h at 4°C with rocking. * This work was supported in part by Grant RPG-98-104-01-TBE from the American Cancer Society and Grant AI 41147 from the National Institutes of Health (to A. L. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The beads were washed in buffer A containing 0.05% Nonidet P-40, resuspended in Laemmli sample buffer, and subjected to 7.5% SDS-PAGE. The gel was Coomassie Blue-stained, dried, and briefly subjected to autoradiography. A band corresponding to 120 kDa was excised from the gel, rehydrated, and subjected to PAA (14). Briefly, constantly boiling HCl (90 l) was added to each sample and allowed to boil for 1.5 h. Samples were dried in a Speed-Vac and washed twice with (50 l) and once with (25 l) glass-distilled water. The samples were resuspended in sample buffer containing unlabeled standard phosphoserine, phosphothreonine, and phosphotyrosine. The samples were spotted on TLC plates and subject to electrophoresis for 30 min at 1000 V in the first dimension (buffer: 2.2% formic acid and 7.8% acetic acid) and to TLC in the second dimension (buffer: 70% isopropyl alcohol and 36.5% hydrochromic acid). TLC plates were allowed to dry and were subjected to autoradiography for 1 week at Ϫ70°C. Standards were visualized with ninhydrin spraying and matched with the spots visualized by autoradiography.
For immunoprecipitation with anti-phosphotyrosine antibody, the monoclonal anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc.) was coupled to protein A-Sepharose beads (20 l, 1:1 slurry) for 1 h at 4°C with rocking. The antibody-coupled beads were washed in buffer A (3 ϫ 0.5 ml), and partially purified TFII-I (15) was added to the antibody-coupled matrix or to the matrix alone and incubated for 30 min at 4°C. The matrix was washed in buffer A (3 ϫ 0.5 ml) and was subjected to 7.5% SDS-PAGE.
For removal of immune complexes from Western-blotted membranes, each blot was incubated with 62.3 mM Tris (pH 6.9), 2% SDS, and 100 mM ␤-mercaptoethanol for 2 h at 55°C with one exchange of buffer after 1 h. The membrane stripped of immune complexes was washed in Tris-buffered saline and re-blocked for subsequent Western analysis.
Phosphatase Treatment of Native TFII-I-Dephosphorylation reactions were performed with alkaline phosphatase-coupled agarose beads (Sigma). The insoluble enzyme was used at 2.95 units/reaction, where 1 unit is defined as the ability of the enzyme to hydrolyze 1.0 mol of p-nitrophenyl phosphate/min at pH 9.8 and 37°C. The coupled beads were equilibrated and resuspended in buffer A, and protein was added as indicated. The dephosphorylation reaction proceeded for 20 min at 37°C. The insoluble beads were centrifuged for 10 s at room temperature, and the supernatant was aspirated and subjected to Western analysis and DNA binding or transcription reactions.
Electrophoretic Mobility Shift Assay (EMSA) and in Vitro Transcription-EMSA and in vitro transcription reactions were performed as described (4). Preparation of immunodepleted nuclear extracts (4) as well as highly purified TFII-I (double-stranded DNA-cellulose) from HeLa nuclear extracts (15) used in these assays was carried out as described.
Eukaryotic Expression of TFII-I and Pervanadate Treatment-COS-7 cells were transfected with pEBII-I by the LipofectAMINE method (Life Technologies, Inc.) as described (7,16). These cells (three plates each for treatment or control) were harvested by scraping, washed in 14 ml of phosphate-buffered saline with 5 mM EDTA, resuspended to 1.5 ml in Dulbecco's modified Eagle's medium, and incubated for 5 min at 37°C. During the time of harvest, the pervanadate solution was prepared by mixing 20 l of 0.1 M Na 3 VO 4 , 372 l of H 2 O, and 8 l of 30% H 2 O 2 and was incubated for 15 min at room temperature (17,18). The pervanadate solution (75 l) was added to the test cells, and Dulbecco's modified Eagle's medium (75 l) was added to the control cells, which were incubated for 10 min at 37°C. Cells were collected by centrifugation for 5 min at 1000 rpm, transferred to a microcentrifuge tube in phosphatebuffered saline without EDTA, and microcentrifuged for 5 s, and the supernatant was aspirated. Whole cell extracts were prepared by addition of lysis buffer (25 mM Tris (pH 8.0 at 25°C), 150 mM NaCl, 1.2% Nonidet P-40, 5 mM NaF, and 2 mM Na 3 VO 4 ) containing the protease inhibitors 4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, phenylmethylsulfonyl fluoride, and soy bean trypsin inhibitor. The cells were incubated on ice for 15 min with periodic vortexing and centrifuged for 10 min at 14,000 rpm and 4°C.
GST Pull Down of Recombinant TFII-I-Whole cell extracts (200 g (see Figs. 4B and 6) and 50 g (see Fig. 4C)) from untransfected or p146-transfected COS cells with or without pervanadate treatment were subjected to precipitation with glutathione-Sepharose (40 l, 1:1 slurry; Sigma) for 90 min with rocking at 4°C. The reactions were centrifuged for 2 min at 9000 rpm and 4°C. The supernatants were aspirated, and the beads were washed three times. For washing, 1 ml of lysis buffer was added to the beads, vortexed for 5 s, and incubated on ice for 5 min. After the final wash, the supernatant was aspirated, and 2ϫ Laemmli buffer was added to the beads and subjected to SDS-PAGE and Western analysis.
In Vitro Mutagenesis-The codons for tyrosine residues at amino acids 248 and 249 in TFII-I cDNA were changed to codons for phenylalanine by PCR-based mutagenesis. In the first round of PCR, the region between the codons encoding amino acids 132 and 254 were amplified using primer 1 (5Ј-ACAGTGGTACCTGTACCATAT-3Ј) and primer 2 (5Ј-TTGAATGTTATATTGAaAAaAATCAGG-3Ј) (Reaction 1). In a separate reaction (Reaction 2), the codons between amino acids 243 and 322 were PCR-amplified using primer 3 (5Ј-TCAGAAGATCCT-GATTtTTtTCAATATAAC-3Ј) and primer 4 (5Ј-GGGTTTACGTAGAT-CAGTGATG-3Ј). The mutated nucleotides for coding phenylalanine are designated in lowercase letters in primers 2 and 3. In the second round of PCR, 0.25 ng each from Reactions 1 and 2 were mixed and used as templates and amplified using primers 1 and 4. The final product was gel-isolated, digested with Acc65I and SnaBI, and then ligated with pEBGII-I. The positive clone obtained was confirmed by sequencing.
Reporter Assays-Construction of the V␤ promoter fused to the luciferase cassette and transient transfection assays in COS-7 cells were carried out as described (7).

TFII-I Is Phosphorylated in Vivo-
A highly interesting feature revealed by analysis of the primary amino acid sequence of TFII-I is the presence of a number of potential phosphorylation sites, including 35 potential serine/threonine phosphorylation and two tyrosine phosphorylation (EDXDY) sites, suggesting that TFII-I has the potential to be differentially regulated in response to distinct physiological stimuli (6). Thus, we first tested the phosphorylation status of native TFII-I in HeLa cells ( Fig. 1) by PAA. TFII-I was immunoprecipitated by a highly specific anti-TFII-I antibody from orthophosphate-labeled HeLa cells and subjected to SDS-PAGE and autoradiography (Fig. 1A). The band corresponding to 120 kDa/TFII-I was excised and subjected to PAA (Fig. 1B). When compared with the ninhydrin standards, PAA revealed that TFII-I was phosphorylated predominantly at serine residues. These assays employed acid hydrolysis of peptide bonds. Because phosphothreonine and phosphotyrosine are acid-labile relative to phosphoserine (14), alternative methods were employed to detect potential phosphorylated threonine and tyrosine residues in TFII-I. The detection of phosphotyrosine residues in HeLaderived TFII-I was attempted by immunochemical analysis subsequent to immunoprecipitation with anti-phosphotyrosine antibody. Because TFII-I phosphorylation decreases upon purification, 3 a two column-purified fraction containing TFII-I derived from HeLa nuclear extracts was used for this analysis (Fig. 1C). TFII-I was detected in immunoprecipitates from this fraction with a monoclonal anti-phosphotyrosine antibody, but not with protein A-Sepharose beads alone. However, the amount of TFII-I in the immunoprecipitate, compared with the supernatant, was low (data not shown), indicating either that TFII-I is weakly phosphorylated at tyrosine residues or that only a fraction of total TFII-I is tyrosine-phosphorylated basally under these conditions.
Role of Phosphorylation in in Vitro Function of TFII-I-We tested whether the basal phosphorylation of TFII-I is necessary and sufficient for its DNA binding and transcription functions.
To determine the functional role of phosphorylation of TFII-I, we dephosphorylated native TFII-I in vitro and tested its DNA binding (Fig. 2) and transcriptional (Fig. 3) properties. EMSA showed that the dephosphorylated protein ( Fig. 2A, lanes 2-4) was capable of binding to the V␤ Inr probe almost as efficiently as its phosphorylated counterpart (lane 1). At the highest phosphatase concentrations (lane 4), there was a slight reduction in DNA binding. To demonstrate that the recovery of TFII-I protein after phosphatase treatment was nearly identical in all lanes, Western blot analysis with anti-TFII-I antibody was performed (Fig. 2B, left panel). The slight reduction in EMSA ( Fig. 2A, lane 4) corresponds to a lower recovery of protein observed in Western blotting with anti-TFII-I antibody (Fig.  2B, left panel, lane 4). To demonstrate that TFII-I was efficiently dephosphorylated under these conditions, the blot was stripped and reprobed with anti-phosphoserine antibody (Fig.  2B, right panel). Untreated native TFII-I (lane 1), but not phosphatase-treated TFII-I (lanes 2-4), was specifically immunoreactive to anti-phosphoserine antibody, suggesting that the dephosphorylation reaction proceeded to completion to the limit of detection by this analysis. Identical results have also been obtained with anti-phosphothreonine and anti-phosphotyrosine antibodies (data not shown). Next we tested whether the DNA binding specificity of TFII-I was altered during dephosphorylation and found that it was indistinguishable from that of the corresponding phosphorylated protein (Fig. 2C). Taken together, these results demonstrate that phosphorylation of TFII-I does not significantly alter its DNA binding properties either qualitatively or quantitatively. Finally, we tested the effect of dephosphorylation of TFII-I on V␤ transcription in Jurkat nuclear extracts immunodepleted of endogenous TFII-I (Fig. 3A, compare lanes 1 and 2). Although native phosphorylated TFII-I was competent to fully restore V␤ transcription (compare lanes 2 and 3), identical amounts of in vitro dephosphorylated TFII-I were incompetent to restore V␤ transcription when added to the TFII-I-depleted extract (compare lanes 3 and 4), indicating that phosphorylation of TFII-I is required for its transcriptional activity. To demonstrate that the lack of transcriptional activity of dephosphorylated TFII-I under our assay conditions is not due to carryover of residual phosphatase leading to nonspecific inhibition of the transcription reaction, control experiments were performed (Fig. 3B). V␤ transcription (lane 1) was not inhibited by addition of either bovine serum albumin alone (lane 2) or alkaline phosphatasetreated bovine serum albumin (lane 3).
Tyrosine Phosphorylation of Ectopic TFII-I in Vivo-TFII-I has been recently cloned by us and by others as a multifunctional transcription factor (6,8) as well as a protein (BAP-135) that associates with Bruton's tyrosine kinase, mutations that are associated with X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice (10). We employed a recombinant form of TFII-I (GST-TFII-I/p146) (7-9) to study its FIG. 1. TFII-I is serine-and tyrosine-phosphorylated in vivo. A, immunoprecipitates of TFII-I, from orthophosphate-labeled HeLa cells with anti-TFII-I antibody, when subjected to SDS-PAGE and autoradiography, demonstrated a heavily labeled protein that migrated with a relative molecular mass of 120 kDa, identical to native TFII-I. B, PAA of the excised band in A demonstrated that the 120-kDa band/TFII-I is phosphorylated at serine residues in vivo. Ninhydrin-stained spots are marked as follows: Y, phosphotyrosine; T, phosphothreonine; S, phosphoserine; Ori, origin; P, free phosphate. The directions for electrophoresis and chromatography are marked. C, Western blotting with anti-TFII-I antibody (␣-TFII-I) indicated the presence of TFII-I in an anti-phosphotyrosine precipitate (␣-P-Tyr ppt.), but not in a control precipitate (Control ppt.), of partially purified TFII-I fractions derived from HeLa nuclear extracts.

FIG. 2. Phosphorylation of TFII-I is dispensable for DNA binding.
Highly purified TFII-I was dephosphorylated in vitro with increasing amounts of alkaline phosphatase-coupled agarose beads (Ptpase). One-fourth of each reaction was loaded in EMSA (A), and three-fourths of the reaction was loaded in SDS-PAGE (B). A, phosphorylated TFII-I (lane 1) bound the V␤ Inr-containing probe to the same extent as phosphatase-treated TFII-I (lanes 2-4). B, phosphorylated (lane 2) and phosphatase-treated (lanes 2-4) TFII-I subjected to SDS-PAGE was blotted and probed first with anti-TFII-I antibody (␣-TFII-I). This blot was stripped and reprobed with anti-phosphoserine antibody (␣-P-Ser). C, dephosphorylated TFII-I bound with the same specificity as the phosphorylated protein. Binding of the dephosphorylated protein to the V␤ Inr-containing probe was competed with wild-type Inrcontaining (W), but not with mutant Inrcontaining (M) or E-box-containing (U), oligonucleotide competitor (Comp). function and phosphorylation status in mammalian cells (COS-7) since these cells express low amounts of endogenous TFII-I (7). To test whether recombinant TFII-I, like native TFII-I, exhibits tyrosine phosphorylation, COS-7 cells ectopically expressing p146 were treated with pervanadate (a potent tyrosine phosphatase inhibitor) (18), and the derived lysates were analyzed by Western blot analysis using a monoclonal anti-Tyr(P) antibody (Fig. 4A, left panel). Compared with both the untransfected and transfected control lysates in the absence of pervanadate, the pervanadate-treated transfected lysate demonstrated several proteins with markedly increased phosphotyrosine content. This blot was stripped and reprobed with anti-TFII-I antibody (Fig. 4A, right panel) to demonstrate that comparable amounts of p146 are expressed in both pervanadate-treated and untreated lysates. To ensure that p146 is among the proteins that exhibit increased tyrosine phosphorylation, it was pulled down with GST-agarose and probed with anti-Tyr(P) antibody (Fig. 4B, left panel). Whereas beads from neither the untransfected control lysate nor the transfected but untreated lysate showed any significant anti-Tyr(P) antibodyreactive bands, beads from the pervanadate-treated lysate showed a 146-kDa anti-Tyr(P) antibody-reactive band. When stripped and reprobed with anti-TFII-I antibody (Fig. 4B, right  panel), the 146-kDa tyrosine-phosphorylated protein comigrated with GST-TFII-I/p146. Moreover, these data show that almost equal amounts of GST-TFII-I/p146 are pulled down in the untreated lysate compared with the pervanadate-treated lysate. In contrast, and as expected, pervanadate treatment did not cause any significant increase in phosphoserine on ectopically expressed GST-TFII-I since TFII-I from both the pervanadate-treated and untreated lysates showed equal anti-phosphoserine reactivity (Fig. 4C, left panel) and equal recovery of p146 under both conditions (right panel). We conclude that ectopically expressed TFII-I exhibits tyrosine phosphorylation in the absence of any apparent extracellular signals. The pervanadate-treated and untreated lysates were also compared in EMSA, and no significant differences were observed (data not shown). mutant with that of the wild-type protein, transient transfection assays were performed using a V␤ reporter construct as described (7). Whereas wild-type TFII-I exhibited nearly a 4-fold induction of V␤ promoter activity, the mutant failed to activate the promoter significantly over and above the control levels ( Fig. 5, upper panel). The residual activity seen with the mutant could be due to tyrosine phosphorylation of site II, which has not yet been mutated. More important, the difference in transcriptional activity between wild-type and mutant TFII-I is not due to a difference in their protein expression since Western blot analysis showed that the expression levels were very similar, and thus, the Y-F mutation does not cause any apparent protein instability (Fig. 5, lower panel). That the Y-F mutation leads to reduced basal tyrosine phosphorylation in TFII-I was demonstrated by GST pull-down assays (Fig. 6). Lysates were prepared from COS-7 cells transiently transfected with either wild-type or Y-F mutant TFII-I and incubated with GST-agarose. The GST beads were boiled and subjected to SDS-PAGE and Western blot analysis with antiphosphotyrosine antibody (left panel). The difference in tyrosine phosphorylation signals was quantitated by densitometry and found to be 2.32-fold higher in the wild-type than in the mutant protein. To show that equivalent quantities of wildtype and mutant proteins were loaded, the blot was stripped and reprobed with anti-TFII-I antibody. The level of mutant TFII-I was 1.45-fold higher than wild-type TFII-I, and thus, the difference in tyrosine signals between the wild-type and mutant proteins is nearly 4-fold. However, although the mutant shows reduced tyrosine phosphorylation, it still shows some residual tyrosine phosphorylation, which could be due to site II. DISCUSSION Regulation of gene-specific transcription factor activity by phosphorylation is not unique (19 -21). However, direct regulation of core promoter activity via phosphorylation of components of the basal transcription machinery has been only recently demonstrated (22)(23)(24). In line with these latter observations, we show here that basal transcription of the TATA Ϫ Inr ϩ V␤ promoter is regulated via phosphorylation of TFII-I. For these studies, we attempted to determine the role of tyrosine phosphorylation in TFII-I. We demonstrate that tyrosine phosphorylation of TFII-I is required for its Inr-dependent transcriptional activity. To our knowledge, this is the first report that demonstrates basal transcriptional regulation via tyrosine phosphorylation. Although tyrosine phosphorylation of TFII-I is required for its basal transcription function, we do not yet know the precise mechanism. Since the DNA binding seems to be unaffected by phosphorylation, it is likely that the protein-protein interactions of TFII-I with the basal machinery may be dependent upon its phosphorylation status. It is also possible that tyrosine phosphorylation of TFII-I is required for its nuclear translocation. However, the latter does not appear to be the case as the Y-F mutant readily translocates to the nucleus upon ectopic expression (data not shown).

Mutation of a Consensus Tyrosine Phosphorylation Motif in TFII-I Affects Its Transcriptional Activity via the V␤
The role of serine phosphorylation in TFII-I is less clear at present. Because there are numerous potential Ser/Thr phosphorylation sites, it is difficult to assess which one of these is utilized inside the cell. It is possible that serine phosphorylation of TFII-I is required for protein stability. Thus, addition of completely dephosphorylated TFII-I to nuclear extract could lead to degradation/proteolysis of the protein. It is also possible that serine phosphorylation may be an obligatory step for tyrosine phosphorylation. Hence, serine phosphorylation may be necessary but not sufficient for transcriptional activity. Since the Y-F mutant retains its serine phosphorylation (data not shown), we cannot rule out this latter possibility. Finally, it remains possible that serine phosphorylation affects its upstream activator functions as opposed to its core promoter functions.
Here we have attempted to analyze the basal phosphoryla-  (7) and with 200 ng of plasmid expressing wild-type TFII-I (WT), Y-F mutant TFII-I, or vector alone. The transfection efficiencies were normalized by including 35 ng of renilla luciferase expression plasmid in each transfectant (pRL-TK, Promega). The -fold activation was calculated by normalizing the wild-type V␤ promoter activity to 1 (7). The results represent an average of three independent experiments. Lower panel, lysates from transfection assays were subjected to Western blot analysis to show the comparable expression of wild-type and Y-F mutant TFII-I. Equal amounts of cell lysates (5 g of protein) from each transfection were subjected to Western blot analysis with anti-TFII-I antibody. tion of TFII-I and its potential effects on DNA binding and transcription. Although TFII-I appears to have both core promoter and upstream activator element-dependent transcription functions, use of the basal V␤ promoter does not allow us to assess the importance of the TFII-I phosphorylation for its upstream element-dependent function. Whereas phosphorylation appears to have no significant role in modulating its DNA binding activity, basal phosphorylation seems to be required for its Inr-dependent transcriptional activity. We would like to emphasize that what we termed as basal phosphorylation may not be so since we cannot rule out cell cycle effects and/or effects of growth factors present in serum that are required to grow the cells. However, since TFII-I is tyrosine-phosphorylated even under very low (0.5%) serum conditions (9), it is likely that additional parameters may contribute to phosphorylation of TFII-I. Although we have not yet addressed whether induced tyrosine phosphorylation of TFII-I (e.g. by growth factors) affects its basal transcription functions, induced tyrosine phosphorylation of TFII-I in response to epidermal growth factor stimulation appears to correlate with its upstream element-dependent transcription functions at the c-fos promoter (9). Thus, we postulate that although induced tyrosine phosphorylation of TFII-I may not have any significant role in affecting its DNA binding capabilities, induced tyrosine phosphorylation could have a significant role in modulating its transcription functions, especially upstream activating functions. Because the TFII-I structure reveals several presumptive serine/threonine and tyrosine phosphorylation sites (6), it is likely that TFII-I phosphorylation is induced in response to a wide variety of signaling pathways and consequently affects a multitude of TFII-I-responsive genes. Consistent with this notion, it appears that mitogen-activated protein kinase may be capable of phosphorylating TFII-I both in vitro and in vivo. 4 Taken together, these observations suggest that the various functions of TFII-I might be regulated by its differential phosphorylation. Structure-function analysis of TFII-I should provide important clues regarding its presumptive role as a mediator between signal transduction and transcriptional cascades.