©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ser-Ser Pair in HeLa Nuclear Protein p21/SIIR Mediates Ser/Thr Phosphorylation and Is Essential for Rous Sarcoma Virus Long Terminal Repeat Repression (*)

(Received for publication, July 24, 1995; and in revised form, August 21, 1995)

Chen-Hsiung Yeh Wei-Xing Zong Aaron J. Shatkin (§)

From the Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Phosphorylation of HeLa SII (or TFIIS)-related nuclear protein p21/SIIR was demonstrated in transfected COS-1 cells. To test for a possible functional link between phosphorylation and the previously described Rous sarcoma virus (RSV) long terminal repeat (LTR) repression (Yeh, C. H., and Shatkin, A. J.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11002-11006), p21/SIIR mutants were constructed and assayed for phosphorylation level and effect on RSV LTR-driven chloramphenicol acetyltransferase (CAT) reporter expression. A major phosphorylation target in p21/SIIR was localized to the Arg/Ser-rich region between amino acids 12 and 49. Deletion of this region impaired the ability of p21/SIIR to down-regulate RSV LTR promoter function. Four serine pairs, all displaying the Arg/Lys-Ser-Ser motif typical of phosphorylation sites, are present in p21/SIIR between positions 31 and 48. Conversion of these individual serine pairs to alanine resulted in decreased phosphorylation in each case. Mutation of the Ser-Ser pair also diminished by severalfold the repression activity of p21/SIIR. The single tyrosine (Tyr) in p21/SIIR was not detectably phosphorylated in transfected COS-1 cells, suggesting that the Ser-Ser pair mediates Ser/Thr phosphorylation of p21/SIIR and is critical for LTR repression function.


INTRODUCTION

Protein phosphorylation is one of the most common forms of post-translational modification in eukaryotic cells. It is involved in the regulation of a multitude of cellular processes(1, 2) . Since many different types of stimuli that affect gene expression also lead to the activation of protein kinases, it is not surprising that transcription factors are directly regulated by phosphorylation. Almost every eukaryotic transcription factor that has been analyzed in detail has proved to be phosphorylated, and global regulation of transcription involves phosphorylation of both general transcription factors and subunits of RNA polymerases(3, 4) . Phosphorylation of transcription factors has also been shown to modulate protein subcellular localization, DNA binding activity, and transactivation function(5) . The precise mechanism of transcriptional control by phosphoproteins and the role of phosphorylation status in these processes have been intensively investigated. A thorough analysis of transcription factor phosphorylation will be essential for complete understanding of the signaling pathways that control cell proliferation and differentiation.

We previously isolated a HeLa cDNA clone that encodes a nuclear protein related to transcription elongation factor SII(6) . Expression of this 157-amino acid protein, p21/SIIR, in COS-1 cells repressed RSV (^1)LTR-driven reporter CAT expression(7) . Examination of the primary sequence of p21/SIIR revealed that it contains an Arg/Ser-rich region (amino acids 4-72) near the N-terminal end. This region includes multiple phosphorylation consensus sites for a variety of cellular kinases including protein kinase C, cAMP-dependent protein kinase, p34 kinase, Ca-calmodulin-dependent kinase II, and glycogen synthase kinase 3(8) . As a first step toward unraveling the upstream signaling pathway involved in p21/SIIR regulation, we set out to identify any site(s) of phosphorylation in p21/SIIR and to test their importance for RSV LTR repression function.

In this communication, we demonstrate that a segment in p21/SIIR encompassing amino acids 12 to 49 represents a major site of phosphorylation that is required for the inhibition of RSV LTR. Further mutation analysis revealed that the Ser-Ser pair is crucial for both phosphorylation and promoter repression function, suggesting that Ser/Thr phosphorylation mediated through this Ser pair plays a role in regulating p21/SIIR activity.


EXPERIMENTAL PROCEDURES

Construction of Expression Vectors and Site-directed Mutagenesis

Vectors for expression of CAT reporter gene (pR-CAT) and HA-tagged p21/SIIR (pR-p21) were constructed as described previously(7) . To obtain truncated proteins of p21/SIIR, synthetic oligonucleotides were used to introduce unique DraI or PvuII sites into p21/SIIR cDNA (in pBluescript) by single-stranded DNA-based mutagenesis(9) . The resulting plasmids were digested with DraI or PvuII to delete defined regions (amino acids 12-49, 50-100, and 101-149), and the plasmids were then religated. Four serine to alanine mutants, S31A/S32A, S36A/S37A, S41A/S42A, and S47A/S48A, were created by the same mutagenesis method. All mutants and ligation junctions were confirmed by DNA sequencing. For expression in mammalian cells, these constructs were inserted into the XhoI and XbaI sites of the expression vector pBC12BI(7) .

Analysis of Expressed and Phosphorylated p21/SIIR Mutants

Recombinant plasmids were Qiagen-column purified and transfected into COS-1 cells by the DEAE-dextran method(7) . COS-1 cells transfected with 5 µg of the indicated expression plasmid were incubated with Dulbecco's modified Eagle's medium (Life Technologies, Inc.) minus phosphate or methionine for 1 h prior to addition of [P]phosphate (800 µCi/ml) or [S]methionine (200 µCi/ml, Amersham). After incubation for 3-4 h, cells were washed and lysed in radioimmunoprecipitation assay buffer (RIPA: 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride). Extracts (500 µl containing 500 µg of protein) were immunoprecipitated using 5 µl of anti-HA monoclonal antibody for 16 h at 4 °C, followed by 40 µl of protein A-agarose beads (Pharmacia) for 1 h at 4 °C. Bound radiolabeled proteins were collected by centrifugation, washed three times in RIPA buffer and one time in phosphate-buffered saline, resuspended in SDS sample buffer and analyzed by 12.5% SDS-PAGE followed by autoradiography.

Western blotting of the anti-HA immunoprecipitated complexes was also performed after 12.5% SDS-PAGE using 1:1000 dilutions of anti-HA monoclonal antibody (7, 10) or anti-phosphotyrosine antibody (Sigma).

Transient Transfections and CAT Assays

COS-1 cells were cotransfected by the DEAE-dextran method with 0.5 µg of CAT reporter and 5 µg of the appropriate vector for expression of p21/SIIR or mutant proteins, or the same amount of control parental vector pBC12BI. Cells were harvested 48 h after transfection, and extracts were assayed for CAT protein level by enzyme-linked immunosorbent assay(7) .


RESULTS AND DISCUSSION

A Major Phosphorylation Site in p21/SIIR Required for Repression of RSV LTR

To determine if p21/SIIR can be phosphorylated in mammalian cells and to localize any phosphorylation site(s), we constructed mutants deleted in amino acids 12-49 (pd12-49), 50-100 (pd50-100), and 101-149 (pd101-149). COS-1 cells were transfected with control vector pBC12BI, a plasmid expressing full-length p21/SIIR, or one of the deletion mutants. Transfected cells were metabolically labeled with either [P]phosphate or [S]methionine, and cell extracts were analyzed by immunoprecipitation and SDS-PAGE. As shown in Fig. 1, cells transfected with the control plasmid did not contain radiolabeled, immunoprecipitable proteins (lane 1). However, P-labeled bands migrating in the positions of full-length p21/SIIR (lane 2), mutant pd50-100 (lane 4), and mutant pd101-149 proteins (lane 5) were obtained from the corresponding transfected cells (Fig. 1, right panel). In contrast, P radiolabeling was eliminated in the pd12-49 mutant (lane 3) indicating that this region of p21/SIIR contains a major phosphorylation site(s). Although the expression of p21/SIIR and all three deletion mutants was confirmed by [S]methionine labeling (Fig. 1, left panel), pd12-49 (lane 3) and pd50-100 (lane 4) were present in 10-15-fold lower amounts than either the full-length protein (lane 2) or mutant pd101-149 (lane 5). (Cross-reacting immunoprecipitated proteins were also evident in positions just above and below the mutant proteins.) After correction for the differences in protein levels and methionine content, mutant pd50-100 had the highest relative level of phosphorylation and pd12-49 the lowest.


Figure 1: Localization of a major phosphorylation site in p21/SIIR. COS-1 cells transfected with the control vector pBC12BI (lane 1), the plasmid for expressing full-length p21/SIIR (lane 2), or three deletion mutants (lanes 3-5) were metabolically labeled with [P]phosphate (right panel) or [S]methionine (left panel). The radiolabeled proteins were immunoprecipitated using anti-HA monoclonal antibody and analyzed by SDS-PAGE followed by autoradiography. Arrows on the left and right margins indicate the positions of full-length p21/SIIR and mutant proteins, respectively.



We also compared the effects of the p21/SIIR deletion mutants on RSV LTR-driven CAT reporter gene expression in cotransfected COS-1 cells. As reported previously(7) , mutant proteins pd12-49 and pd50-100 lost 84% and 94%, respectively, of the inhibitory activity of full-length p21/SIIR (data not shown). In contrast, the inhibitory activity of pd101-149 was essentially unchanged, and, like the full-length protein, this mutant repressed CAT expression by 15-fold (data not shown). These results indicate that sequences in p21/SIIR between residues 12 and 100, which includes the major phosphorylation site, are necessary for RSV LTR repression. Since either a relatively high degree of phosphorylation (pd50-100) or negligible phosphorylation (pd12-49) both resulted in almost complete loss of LTR repression, phosphorylation/dephosphorylation has an important role in regulating the LTR promoter repression function of p21/SIIR. Excess phosphorylation of p21/SIIR may induce an inappropriate conformation resulting in an inactive protein, while a single (or few) phosphorylation event(s) may be insufficient to activate p21/SIIR.

The Ser-SerPair Is Critical for Phosphorylation and Repression Function of p21/SIIR

We next sought to identify in the major phosphorylation site (amino acids 12-49) any specific residues necessary for phosphorylation and possibly also the LTR repression activity of p21/SIIR. Four potentially phosphorylated serine pairs are present in this region, and they display multiple phosphorylation consensus sequences for cAMP-dependent protein kinase (Arg-Arg-Ser-Ser, Arg-Arg-Ser-Ser), protein kinase C (Pro-Arg-Ser-Ser, Arg-Arg-Ser-Ser, Arg-Arg-Ser-Ser), p34 kinase (Ser-Ser-Pro-Pro-Arg, Ser-Ser-Pro-Arg-Arg), and glycogen synthase kinase 3 (Ser-Ser-Pro-Pro-Arg-Ser-Ser, Ser-Ser-Leu-Arg-Arg-Ser-Ser) (Fig. 2)(8) . Each serine pair was mutated to alanine, and the resulting mutant proteins were tested for phosphorylation level and effect on RSV LTR-driven reporter CAT expression in COS-1 cells. As shown in Fig. 3A, right panel, mutation of individual serine pairs resulted in a decreased level of phosphorylation in each case.


Figure 2: Diagram of the region in p21/SIIR containing the major phosphorylation site. HA-tagged p21/SIIR is depicted schematically, including two bipartite nuclear localization signals (BNLS), the Arg/Ser-rich sequence with homology to the RS domain of pre-mRNA splicing factor SC-35(16) , the zinc finger-like motif, and the helix-turn-helix (HTH) structure. Influenza virus HA epitope was added to the C terminus of p21/SIIR as indicated. The amino acid sequence of the region containing the major phosphorylation site between residues 12 and 49 is shown below the diagram, and the four serine pairs displaying the Arg/Lys-Ser-Ser motif (underlined) were mutated to alanine as indicated.




Figure 3: Decreased phosphorylation level and repression function of p21/SIIR S36A/S37A mutant. A, COS-1 cells were metabolically labeled with [P]phosphate (right panel) or [S]methionine (left panel) after transfection with the control vector pBC12BI (lane 1) or expression plasmids for p21/SIIR (lane 2) or the four serine to alanine mutants (lanes 3-6). Cell extracts were immunoprecipitated with anti-HA monoclonal antibody and analyzed by SDS-PAGE. B, the intensity of each band shown in A was quantitated by PhosphorImager analysis. After correction for the differences in protein expression, the relative phosphorylation level of each mutant was normalized to the wild-type p21/SIIR level set as 100% (right panel). To assay for repression of RSV LTR, COS-1 cells were transfected with 0.5 µg of CAT reporter plasmid and 5 µg of either pBC12BI or expression vector for the indicated p21/SIIR constructs. Results were normalized to the CAT value obtained from pBC12BI-transfected samples and expressed as the fold repression of pR-CAT (left panel).



By normalization to [S]methionine incorporation (Fig. 3A, left panel), we found that serine to alanine mutants S31A/S32A and S47A/S48A were phosphorylated at 40% and 45% of the p21/SIIR level, respectively, and in the S41A/S42A and S36A/S37A mutants phosphorylation was decreased 75% and 83%, respectively, relative to p21/SIIR (Fig. 3B, right). These data suggest that phosphorylation of p21/SIIR is a complex process, and one phosphorylation event may influence another. As in hierarchal protein phosphorylation, which usually involves different protein kinases(11) , there may be distinctive primary phosphorylation(s) of p21/SIIR which affect the course of subsequent secondary phosphorylation(s). It is reasonable that if the actions of several kinases influence the functional status of a target protein, its regulation would be more complex relative to that of a single kinase.

Comparison of the effects of p21/SIIR mutants on RSV LTR promoter demonstrated that alanine replacements at Ser-Ser had no adverse effect on p21/SIIR repression of pR-CAT expression (Fig. 3B, left), and mutation at Ser-Ser or Ser-Ser resulted in only a slight decrease in inhibitory activity (14-fold and 13-fold repression of pR-CAT, respectively, compared to 16-fold by wild-type p21/SIIR). However, the S36A/S37A mutation resulted in a substantial loss of LTR repression function (2.4-fold compared to 16-fold inhibition) (Fig. 3B, left). Thus, both promoter repression activity and phosphorylation level were decreased in the S36A/S37A mutant, suggesting that RSV LTR repression by p21/SIIR may be regulated by the phosphorylation events mediated by the Ser-Ser pair. It will be of interest to test if p21/SIIR can be phosphorylated on Ser and/or Ser by PKC and/or glycogen synthase kinase 3.

Tyrosine phosphorylation usually precedes Ser/Thr phosphorylation in the signal transduction network(12, 13, 14) . As a first step to determine if phosphorylation of the single p21/SIIR tyrosine (Tyr) is involved in regulating repression activity, extracts were prepared from COS-1 cells transfected with p21/SIIR expression plasmid or the control vector pBC12BI. Samples were either immunoprecipitated with anti-HA antibody or not and then immunoblotted with either anti-HA or anti-phosphotyrosine antibody. No phosphotyrosine was detected in p21/SIIR either with or without immunoprecipitation (Fig. 4, lanes 6 and 8), suggesting that regulation of p21/SIIR activity involves Ser/Thr but not Tyr phosphorylation.


Figure 4: Absence of detectable phosphorylation of the single tyrosine (Tyr) in p21/SIIR expressed in transfected COS-1 cells. Cell extracts prepared from COS-1 cells that had been transfected with pBC12BI (lanes 1, 3, 5, and 7) or p21/SIIR expression plasmid (lanes 2, 4, 6, and 8) were immunoprecipitated (IP: +) (lanes 3, 4, 7, and 8) or directly analyzed (IP: -) (lanes 1, 2, 5, and 6) by 12.5% SDS-PAGE. Resolved proteins were electrotransferred to nitrocellulose membranes which were then immunoblotted (IB) with either anti-HA (lanes 1-4) or anti-phosphotyrosine (anti-PY) antibody (lanes 5-8). p21/SIIR is indicated by the arrow, and immunoglobulin heavy and light chains and protein A by .



The results presented here indicate that the major phosphorylation site associated with p21/SIIR repression activity resides within a region of 38 amino acids between residues 12 and 49. A frequent hallmark of regulatory phosphorylation is the clustering of phosphorylation sites (11, 15) , and this region of p21/SIIR contains four serine pairs with the motif Arg/Lys-Ser-Ser. These observations make the serine pairs good candidates for regulating p21/SIIR activity. Indeed, we have identified Ser-Ser as critical residues for mediating phosphorylation and repression activity of p21/SIIR. Moreover, p21/SIIR mutants that had either a high (pd50-100) or relatively low level of phosphorylation (pd12-49, S36A/S37A) were less effective inhibitors of the RSV LTR promoter, implying that multisite phosphorylation may provide a means for fine-tuning p21/SIIR repression activity.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. Tel.: 908-235-5311; Fax: 908-235-5318.

(^1)
The abbreviations used are: RSV, Rous sarcoma virus; LTR, long terminal repeat; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; pd, putative domain; PAGE, polyacrylamide gel electrophoresis.


REFERENCES

  1. Hunter, T. (1995) Cell 80, 225-236 [Medline] [Order article via Infotrieve]
  2. Campbell, J. S., Seger, R., Graves, J. D., Graves, L. M., Jensen, A. M., and Krebs, E. G. (1995) Recent Prog. Horm. Res. 50, 131-159 [Medline] [Order article via Infotrieve]
  3. Hill, C. S., and Treisman, R. (1995) Cell 80, 199-211 [Medline] [Order article via Infotrieve]
  4. Shiekhattar, R., Mermelstein, F., Fisher, R. P., Drapkin, R., Dynlacht, B., Wessling, H. C., Morgan, D. O., and Reinberg, D. (1995) Nature 374, 283-287 [CrossRef][Medline] [Order article via Infotrieve]
  5. Hunter, T., and Karin, M. (1992) Cell 70, 375-387 [Medline] [Order article via Infotrieve]
  6. Yeh, C. H., and Shatkin, A. J. (1994) Gene (Amst.) 143, 285-287 [Medline] [Order article via Infotrieve]
  7. Yeh, C. H., and Shatkin, A. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11002-11006 [Abstract/Free Full Text]
  8. Kennelly, P. J., and Krebs, E. G. (1991) J. Biol. Chem. 266, 15555-15558 [Free Full Text]
  9. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382 [Medline] [Order article via Infotrieve]
  10. Yeh, C. H., and Shatkin, A. J. (1995) J. Biol. Chem. 270, 15815-15820 [Abstract/Free Full Text]
  11. Roach, P. J. (1991) J. Biol. Chem. 266, 14139-14142 [Abstract/Free Full Text]
  12. Kozma, S. C., and Thomas, G. (1992) Rev. Physiol. Biochem. Pharmacol . 119, 123-155 [Medline] [Order article via Infotrieve]
  13. Cantly, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302 [Medline] [Order article via Infotrieve]
  14. Fu, X. Y. (1992) Cell 70, 323-335 [Medline] [Order article via Infotrieve]
  15. Roach, P. J. (1990) FASEB J. 4, 2961-2968 [Abstract]
  16. Fu, X. D., and Maniatis, T. (1992) Science 256, 535-538 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.




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