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