(Received for publication, March 21, 1994; and in revised form, November 9, 1994)
From the
The DNA-dependent protein kinase (DNA-PK) phosphorylates RNA polymerase II and a number of transcription factors. We now show that the activity of DNA-PK is directly stimulated by certain transcriptional activator proteins, including the human heat shock transcription factor 1 (HSF1) and a transcriptionally active N-terminal 147 amino acid GAL4 derivative. Stimulation of DNA-PK activity required specific sequences in the activator proteins outside the minimal DNA binding domains. The stimulation of DNA-PK activity also required DNA and was greater with DNA containing relevant activator binding sites. Comparison of different HSF binding fragments showed that optimal stimulation occurred when two HSF binding sites were present. Stimulation with HSF and GAL4 was synergistic with Ku protein, another regulator of DNA-PK activity. DNA-PK is tightly associated with the transcriptional template, and an increase in its activity could potentially influence transcription through the phosphorylation of proteins associated with the transcription complex.
Protein phosphorylation is a widespread mechanism for regulation
of messenger RNA synthesis(1, 2) . Phosphorylation can
affect transcription factors by changing the oligomeric state, the
subcellular distribution, the DNA binding activity, or the ability to
interact with other components of the transcription
apparatus(3, 4, 5, 6, 7) .
In addition, phosphorylation of RNAP ()II at multiple sites
within the C-terminal domain of its largest subunit may facilitate the
transition from the initiation to the elongation phase of the
transcription reaction(8, 9) .
Among the many protein kinases that phosphorylate the transcriptional apparatus in vitro, the DNA-dependent protein kinase is of particular interest because of its tight association with the transcriptional template and its ability to phosphorylate many proteins that are relevant to transcription, including RNAP II, Sp1, p53, SV40 large T antigen, Myc, Oct-1, Fos, Jun, SRF, and histone H1(10, 11, 12, 13, 14, 15, 16, 17, 18) . The activity of DNA-PK is regulated in a unique way. It is strongly dependent on double stranded DNA, and there is a marked preference for substrates that are colocalized to the same DNA fragment(19, 20, 21, 22, 23) . DNA-PK phosphorylates RNAP II, but only in the presence of transcription factors TBP, TFIIB, and TFIIF(16) , presumably because these factors are required for binding of RNAP II to promoter DNA. DNA-PK has a 350-kDa catalytic component (11, 15) and a separate regulatory component, identical to Ku autoantigen, which recruits p350 into a stable complex on the DNA(16, 21, 22) .
In the current study, we report an additional property of DNA-PK, which is that activity is directly stimulated by certain transcriptional activators in vitro. We find that DNA-PK exhibits behavior consistent with that of a signal transduction kinase, responding to certain proteins by phosphorylating others. This suggests that DNA-PK could be part of a signaling pathway which is initiated by certain transcriptional activators and results in the phosphorylation of other nuclear proteins.
HSF1-DNA binding reactions contained
0.1 nM of a singly endlabeled DNA fragment, as indicated in
figure legends. Reactions also contained HSF1 derivatives, as described
in the figure legends, and 0.5 TZ buffer, in a volume of 50
µl. DNase I footprinting was performed as described(28) .
The phosphorylation reactions shown in Fig. 1contained
purified DNA-PK catalytic subunit, a DNA fragment with five GAL4 sites
and a core promoter, and a model substrate with sequences from the CTD
of the large subunit of RNAP II. The basal level of DNA-PK activity in
this system was very low, and phosphorylation increased 20-fold in the
presence of GAL4-(1-147). Input substrate (CTD) was
shifted to a lower mobility form (CTD
), indicative of
multiple phosphorylation. Although the substrate used here contained a
DNA binding domain from the transcription factor Sp1, separate
experiments showed that its DNA binding activity was very weak (not
shown), and the same substrate without Sp1 sequences responded
similarly to activators (see Fig. 3).
Figure 1:
Stimulation of DNA-PK by GAL4
derivatives. A, effect of GAL4-(1-147). Phosphorylation
reactions contained Sp1CTD fusion protein, purified DNA-PK catalytic
subunit, a DNA fragment containing five GAL4 binding sites, and
variable amounts (µM) of GAL4-(1-147) activator
protein. Radiolabeled products were analyzed by 8% SDS-PAGE and
visualized by autoradiography. Arrows indicate the position of
the input CTD and multiply phosphorylated CTD
forms of the Sp1CTD substrate. B, comparison of
GAL4-(1-147) and GAL4-(1-94). Reactions in A and
parallel reactions containing GAL4-(1-94) were analyzed using a
Molecular Dynamics PhosphorImager. Data were normalized to the amount
of radiolabeling in the absence of GAL4 derivatives. C, DNA
binding by GAL4-(1-94) and GAL4-(1-147). Radiolabeled DNA
probe containing five GAL4 binding sites were incubated with various
amounts (µM) of GAL4-(1-147) or GAL4-(1-94) as
indicated. Protein-DNA complexes were analyzed by 5% native PAGE and
visualized by autoradiography. Arrows indicate probe with five
GAL4 binding sites occupied.
Figure 3:
Transcriptional activators promote
phosphorylation of multiple DNA-PK substrates. A, effect of
GAL4-(1-147). Phosphorylation was assayed as described in the
legend to Fig. 1except with 20 ng of GCTD fusion protein, 400
ng of HSP 90, or 200 ng of -casein as substrate. Concentrations of
GAL4-(1-147) are indicated in µM. Products were
analyzed by 10% SDS-PAGE and visualized by autoradiography. Arrows indicate the position of the phosphorylated substrates. B, effect of HSF1. Phosphorylation was assayed as in A, except with concentrations of HSF1 as indicated in
nM.
DNA binding assays were
performed using GAL4-(1-147) and a subfragment of the
GE4T DNA used in the phosphorylation assays. The amount of
GAL4-(1-147) required to stimulate the kinase approximately
correlated with the amount required to saturate the binding sites in
the probe (compare Fig. 1, B and C).
Specific GAL4 sequences outside the DNA binding domain influenced the ability to stimulate the kinase. GAL4-(1-94), which contains only a minimal DNA binding and dimerization domain, showed a reduced ability to stimulate the kinase (Fig. 1B). The difference between the two GAL4 derivatives could not be attributed to differences in DNA binding affinity, as binding approached saturation with the same amount of each GAL4 derivative (Fig. 1C).
Separate experiments confirmed previous reports that GAL4-(1-147) strongly activates transcription in vitro, and GAL4-(1-94) does not(24, 29) . Thus, in vitro transcription and kinase stimulatory activities are both influenced by sequences within a 53-amino acid region of GAL4. The correlation between transcription and kinase stimulatory activities is not perfect; however, since GAL4-(1-94) showed some residual activity in the kinase assay at the highest concentration tested, whereas no activity was detected in an in vitro transcription assay (data not shown).
We next tested the effect of a different factor, the human heat shock transcription factor, HSF1, in similar experiments. Whereas GAL4 had been identified serendipitously, HSF1 was chosen for study because of prior evidence that protein phosphorylation plays a role in the heat shock transcription response. Heat shock increases total CTD kinase activity in cell extracts(30) , and it has been proposed that CTD phosphorylation may be involved in release of RNAP II arrested at heat shock promoters(30, 31) . Recent cytologic and biochemical studies show that phosphorylation of Drosophila RNAP II accompanies heat shock transcriptional induction in vivo(32, 33) .
The phosphorylation reactions in Fig. 2contained bacterially expressed HSF1, which is constitutively active for DNA binding and transcription, a DNA fragment containing two HSF binding sites, and the same CTD model substrate as before. HSF1 increased phosphorylation about 6-fold. The concentration of HSF1 required for maximal stimulation was at least 15-fold lower than with GAL4-(1-147), probably reflecting the higher affinity of HSF1 for DNA. In this experiment, somewhat higher concentrations of HSF1 were required to activate the kinase than to saturate the binding sites in the probe. However, this result appears to be anomalous, as later experiments demonstrated a closer correlation (see Fig. 4). The variability in activation at low concentrations of HSF1 has not been explained, but could reflect nonspecific loss of HSF1 at low protein concentrations, as no carrier protein was present in the assay.
Figure 2:
Stimulation of DNA-PK by HSF1. A,
effect of wild-type HSF1. Phosphorylation of Sp1CTD fusion protein was
analyzed as described in the legend to Fig. 1with variable
amounts (nM) of HSF1. Arrows indicate the position at
CTD, CTD
, and HSF1. B, comparison of
wild type and M3 mutant HSF1. Phosphorylation reactions were performed
as in A except with GCTD substrate. Data were analyzed as
described in the legend to Fig. 1. C, DNA binding by
wild type and mutant HSF1. Radiolabeled probe containing two idealized
HSEs was incubated with variable amounts (nM) of HSF1 and HSF1
M3 as indicated. DNase I footprinting was performed as described under
``Materials and Methods.'' Probe DNA was a Kas/DdeI
fragment of pUCHSEhsp70 (created by insertion of the KpnI/XbaI fragment of pHSP
50HSE
into
pUC19), singly end-labeled at the DdeI site, downstream of the
HSEs.
Figure 4:
Dependence of DNA-PK stimulation on HSF1
binding sites. A, schematic diagram of HSF1 binding DNA
fragments, constructed as described under ``Materials and
Methods.'' Arrows indicate the orientation of HSEs,
relative to pHSP50HSE
. B, phosphorylation of
GCTD fusion protein was assayed in the presence of 439-565-bp KpnI/XbaI fragments derived from constructs shown in A. Reactions were performed with variable amounts of HSF1 as
indicated. Radiolabeled products were analyzed as described in the
legend to Fig. 3. C, binding of HSF1 to DNA fragments
derived from constructs used in A. DNase footprinting was as
described under ``Materials and Methods.'' Probe DNAs were a BglI/EspI fragments singly end-labeled at the EspI site in the hsp70 gene, downstream of the
HSEs.
HSF1 was itself phosphorylated by DNA-PK (Fig. 2A). HSF1 is phosphorylated during the heat shock response in vivo(34, 35) , although the sites of phosphorylation have not been mapped and it is not known if they are the same as are used by DNA-PK in vitro.
As with GAL4,
specific sequences in HSF1 were required for stimulation of the kinase.
A 202-amino acid derivative, HSF1 M3, contains only the minimal DNA
binding and trimerization domains (25) and lacks
transcriptional activity(36) . ()This mutant failed
to stimulate the kinase (Fig. 2B). Although wild type
HSF1 had a 2-fold greater affinity for DNA than HSF1 M3 (Fig. 2C), this could not account for the difference in
activity, since HSF1 M3 worked poorly even at concentrations sufficient
to drive DNA binding to completion.
Additional experiments showed that stimulation of DNA-PK activity is not a universal property of transcriptional activators(36) . It is now recognized that activators fall into a number of classes, which may work by different mechanisms. The acidic class is the best studied. We tested several fusion proteins containing GAL4 sequences joined to the prototypical acidic activation domain from herpes virus VP16. The kinase stimulatory activity of the GAL4-VP16 fusion proteins was no greater than that of GAL4-(1-147) and in some constructs was less, suggesting that the VP16 sequences hindered the ability of the GAL4 domain to activate the kinase(36) . VP16 binds a number of transcription factors in vitro(37, 38) ; its inability to stimulate the kinase underscores the specificity of the activator-kinase interaction.
Neither the HSE nor the
HSE
derivative was effective in promoting DNA-PK activity,
despite the fact that HSF1 bound relatively well to the HSE
construct. By contrast, HSE
was effective in
promoting DNA-PK activity. Maximal activation of the kinase appeared to
correlate with occupancy of the second HSE (compare Fig. 4, B and C). Together, these data suggest that two
DNA-bound HSF molecules are required for a high level of stimulation
and therefore that the interaction of HSF and DNA-PK may be
cooperative.
Interestingly, HSE, HSE
, and
HSE
were less able to promote DNA-PK activity than
HSE
. This was unexpected, and the explanation has not yet
been determined. It could be that in some instances a long array of
tandem sites rendered the DNA less able to assume a conformation
required for interaction with the bulky 350-kDa DNA-PK catalytic
subunit. The ability to support activation was not critically dependent
on the orientation of the HSEs. A construct similar to HSE
with the central two HSEs inverted behaved similarly to HSE
in a phosphorylation assay (data not shown).
The dependence of GAL4-(1-147) activation on GAL4 DNA binding sites was also investigated, although not in as much detail as for HSF1. Stimulation was 5-fold greater when DNA containing GAL4 sites was used in the reaction, compared with DNA without GAL4 sites. There was also virtually no stimulation when DNA was omitted (data not shown). Taken together, the dependence on factor binding sites, seen with both HSF1 and GAL4, implies that binding of the activators to DNA is necessary to activate the kinase.
Figure 5:
GAL4-(1-147) protein cooperates with
Ku protein to stimulate DNA-PK. Phosphorylation of Sp1CTD fusion
protein was assayed as described in the legend to Fig. 1A in the presence of variable amounts of Ku protein in the absence (solid bars) or presence (stippled bars) of 1
µM GAL4-(1-147) protein. Products of the reaction
were analyzed as described in the legend to Fig. 1, and
incorporation of radiolabel into the CTD form was
quantitated by Molecular Dynamics and PhosphorImager
analysis.
In the present study, we have shown that two proteins that activate RNAP II transcription also stimulate the activity of a template-associated protein kinase, DNA-PK. Our findings reveal a previously unrecognized behavior of DNA-PK. DNA-PK acts as a molecular transducer, in that it responds to one kind of signal, the presence of certain DNA-bound activator proteins, by generating another kind of signal, the modification of target proteins by phosphorylation.
The effect of HSF1 on DNA-PK is of special interest because of previous suggestions that the heat shock transcription response involves phosphorylation of RNAP II(30, 31, 32, 33) . A protein kinase with the properties of DNA-PK is a strong candidate to be involved in the CTD phosphorylation event known to accompany induction of transcription in vivo in certain organisms(32) . Caution is warranted, however, since several other protein kinases are activated during heat shock, and the situation in vivo may be complex(39, 40, 41) . It is possible that transcription factor-mediated activation of DNA-PK stimulates localized phosphorylation of proteins bound at nearby promoter sites. Alternatively, activation could be a more generalized phenomenon, stimulating phosphorylation of proteins throughout the nucleus. These alternatives have not yet been distinguished experimentally.
There are several plausible mechanisms by which an increase in DNA-PK activity could influence transcription. We emphasize that none of these mechanisms has been definitively tested, and some may operate only in vivo, not with in vitro transcription systems. DNA-PK phosphorylates the CTD of RNAP II, and an increase in CTD phosphorylation could facilitate the transition to the elongation phase of the transcription reaction(42) . A similar model has been invoked to explain the ability of another DNA-bound CTD kinase, c-abl, to enhance GAL4-VP16-mediated transactivation in vivo(43) . Alternatively, DNA-PK phosphorylates a number of transcriptional activator proteins. At certain promoters, one activator may induce phosphorylation of another, perhaps altering the ability of the second activator to interact with the transcriptional machinery. Finally, DNA-PK phosphorylates histone H1(15) , which could affect the ability of histone H1 to repress transcription. To better address which targets are relevant to the physiological function of DNA-PK, it will be helpful to establish systems in which the role of DNA-PK and its different substrates can be studied more directly.