Stimulation of p300-mediated Transcription by the Kinase MEKK1*

Raymond H. See, Dominica CalvoDagger, Yujiang Shi, Hidehiko Kawa§, Margaret Po-Shan Luke, Zhimin Yuan§, and Yang Shi

From the Department of Pathology, Harvard Medical School and § Department of Radiation Biology, Harvard School of Public Health, Boston, Massachusetts 02115

Received for publication, September 5, 2000, and in revised form, January 29, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p300 and CREB-binding protein (CBP) are related transcriptional coactivators that possess histone acetyltransferase activity. Inactivation of p300/CBP is part of the mechanism by which adenovirus E1A induces oncogenic transformation of cells. Recently, the importance of p300/CBP has been demonstrated directly in several organisms including mouse, Drosophila, and Caenorhabditis elegans where p300/CBP play an indispensable role in differentiation, in patterning, and in cell fate determination and proliferation during development. CBP/p300s are modified by phosphorylation during F9 cell differentiation as well as adenovirus infection, suggesting that phosphorylation may play a role in the regulation of p300/CBP activity. Here we show that the mitogen-activated/extracellular response kinase kinase 1 (MEKK1) enhances p300-mediated transcription. We identify several domains within p300 that can respond to MEKK1-induced transcriptional activation. Interestingly, activation of p300-mediated transcription by MEKK1 does not appear to require the downstream kinase JNK and may involve either a direct phosphorylation of p300 by MEKK1 or by other non-JNK MEKK1-directed downstream kinases. Finally, we present evidence that p300 is important for MEKK1 to induce apoptosis. Taken together, these results identify MEKK1 as a kinase that is likely to be involved in the regulation of the transactivation potential of p300 and support a role of p300 in MEKK1-induced apoptosis.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p300 was initially identified as an adenovirus E1A-associated cellular protein in coimmunoprecipitation experiments (1, 2). The ability of adenovirus E1A to immortalize cells and to induce full morphological transformation is dependent on its ability to interfere with the functions of p300 and its related protein CBP1 (3-5) and members of the retinoblastoma family members (6-9). In addition, inactivation of p300 and CBP is necessary for E1A to inhibit differentiation (10-12). These experiments suggest that p300/CBP play an important role in cell proliferation and differentiation. Recent experiments in mouse, Drosophila, and Caenorhabditis elegans have provided direct evidence that p300/CBP are crucial in both differentiation and cell proliferation during development (13-15).

p300 and CBP are both transcriptional cofactors that can acetylate histones and transcription factors due to their histone acetyltransferase (HAT) activity (16-19). This suggests that p300 and CBP may regulate transcription in part by modifying the chromatin structure as well as activities of transcription factors (18-20). Consistent with this, a C. elegans homolog, CBP-1, has been shown to promote endoderm differentiation by antagonizing the histone deacetylase activity, highlighting the importance of the biological significance of the HAT activity of CBP-1 (13). Additional mechanisms that account for p300/CBP-mediated transcriptional activation involve direct interactions with the basal factors (4, 21, 22) as well as with the RNA polymerase II complex (23).

Although a great deal is known about p300/CBP as transcriptional regulators, very little information is available about mechanisms that regulate their transcriptional activity. Several lines of evidence suggest that phosphorylation may regulate p300/CBP activity. First, p300/CBP have been shown to be phosphoproteins whose phosphorylation state alters during F9 cell differentiation and in adenovirus infection (24). Second, kinases such as protein kinase A and mitogen-activated kinases have been shown to activate CBP-mediated transcription (25, 26). Third, p300 has been reported to be phosphorylated in vitro by cyclin-dependent kinases such as cdc2 and cdk2 (27). In this report, we provide evidence that the mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) stimulates p300-mediated transcription in vivo in a manner that is independent of its downstream kinase JNK.

MEKK1 is a 196-kDa protein which, when cleaved by caspases in response to genotoxic agents, is capable of inducing apoptosis (28, 29). MEKK1 propagates the signal by activating its downstream kinase, MKK4, which in turn activates JNK (30, 31). Activated JNK translocates into the nucleus where it modifies the activity of transcription factors such as AP-1 (32, 33). MEKK1 has also been shown to activate NFkappa B- and c-Myc-mediated transcription, the former involving the activation of the Ikappa Balpha kinase (34). We find that the activated MEKK1 can significantly increase GAL4-p300-mediated transcription. Consistently, nocodazole, which is an extracellular stimulus of MEKK1 (35, 36), can also stimulate p300-dependent transcription. Interestingly, this activation appears to be independent of the downstream kinase JNK since a dominant negative JNK fails to abrogate MEKK1-induced, p300-mediated transcription. In addition, mutations of all the possible JNK sites within subdomains of p300 responsive to MEKK1 had no effect on their response to MEKK1. Although the mechanisms by which MEKK1 induces p300-mediated transcription are still unclear, one possible scenario is a direct phosphorylation of p300 by MEKK1. Consistent with this possibility, we find that MEKK1 can robustly phosphorylate p300 directly in vitro. Significantly, we also find that the activated form of MEKK1 is present exclusively in the nucleus, overlapping with the endogenous p300. In addition, we find the transfected inactive MEKK1 in the cytoplasm, and it appears to localize some of the p300 proteins to the punctate MEKK1 signals. Finally, our results also show that MEKK1-induced apoptosis is impaired by the inhibition of p300 expression. Taken together, our results suggest that phosphorylation of p300 induced by MEKK1 modifies its transcriptional activity, which may be an important event in the apoptosis induced by MEKK1.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Cell Culture-- HeLa, U2OS, and MCF-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics.

Plasmids-- pGAL4-p300 aa 1-596, pGAL4-p300 aa 744-1571, and pGAL4-p300 aa 1257-2414 were described before (22). pGAL4-Sp1Q2 was obtained from Robert Tjian (University of California, Berkeley). p12SE1A expression plasmid was described previously (37). pGAL4-p300 aa 19-2414, pGAL4-p300 aa 1737-2414, and pGAL4-p300 aa 1945-2414 were kindly provided by Antonio Giordano (Jefferson Cancer Institute, Philadelphia, PA). pCDNA3-FLAG MEKK1Delta and pCDNA3-FLAG MEKK1Delta (K432M), consisting of the C-terminal 321 residues of full-length MEKK1, were obtained from Tom Maniatis (Harvard University, Boston). pCDNA3 HA-tagged JNK (APF) was obtained from Tilang Deng (University of Florida, Gainesville). GAL4-cJun aa 1-223 and GAL4-luciferase were gifts from Michael Karin (University of California, San Diego). GST-p300 aa 1709-1913 was provided by David Livingston (Dana Farber Cancer Institute, Boston). pGAL4-p300 aa 302-667 was constructed by subcloning the region in frame with PSG 424 GAL4 expression vector. GST-p300 aa 2-337 and GST-p300 aa 302-667 were made by subcloning the corresponding regions into a compatible pGEX vector (Amersham Pharmacia Biotech) to create an in-frame fusion. GAL4-p300 aa 302-667 Ala-317/499/512/524/594 and GAL4-p300 aa 1709-1913 Ala-1726/1849/1851/1854/1857/1865/1868/1878/1906/1909 were created by mutation of serines and threonines to alanines using the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were verified by sequencing. GST-p300 alanine mutants were constructed by cloning the regions into a compatible pGEX vector (Amersham Pharmacia Biotech) to create an in-frame fusion.

Luciferase Assay-- HeLa cells were transfected using Fugene 6 transfection reagent (Roche Molecular Biochemicals) with indicated reporters and cDNA expression vectors. After 40 h, luciferase activity was determined as described (38). The luciferase activities were normalized on the basis of beta -galactosidase activity of cotransfected Rous sarcoma virus beta -galactosidase vector.

Assay for the Effect of Nocodazole on p300-dependent Transcription-- U2OS cells were transfected with mammalian expression plasmids encoding either GAL4 DNA-binding domain (G4, 1 µg) alone or G4-p300 (1 µg) together with the target reporter G4-luciferase (0.5 µg). One hour before harvest, the cells were treated with two different doses (0.5 and 1.0 µg/ml) of nocodazole. Cell extracts were prepared and used for luciferase assay described above.

Expression and Purification of Recombinant Proteins-- GST-p300 fusion proteins were induced and purified as described (34). His6 MEKK1Delta was purified from baculovirus-infected Sf9 cell lysates using nickel-nitrilotriacetic acid-agarose as described previously (34).

In Vitro Kinase and Apoptosis Assays-- About 5 µg of purified GST fusion proteins were suspended in 30 µl of kinase buffer (20 mM MOPS, pH 7.2, 2 mM EDTA, 10 mM MgCl2, 0.1% Triton X-100) and incubated with either 0.5 µg of His6 MEKK1Delta or 1 µg of recombinant JNK (Stratagene) and 10 µCi of [gamma -32P]ATP at 37 °C for 30 min. The kinase reactions were terminated by the addition of Laemmli sample buffer. Proteins were resolved using 12% SDS-PAGE gels and dried for autoradiography.

Nocodazole stimulation of MEKK1 kinase activity was measured by the activation of JNK. Briefly, proliferating U20S cells were treated with either Me2SO solvent control or nocodazole at the indicated concentrations. Cell lysates were prepared 1 h post-treatment, and the activation of MEKK1 was analyzed by Western blotting with an anti-JNK1 antibody (SC-474, Santa Cruz Biotechnology) as described previously (39).

Apoptosis assays were performed using MCF-7 cells that express either an active p300 or inactive p300 ribozymes as described previously (40). Activated MEKK1 was cotransfected into MCF-7 cells along with green fluorescent protein (GFP) cDNA. Apoptotic cells were scored on the basis of GFP and Hoecht stain 48 h after transfection.

Cell Fixation and Immunofluorescence Microscopy-- U2OS cells were transiently transfected with full-length HA-MEKK1 or FLAG-MEKK1Delta cDNA. Thirty six hours post-transfection, the cells were fixed in 3.7% paraformaldehyde at 25 °C for 10 min. After 5 min of washing in PBS, the fixed cells were permeabilized in 0.2% Triton X- 100 (25 °C for 15 min) followed by 5 min of washing in PBS. The cells were then incubated with primary antibody for 1 h at 25 °C. Anti-HA epitope tag monoclonal antibody (Babco) and anti-FLAG epitope tag monoclonal antibody (Sigma) were used at a 1:200 dilution; anti-p300 polyclonal antibody C-20 (Santa Cruz Biotechnology) was diluted 1:100. After three 10-min PBS washes, coverslips were incubated in secondary antibody for 1 h at 25 °C. Fluorescein isothiocyanate-conjugated goat anti-mouse (Jackson ImmunoResearch) and Cy3 Red-conjugated goat anti-rabbit (Jackson ImmunoResearch) secondary antibody were used at a dilution of 1:400. For the DAPI staining, the coverslips were also incubated in 1× PBS with 1:10,000 dilution of DAPI for 10 min. Vectashield 1200 media was used for slide mounting (Vector Labs). Epifluorescence microscopy was conducted using a Nikon microscope. Images were acquired by a Sony camera and analyzed using Adobe Photoshop version 5.0.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MEKK1Delta Activates GAL4-p300-mediated Transcription-- To determine whether MEKK1 affects p300-mediated transcription, a constitutively activated MEKK1 mutant, MEKK1Delta (34), was cotransfected into HeLa cells along with GAL4-p300 and a GAL4 luciferase reporter construct. Fig. 1A shows that MEKK1Delta augmented GAL4-p300-mediated transcription about 20-fold compared with vector control. In contrast, a catalytically inactive mutant of MEKK1, MEKK1Delta K432M (lysine 432 converted to methionine), had little effect on GAL4-p300-mediated transcription (Fig. 1B). This indicates that stimulation of p300-mediated transcription by MEKK1 is dependent on the kinase activity of MEKK1. Furthermore, MEKK1Delta did not affect transcription mediated by GAL4-Sp1Q2 (Fig. 1A), a previously characterized glutamine-rich activation domain of Sp1, suggesting that the ability of MEKK1Delta to up-regulate p300-mediated transcription was not through a general enhancement of the basal transcription machinery. The ability of MEKK1Delta to activate GAL4-p300-mediated transcription was not restricted to HeLa cells as similar results were obtained in COS-7 cells (data not shown). These findings suggest that a kinase pathway involving MEKK1 can regulate GAL4-p300-mediated transcription.


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Fig. 1.   Response of GAL4-p300 to activated MEKK1. A, GAL4-p300 or GAL4-Sp1Q2 (1 µg) was cotransfected into HeLa cells along with a GAL4-luciferase reporter in the presence or absence of activated MEKK1 (MEKK1Delta ). Luciferase activity was assayed after 48 h. Results, representative of three experiments, describe the mean ± S.D. Luciferase activity in the absence of MEKK1Delta was normalized to one. B, same as A except that catalytically inactive MEKK1 (MEKK1Delta K432M) was cotransfected into HeLa cells instead of activated MEKK1Delta .

Nocodazole Stimulates p300-dependent Transcription-- To determine whether extracellular stimuli known to activate MEKK1 can stimulate p300-dependent transcription, we examined the ability of nocodazole (35, 36) to regulate GAL4-p300-mediated transcription. As shown in Fig. 2A, MEKK1 kinase activity is stimulated by nocodazole in a dose-dependent manner as shown by its ability to induce JNK phosphorylation (compare lanes 2 and 3 with lane 1). When the kinase activity of MEKK1 is stimulated, we find consistent enhancement of GAL4-p300-mediated transcription by nocodazole in a dose-dependent manner, although at a modest level (Fig. 2B, lanes 5 and 6). Taken together, activated MEKK1 provided either exogenously (transfection) or endogenously (conversion of an inactive MEKK1 to an active one by nocodazole) can stimulate GAL4-p300-mediated transcription.


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Fig. 2.   Nocodazole stimulates GAL4-p300-mediated transcription. A, nocodazole stimulation of MEKK1 kinase activity as measured by the activation of JNK. Proliferating U20S cells were treated with either the solvent Me2SO (lane 1) or nocodazole at 0.5 µg/ml (lane 2) or 1.0 µg/ml (lane 3). Cell lysates were prepared 1 h post-treatment and analyzed by Western blotting with an anti-JNK1. The JNK1 and phosphorylated JNK1 are indicated by arrows on the right and labeled as such. The concentration of nocodazole used is indicated. B, nocodazole stimulates GAL4-p300-mediated transcription. Cells were transfected as G4-Luc reporter plasmid together with either the GAL4 (lanes 1-3) or GAL4-p300 (lanes 4-6) expression plasmids and were treated with either Me2SO (lanes 1 and 4) or nocodazole (lanes 2 and 3, and 5 and 6) 1 h before harvest. The y axis represents luciferase activity. The data represent means ± S.D. of triplicates from two independent experiments. The concentration of nocodazole used is indicated.

Identification of Domains of p300 Involved in Its Response to MEKK1-- Previously, we and others (21, 22) have shown that at least two independent activation domains are present in the N- and C-terminal portions of p300. We wished to determine whether the transcriptional activity of these two p300 domains could be regulated by MEKK1Delta . Fig. 3 shows that the transcriptional activity of the N-terminal (aa 1-596) and the C-terminal regions (aa 1257-2414), when fused to GAL4, were highly responsive to MEKK1Delta . In contrast, the middle region of p300 (aa 744-1571), which itself has no detectable transcriptional activity (22), did not respond at all to MEKK1Delta .


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Fig. 3.   Mapping of the p300 domain that responds to activated MEKK1Delta . Diagram shows p300 and its deletion mutants fused to the GAL4 DNA binding domain. The black boxes indicate either the bromodomain (B) or the cysteine/histidine (C/H) rich regions. The gray box indicates the GAL4 DNA binding domain at the N terminus. HeLa cells were transfected with GAL4-luciferase and 1 µg of GAL4-p300 deletion mutants in the presence or absence of MEKK1Delta . Results represent the fold activation of the luciferase activity in the presence of MEKK1Delta . Luciferase activity in the absence of MEKK1Delta was normalized to one. Results are representative of four experiments and represent the mean ± S.D.

We next sought to identify a minimal domain within the N-terminal and C-terminal regions of p300 that were responsible for the transcriptional activation by MEKK1Delta . As shown in Fig. 3, the response of the large N-terminal region of p300 to MEKK1Delta was mediated by two subdomains, aa 2-337 (N1, 32-fold) and aa 302-667 (N2, 89-fold) The N2 subdomain contains the C/H1 domain (aa 347-411) and is three times more responsive than N1. Further deletions that remove aa 302-406 (inclusive of the C/H1 domain) and aa 567-667 from N2 did not significantly reduce its response to MEKK1, suggesting that p300 aa 407-566 (N3) is sufficient to mediate part of the response of p300 to MEKK1Delta . Taken together, these results suggest that there are at least two transactivation domains within the N terminus that are responsive to MEKK1Delta , p300 aa 2-337 and p300 aa 407-566, and that the C/H1 domain is not involved in the N-terminal response of p300 to MEKK1Delta .

The C-terminal deletion mutants of p300 were also analyzed for the same purpose. Fig. 3 shows that MEKK1Delta enhanced the transcriptional activity of GAL4-p300 (aa 1737-2414) (C1) 32-fold. In contrast, MEKK1Delta only weakly activated the transcriptional activity of GAL4-p300 (aa 1945-2414) (C2). These results suggest that p300 aa 1737-1945 is an important domain within the C-terminal half of p300 for responding to MEKK1Delta . Amino acids 1737-1945 of p300 contain the carboxyl portion of the C/H3 domain (aa 1653-1817) that is rich in cysteines and histidines (12). The C/H3 domain has been shown to be an important surface that mediates a number of important protein-protein interactions (12, 21, 41-43). The above data suggest that the C-terminal domain of p300 that mediates the response to MEKK1 may reside somewhere between aa 1737 and 1945. We therefore tested a GAL4-p300 construct that includes this region (GAL4-p300 aa 1709-1913) (C3). As shown in Fig. 3, MEKK1Delta activates transcription mediated by GAL4-p300 C3 (aa 1709-1913) by 20-fold. This is a significant transcriptional induction, although slightly less than the fold of activation (32-fold) observed for GAL4-p300 C1 (aa 1737- 2414) in response to MEKK1Delta . Thus, a region including part of the C/H3 domain of p300 is involved in the transcriptional response of the C-terminal region of p300 to MEKK1Delta .

MEKK1 Stimulation of GAL4-p300-mediated Transcription Does Not Require JNK-- It has been well documented that MEKK1 activates the JNK family members of kinases (30, 31, 44). To determine whether JNK1 was involved in the activation of GAL4-p300-mediated transcription induced by MEKK1, we first asked whether JNK is necessary for the MEKK1 response mediated by the subdomains of p300 described above. We tested a dominant negative inhibitor of JNK1 (JNK1 APF) (32) for its ability to block MEKK1Delta -mediated activation of GAL4-p300-mediated transcription. As shown in Fig. 4, JNK1 (APF) was unable to block MEKK1Delta induction of transcription by either GAL4-p300N1 (aa 2-337), GAL4-p300N2 (aa 302-667), or GAL4-p300C1 (aa 1737-2414). In contrast, the dominant negative JNK1 mutant APF potently inhibited GAL4-cJun-mediated transcription 8-fold as expected (Fig. 4). These finding therefore suggest that JNK is not necessary for MEKK1 to enhance p300-mediated transcription. MEKK1 has also been shown to activate the Ikappa Balpha kinases (34). By using a dominant negative form of the Ikappa Balpha kinase (S32A/S36A), we found no evidence that this kinase is involved in mediating the ability of MEKK1Delta to enhance p300-mediated transcription (data not shown).


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Fig. 4.   Effect of dominant negative JNK on the ability of MEKK1Delta to induce GAL4-p300-mediated transcription. HeLa cells were transfected with 1 µg of MEKK1Delta and GAL4-luciferase along with either GAL4-p300 aa 2-337, GAL4-p300 aa 302-667, GAL4-p300 aa 1737-2414, or GAL4-cJun 1-223 (1 µg each) in the presence or absence of JNK APF. Results are representative of three experiments, and the mean ± S.D. is indicated.

The above results predict that these subdomains of p300 are either not substrates for JNK1, or the JNK phosphorylation sites residing in these subdomains are not important for MEKK1-induced, p300-mediated transcription. To determine whether JNK1 can phosphorylate the MEKK1-responsive p300 subdomains, we performed in vitro kinase assays using the various p300 domains fused to GST as substrates for recombinant JNK. Equal amounts of GST proteins were used for the in vitro kinase assays (data not shown). Fig. 5 shows that recombinant JNK phosphorylates GST-p300 aa 1709-1913 (C3) very strongly (lane 4). In contrast, recombinant JNK barely phosphorylated GST-p300 aa 2-337 or GST-p300 aa 302-667 (Fig. 5, lanes 2 and 3). Similar results were found in JNK immune complex kinase assays using the same GST-p300 substrates (data not shown). These results indicate that only the C3 domain of p300 was a potential substrate for JNK, whereas the N-terminal domains were not. The fact that the N-terminal domains of p300 are poor JNK substrates is consistent with the above finding (Fig. 5) that JNK is not involved in MEKK1-induced transcription mediated by the N-terminal domains of p300.


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Fig. 5.   Phosphorylation of GST-p300 domains in vitro by recombinant JNK. GST-p300 aa 2-337, GST-p300 aa 302-667, and GST-p300 aa 1709-1913 were purified as described under "Experimental Procedures" and standardized according to protein concentration. Recombinant JNK was then used to phosphorylate GST-p300 fragments in the presence of [gamma -32P]ATP at 37 °C. Reactions were terminated after 30 min by adding sample loading buffer and running on SDS-PAGE. The gel was then dried and autoradiographed. * indicates the positions of the GST or GST-p300 fusion proteins.

The finding that JNK phosphorylates GST-p300 aa 1709-1913, which can also mediate a MEKK1 response, appears to be contradictory to the observation that the p300 C-terminal domain-mediated MEKK1 response is unaffected by a dominant negative form of JNK (Fig. 4). To resolve this issue, we determined whether JNK phosphorylation of this domain is correlated with its ability to mediate a MEKK1 response. We therefore mutated all potential JNK sites with the consensus sequence serine/proline or threonine/proline within this domain. Because of the large number of proline-directed serines and threonines within this region, we made a series of cluster mutations designated A-D shown in Fig. 6A. Equal amounts of the mutant GST fusion proteins (Fig. 6C) were then tested for their ability to be phosphorylated by recombinant JNK. Fig. 6B shows that compared with wild-type p300 aa 1709-1913, mutation of proline-directed serines and threonines to alanines within either clusters A or B partially abolished the ability of recombinant JNK to phosphorylate GST-p300 aa 1709-1913 (lanes 2 and 3). Mutation of serines and threonines to alanines within clusters C or D did not inhibit the ability of JNK to phosphorylate GST-p300 aa 1709-1913 (data not shown). However, mutant GST-p300 aa 1709-1913, which combines the mutations in both clusters A and B (mut A/B), totally lost the ability to respond to JNK-induced phosphorylation (Fig. 6B, lane 4).


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Fig. 6.   A, description of GST-p300 1709-1913 alanine mutants. Proline-directed serines or threonines of GST-p300 aa 1709-1913 were mutated to alanines in clusters (designated A-D) as described under "Experimental Procedures." The position of the serines (S) or threonines (T) mutated are indicated by the asterisks. Numbers represent the amino acid residue. B, phosphorylation of wild-type and mutant p300. Phosphorylation of wild-type (WT) GST-p300 aa 1709-1913 (lane 1), GST-p300 aa 1709-1913 Ala-1726/1849/1851/1854/1857 (Mut A, lane 2), GST-p300 aa 1709-1913 Ala-1865/1868 (Mut B, lane 3), or GST-p300 aa 1709-1913 Ala-1726/1849/1851/1854/1857/1865/1868 (Mut A/B, lane 4) by recombinant JNK in vitro. GST-p300 mutants were purified as described under "Experimental Procedures" and phosphorylated by recombinant JNK in the presence of [gamma -32P]ATP at 37 °C. Equal amounts of GST-p300 fusion proteins were used as substrates. Reactions were terminated by adding sample loading buffer and running on SDS-PAGE. The gel was then dried and autoradiographed. C, Coomassie Blue-stained gel of GST-p300 aa 1709-1913 wild-type or alanine mutants. D, activation of GAL4-p300 aa 302-667 and GAL4-p300 aa 1709-1913 ala mutants by MEKK1Delta . HeLa cells were transfected with GAL4-luciferase, wild-type GAL4-p300 aa 302-667, wild-type GAL4-p300 aa 1709-1913, GAL4-p300 aa 302-667 Ala-317/499/512/524/594, or GAL4-p300 aa 1709-1913 Ala-1726/1849/1851/1854/1857/1865/1868/1878/1906/1909 (1 µg each) in the presence or absence of MEKK1Delta . Results are representative of three experiments, and the mean ± S.D. is indicated. Luciferase activity in the absence of MEKK1Delta was normalized to one.

We next analyzed the ability of this JNK phosphorylation-defective mutant for its ability to mediate an MEKK1 response. As shown in Fig. 6D, mutation of all potential JNK sites (clusters A-D) did not inhibit the response of this GAL4-p300 aa 1709-1913 mutant to MEKK1Delta compared with its wild-type counterpart. Although the N- terminal subdomain of p300, N2 (aa 302-667), is not phosphorylated by JNK1 in vitro (Fig. 5), we nevertheless mutated all possible JNK sites in this domain and analyzed the effect the mutations. As expected, mutation of the potential JNK sites in the N2 subdomain had little effect on its ability to mediate MEKK1-induced transcriptional activation (Fig. 6D). Taken together, these results support the notion that the downstream kinase of MEKK1, JNK1, is not essential for MEKK1-stimulated transcriptional activation mediated by p300.

MEKK1 Can Phosphorylate the N Terminus of p300 in Vitro-- Given the fact that JNK1 is not involved in the response of p300 to MEKK1, nor are the other potential downstream kinases such as p38 and Ikappa Balpha , we considered the possibility that MEKK1Delta may be able to directly phosphorylate p300. Baculovirus MEKK1Delta was purified as described previously (34) and used in kinase assays with GST-p300 fragments as substrates. Fig. 7A shows that baculovirus MEKK1Delta strongly phosphorylated GST-p300 aa 302-667 (lane 3) as well as GST-p300 aa 300-528 (data not shown). As a control, MEKK1Delta did not phosphorylate GST alone as shown in lane 1 (Fig. 7A). The ability of MEKK1 to phosphorylate GST-p300 aa 302-667 was also observed in immunocomplex kinase assays with MEKK1 (data not shown). In contrast, GST-p300 aa 2-337 (N1) (lane 2) and GST-p300 aa 1709-1913 (C3) (lane 4) were weakly phosphorylated by MEKK1Delta (compare lanes 2 and 4 with lane 3). The phosphorylation of GST-p300 aa 302-667 as opposed to GST-p300 aa 2-337 and GST-p300 aa 1709-1913 was not due to differences in protein amount as Fig. 7B shows relatively equal levels of protein by Coomassie Blue staining. These results are consistent with the possibility that stimulation of GAL4-p300 aa 302-667-mediated transcription by MEKK1Delta may occur via direct phosphorylation of p300 aa 302-667 by MEKK1. Furthermore, the inability of MEKK1 to phosphorylate GST-p300 N1 and GST-p300 C3 (Fig. 7A, lanes 2 and 4) suggests that a yet unidentified MEKK1-directed kinase other than JNK may be involved in activating GAL4-p300-mediated transcription.


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Fig. 7.   A, in vitro phosphorylation of p300 domains by purified MEKK1Delta . GST, GST-p300 aa 2-337, GST-p300 aa 302-667, and GST-p300 aa 1709-1913 were purified as described under "Experimental Procedures" and standardized according to protein concentration. Purified baculovirus MEKK1Delta (0.5 µg) was then used to phosphorylate GST-p300 fragments in the presence of [gamma -32P]ATP at 37 °C. Reactions were terminated by adding sample loading buffer and running on SDS-PAGE. The gel was then dried and autoradiographed. * indicates the positions of the GST or GST-p300 fusion proteins. Autophosphorylated MEKK1Delta is indicated by the arrow. B, Coomassie Blue-stained gel of GST or GST-p300 fusion proteins. * indicates positions of the GST or GST-p300 fusion proteins. Lower bands represent breakdown products of the GST-p300 fusion proteins.

Analyses of Subcellular Localization of MEKK1 and p300-- The model that MEKK1 may regulate p300 activity by directly phosphorylating p300 predicts that the active form of MEKK1 resides in the nucleus, and these two proteins may physically interact. To address this question, we analyzed the localization of endogenous p300 in the presence and absence of either active or inactivate MEKK1. As expected, we find MEKK1Delta present almost exclusively in the nucleus in a punctate pattern similar to that of the endogenous p300 (compare Fig. 8, E with F). In fact, upon close inspection of the individual and merge images, the active MEKK1 signal appears to overlap with that of p300, suggesting that these two proteins my colocalize in the nucleus. This finding is consistent with a direct regulatory role of MEKK1Delta on p300 in the nucleus.


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Fig. 8.   Analyses of the subcellular localization of MEKK1 and p300. U2OS cells were transfected with 10 µg of HA-MEKK1 (A-D) or FLAG-MEKK1Delta (E-H) and double-stained with either anti-HA monoclonal plus anti-p300 polyclonal (A and B) or anti-FLAG monoclonal plus anti-p300 polyclonal (E and F) antibodies as indicated. The positive transfectants (white arrowhead) and untransfected neighboring cells (open arrows) are indicated. A, untransfected (open arrow) and neighboring cells transfected with HA-MEKK1 (arrowhead) were stained with an HA epitope antibody. B, the same cells as in A stained with anti-p300 polyclonal antibodies. C, merged image of A and B in which an arrow indicates a cell in which p300 is found in the cytoplasm with a punctate pattern identical to that of MEKK1. E, untransfected (open arrow) and a neighboring cell transfected with FLAG-MEKK1Delta (arrowhead) were stained with a anti-FLAG epitope antibody. F, the same cells as in E stained with p300 polyclonal antibodies. G, merged image of E and F. D and H are DAPI stainings that identify the nuclei of the cells.

Inactive MEKK1 resides predominantly in the cytoplasm (see Ref. 29 and Fig. 8A). As shown in Fig. 8A, HA-tagged full-length MEKK1 (inactive form) is present in both diffused and punctate patterns in the cytoplasm (Fig. 8A). On the other hand, endogenous p300 is present in the nucleus (see Ref. 12, and Fig. 8B, open arrow, indicates an untransfected cell in which p300 is present in the nucleus). Interestingly and strikingly, in the cells that overexpress MEKK1 (Fig. 8, arrowheads), p300 staining shows a significant alteration. In addition to the nuclear staining, it is also present in a punctate pattern in the cytoplasm, identical to the punctate HA-MEKK1 pattern (Fig. 8B, HA- MEKK1-transfected cell indicated by a closed arrow). This suggests that some of the p300 molecules may have been retained by HA-MEKK1 in the cytoplasm, perhaps through protein-protein interactions. Taken together, analyses of the subcellular localization of p300 as well as MEKK1 (both active and inactive forms) lend further support to the idea that MEKK1 can regulate p300 activity directly, independent of its downstream JNK kinase.

MEKK1-induced Apoptosis Is Inhibited in a p300-active Ribozyme Cell Line-- It has been documented that MEKK1 activation can induce cells to undergo apoptosis (29, 45, 46). To determine whether p300 is involved in the MEKK1 apoptotic pathway, we made use of an MCF-7 cell line that expresses a much reduced level of p300 due to the presence of an active p300 ribozyme (40). As shown previously, these cells, but not MCF-7 cells containing an inactive p300 ribozyme, are deficient in the apoptotic response of cells to ionizing radiation (40). We asked if activated MEKK1 induces apoptosis in MCF-7 cells in a manner that may be dependent on p300. As shown in Fig. 9, compared with the inactive p300 ribozyme cell line which contains wild-type level of p300, the active p300 ribozyme cell line showed a 50% reduction in apoptosis induced by MEKK1Delta (36% apoptotic cells for inactive ribozyme MCF-7 versus 16% apoptotic cells for p300 active ribozyme MCF-7). This suggests that p300 may be part of the pathway by which MEKK1 induces apoptosis and suggests that the modification of the transcriptional activity of p300 by MEKK1 may be important for this process.


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Fig. 9.   Effect of p300 ribozyme on the ability of MEKK1Delta to induce apoptosis in MCF-7 cells. 10 µg of pCDNA3 vector, pCDNA3 MEKK1Delta , or pCDNA3 MEKK1Delta K432M were cotransfected along with GFP into MCF-7 cells containing an inactive or active p300 ribozyme. Forty eight hours after transfection, 400 cells were counted, and the percentage of apoptotic cells were scored as described under "Experimental Procedures." Results are representative of two experiments, and the mean ± S.D. of triplicate cultures is indicated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p300/CBP participate in the integration of a diverse array of signals with transcriptional events that govern gene expression. In this report, we demonstrate that a kinase in the mitogen-activated kinase pathway, MEKK1, can robustly enhance transcription mediated by p300 when fused to the GAL4 DNA binding domain. This is likely to be physiologically relevant as nocodazole, an extracellular stimulus of MEKK1, can also enhance p300-dependent transcription. MEKK1 has been shown to be important for the apoptotic response of cells to ionizing radiation (29). Significantly, the dynamic interaction between MEKK1 and p300 may be important for MEKK1-induced apoptosis.

MEKK1 has been shown to activate the JNK family of the mitogen-activated kinase members by activating MKK4 (30, 31, 44), an immediate upstream activator of JNK. Surprisingly, our results show that JNK is not involved in the activation of p300-dependent transcription. First, dominant negative JNK1 does not block MEKK1-induced activation of GAL4-p300-mediated transcription. Second, mutations of all potential JNK sites (proline-directed serines or threonines) within the N2 or C3 region do not impair the ability of MEKK1 to enhance p300-mediated transcription. Since MEKK1 can phosphorylate p300 in vitro, these results taken together raise the possibility that MEKK1 may induce p300-mediated transcription by directly phosphorylating p300, particularly within the p300 N2 region. However, since p300 N1 and p300 C3 regions are only weakly phosphorylated by MEKK1 itself (Fig. 7A), it is possible that the ability of MEKK1 to activate GAL4-p300-mediated transcription may also involve additional unidentified MEKK1 downstream kinases. Taken together, our data suggest that stimulation of p300-mediated transcription by MEKK1 may occur via both direct and indirect mechanisms.

Since p300 is a large platform protein and can interact with many different transcription factors, our results cannot rule out the possibility that the effect of MEKK1 on p300 may be mediated by modifications of the interacting transcription factors. For instance, p300 interacts with NFkappa B (47) and MEKK1 activates Ikappa Balpha kinase which activates NFkappa B. We thus considered whether MEKK1 merely activated this pathway that facilitated the interaction of NFkappa B with GAL4-p300 and somehow resulted in transcriptional activation. We tested this possibility using the dominant negative form of the Ikappa Balpha kinase (34) and found no evidence for the involvement of NFkappa B in the stimulation of p300-mediated transcription by MEKK1 (data not shown).

The ability of MEKK1 to regulate transcription directly is not unprecedented. MEKK1 has been shown to increase the transcriptional activity of GAL4-c-myc and GAL4-Elk-1 (46). The activation of GAL4-c-myc-dependent transcription is believed to also occur in a JNK-independent manner (46). Together with the results described here, these findings suggest that direct phosphorylation of transcriptional regulators by MEKK1 may be a common mechanism by which MEKK1 regulates transcription. Consistent with this idea, we find the active form of MEKK1 (MEKK1Delta ) in the nucleus of transfected cells and its localization appears to coincide with that of the endogenous p300 (Fig. 8, E and F). Furthermore, endogenous p300 can be found colocalizing with the punctate cytoplasmic MEKK1 signals in cells overexpressing the full-length inactive form of MEKK1, which resides in the cytoplasm, suggesting a possible physical interaction. Collectively, these findings are consistent with the possibility of direct regulation of p300 by MEKK1.

At present, the phosphoacceptor sites on p300 targeted by MEKK1 or its unknown downstream kinases are not defined. We have mutated all potential JNK sites that consist of proline-directed serines and threonines within p300 aa 302-667 and aa 1709-1913 without causing alterations in the response to MEKK1. In addition, mutations of serines in a putative MEKK1 phosphorylation motif (DSXXXS) (48) located within p300 aa 302-667 did not abolish the ability of this region to respond to MEKK1 (data not shown). Further studies are necessary to identify the amino acid residues that are either directly or indirectly phosphorylated by MEKK1.

How does phosphorylation of p300 leads to an enhancement of its transcriptional activity? p300 has been shown to interact with members of the basal transcriptional machinery such as TFIIB and TATA-binding protein (4, 21, 22, 26) as well as the RNA polymerase II complex (23). Perhaps one mechanism by which MEKK1 increases the transactivation potential of p300 is to promote the interaction of p300 with these members of the transcriptional basal machinery. An alternative but not mutually exclusive possibility is that phosphorylation of p300 by MEKK1 may enhance the HAT activity of p300. It should be interesting in the future to test directly these possibilities.

Activated MEKK1 has been shown to induce cell death (28, 45, 46, 49). We provide evidence here that p300 may be important in mediating MEKK1 induction of the apoptotic pathway in MCF-7 cells. Impaired expression of p300 in MCF-7 cells resulted in significantly lower levels of apoptosis induced by MEKK1 (Fig. 9). As discussed above, the regulation of p300 activity by MEKK1 does not require its downstream kinase JNK. Interestingly, the MCF-7 cell death response to MEKK1 also appears to be JNK-independent (29, 46). Collectively, these findings suggest that the ability of MEKK1 to induce apoptosis in MCF-7 cells may involve an increase in the transcriptional activity of p300.

MEKK1-induced apoptosis appears to involve stabilization of p53 as well as an increase in its transcriptional activity (50). Given the fact that p300 is a coactivator for p53 (41, 51, 52), it is possible that MEKK1-induced phosphorylation of p300 may be important for p300 to serve as a coactivator for p53. It is interesting to note that p300 appears to be essential for the stabilization of p53 as well as increase in p53-dependent transcription in response to ionizing radiation (40). Therefore, it is also possible that phosphorylation of p300 by MEKK1 may contribute to the interaction of p300 with p53 and its stabilization, resulting in changes of p53-dependent gene expression and apoptosis in MCF-7 cells.

In summary, our data show that MEKK1 can significantly enhance transcription mediated by p300. We have identified several regions within p300 that are responsive to MEKK1. The MEKK1-induced transcription mediated by p300 does not appear to require its known downstream kinase JNK. Instead, regulation of p300 activity by MEKK1 may occur via a direct phosphorylation of p300 by MEKK1 and by other currently unknown MEKK1 downstream kinases. Biologically, impaired expression of p300 in MCF-7 cells significantly inhibits the amount of apoptosis induced by MEKK1Delta , suggesting that p300 may be an important component by which MEKK1Delta induces apoptosis. Our findings thus identify MEKK1 as a kinase that may regulate the transcriptional as well as the biological activities of p300.

    ACKNOWLEDGEMENTS

We thank Michael Greenberg and Azad Bonni for critical reading of the manuscript and Melanie Cobb for helpful discussion and suggestions. We thank Hans Peter Hefti and MyungSoo Joo for making GAL4-p300 aa 407-566 and GAL4-p300 aa 302-667, respectively. We thank Kenneth Liu for computer assistance. We also thank many colleagues for providing reagents used in this study.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM58012 (to Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a Spanish Government fellowship and by a Taplin Postdoctoral fellowship.

To whom correspondence should be addressed: Dept. of Pathology, Harvard Medical School, Warren Alpert Bldg., Rm. 120, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-4318; Fax: 617-432-1313; E-mail: yang_shi@hms.harvard.edu.

Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M008113200

    ABBREVIATIONS

The abbreviations used are: CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; JNK, c-Jun N- terminal kinase; MEKK1, mitogen-activated/extracellular response kinase kinase; aa, amino acid; GST, glutathione S-transferase; C/H, cysteine/histidine; HAT, histone acetyltransferase; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; HA, hemagglutinin; DAPI, 4,6-diamidino-2-phenylindole; MOPS, 3-(N-morpholino)propanesulfonic acid; aa, amino acid.

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