Previous overexpression studies in intact cells and in vitro reconstitution assays using chromatinized templates have shown that CARM1 can interact and synergize with both p/CIP and CBP/p300 to activate NR-mediated transcription (6
). This synergy is dependent on the methyltransferase activity of CARM1 and has been attributed to the ability of CARM1 to methylate specific arginines on histone H3 tails (50
). Our data suggest that recruitment of CARM1 by p/CIP may play a more complex role in transcription than previously anticipated. In the present study we demonstrate that the transcriptional coactivator p/CIP/SRC-3 is methylated by CARM1. Using mass spectrometric analysis, we have shown that the predominant CARM1-dependent methylation sites in p/CIP are localized to a glutamine-rich region in the carboxy terminus, which we refer to as MD1.
PRMT1, a related methyltransferase, has also been shown to synergize with SRC and CBP/p300 proteins to activate NR signaling in transient transfection assays (26
). The substrate specificity of CARM1 and PRMT1 is different, suggesting that the mechanism of coactivation of these two related methyltransferases may also differ (50
). Indeed, our data suggest that PRMT1 is unable to methylate p/CIP in vitro, suggesting that p/CIP is a specific substrate for CARM1. Additionally, p/CIP could not be methylated in vitro by extracts derived from CARM1−/−
MEFs which still express PRMT1. This indicates that CARM1 is required for methylation of p/CIP in vivo. Importantly, immunoprecipitation of p/CIP from cell extracts, followed by Western blotting using the αR17H3 antibody which recognizes a dimethylated CARM1 recognition site on histone H3, clearly recognized methylated p/CIP. Taken together, these results, in conjunction with the mass spectrometry data, demonstrate that p/CIP is a direct substrate for CARM1 in vivo.
By in vitro mapping experiments we have also identified a second region in p/CIP that can be methylated (R839
) which we refer to as MD2. Although methylation of MD2 is extremely robust using peptide substrates and recombinant GST proteins, the functional significance of this finding is currently unclear as we did not detect methylation at this site using MS. Furthermore, deletion of MD2 did not have a significant impact on overall methylation of p/CIP. Our inability to detect methylation at R839
through mass comparisons may be due to the fact that R839
in Sf9 cells is already occupied by methyl groups and would not be accessible to CARM1-dependent methylation in Sf9 cells or in vitro using baculovirus-generated protein. Additionally, we have shown that phosphorylation of S847
antagonizes methylation at R839
. This finding is significant as this site has recently been shown to be phosphorylated on endogenous SRC-3 and is functionally required for coactivation in response to hormonal signaling (60
). Perhaps p/CIP produced in Sf9 cells may already be phosphorylated at S847
, which may render R839
inaccessible to subsequent methylation by CARM1.
A major finding of this study is that methylation regulates the stability of p/CIP. p/CIP mutants containing substitutions of arginine to alanine accumulate to a much greater degree when overexpressed in various cell types compared to wild-type p/CIP. A significantly higher concentration of p/CIP was also detected in the CARM1−/−
MEFs relative to the wild-type MEFs. This finding is also reflected in the pulse-chase experiments, which indicate that the methylation status of p/CIP is an important determinant regulating its stability, both in the CARM1−/−
MEFs and in cells expressing p/CIP methylation site mutants. Furthermore, we demonstrate that reconstituting hypomethylated p/CIP with extracts derived from wild-type MEFs resulted in a more rapid degradation of p/CIP compared to reconstitution of p/CIP using CARM1−/−
MEF extracts. These results suggest that the methylation status of p/CIP may serve as a prerequisite for rapid degradation. A critically unanswered question is the mechanism through which methylation affects p/CIP stability. Studies have shown that arginine methylation promotes protein-protein interactions in a variety of signal transduction pathways (3
). Thus, one possibility is that methylation of p/CIP by CARM1 allows specific proteins to interact selectively with p/CIP, which, in turn, facilitates proteolytic degradation.
An additional finding in this study is that p/CIP methylation may play a role in the transcriptional process by regulating complex assembly. This assertion is based on the observation that extension of the carboxy terminus AD1 to include the MD1 resulted in a significant decrease in transcriptional activity when tethered to the Gal4 DBD. Conversely, mutation of the CARM1 recognition sites to nonmethylatable alanines enhanced the transcriptional response of the carboxy terminus when tethered to Gal4. Under these experimental conditions, however, we did not observe a significant difference in the expression levels of the various fusion proteins. This suggested that in addition to its role in protein stability, methylation may promote a conformational change that weakens the association of accessory proteins such as CBP/p300 and consequently contributes to complex dissociation. In support of this hypothesis, we observed that CBP forms a stable association with p/CIP and copurified with p/CIP from extracts derived from CARM1−/−
MEFs, compared to CARM+/+
MEFs where no CBP was detected by Western blotting. Interestingly, a recent study has shown that CARM1-dependent methylation of the GRIP1-binding domain within CBP may also promote coactivator complex disassembly (31
). Alternatively, methylation may result in a conformational change in p/CIP which allows another protein to bind, thereby preventing interaction with CBP/p300.
The increase in p/CIP turnover and dissociation of CBP in response to methylation by another transcriptional coactivator seem paradoxical as these activities would typically be associated with the negative control of transcription. However, they are consistent with the notion that transcriptional activation by nuclear hormone receptors is a dynamic process involving repetitive cycles of association and dissociation of receptors and a large number of coregulators with target genes (38
). A number of mechanisms may contribute to this process, such as interaction with molecular chaperones which can associate with liganded nuclear receptors and disassemble nuclear receptor/coregulator complexes (14
). In addition, several studies have established a link between ligand-dependent transcriptional activation by nuclear hormone receptors and the ubiquitin-proteasome pathway (25
). For example, it has been shown that the degradation of ERα, in response to β-estradiol, is dependent on the presence of AIB1 (48
). Coactivator turnover is also dependent on interactions with the proteasome and may be directly regulated by phosphorylation. The phosphorylation of GRIP1 by protein kinase A induces its degradation via the ubiquitin-proteasome pathway, and a recent study has shown that SRC-3 is phosphorylated and then degraded in response to retinoic acid (16
). Interestingly, Li et al. have identified a novel interaction between the carboxy terminus of SRC-3 and Regγ, an activator of the 20S proteasome. Loss of Regγ results in the accumulation of SRC-3 protein levels (33
). Studies examining promoter occupancy have also established a link between proteasomal activity and transcriptional activation (25
). It has been shown that recruitment of the proteasomal machinery is required for gene activation, presumably to allow cyclic clearance and continuous recycling of NR and coregulators at target genes (46
). The exact mechanism for coordinating the events involved in coactivator association and dissociation are poorly understood although increasing evidence suggests that posttranslational modifications may be instrumental to the overall process. For example, treatment of cells with hormones or various growth factors stimulates the phosphorylation of p/CIP/SRC-3 at specific sites and appears to be a prerequisite for hormone-dependent transcription activation that facilitates binding to CBP (13
). Acetylation of SRC-3 also appears to play a role in influencing transcription by facilitating coactivator release from hormone-bound NR (9
The human homologue of p/CIP was first isolated from human breast tumors, and it is located within a region of chromosome 20 that is often amplified in breast and ovarian cancer (2
). Several follow-up studies have confirmed that p/CIP is amplified in a fraction of breast tumors, with amplification frequencies ranging from 5 to 10% (18
). Moreover, compared to normal breast epithelium, p/CIP is overexpressed at the RNA level in 31 to 64% of breast tumors examined. One question arising from the present study is whether deregulated arginine methylation of p/CIP plays a role in cellular transformation. Based on the data presented, we would predict that the methylation status of p/CIP is an important determinant of p/CIP transcriptional activity and may affect its oncogenic capacity. Alternatively, the accumulation of hypomethylated p/CIP could also adversely affect the cycling process inherent in NR-mediated transcription and impair transcription at selective promoters.
In summary, the findings presented in this study link CARM1-dependent methylation of p/CIP to both protein turnover and coactivator complex dissociation, establishing arginine methylation as a critical regulatory signal in p/CIP-mediated transcription. In addition, this type of regulation could serve to integrate diverse signaling pathways at the level of coactivator proteins and may have important implications for understanding the role of p/CIP in breast cancer. Further delineation of the role of p/CIP methylation and its downstream physiological effects is essential to decipher the complex regulatory circuits in nuclear receptor signaling.