Transcriptional Regulation by Steroid Receptor Coactivator Phosphorylation
http://www.100md.com
内分泌进展 2005年第3期
Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Abstract
The basic mechanisms underlying ligand-dependent transcriptional activation by nuclear receptors (NRs) require the sequential recruitment of various coactivators. Increasing numbers of coactivators have been identified in recent years, and both biochemical and genetic studies demonstrate that these coactivators are differentially used by transcription factors, including NRs, in a cell/tissue type- and promoter-specific manner. However, the molecular basis underlying this specificity remains largely unknown. Recently, NRs and coregulators were shown to be targets of posttranslational modifications activated by diverse cellular signaling pathways. It is argued that posttranslational modifications of these proteins provide the basis for a combinatorial code required for specific gene activation by NRs and coactivators, and that this code also enables coactivators to efficiently stimulate the activity of other classes of transcription factors. In this review, we will focus on coactivators and discuss the recent progress in understanding the role of phosphorylation of the steroid receptor coactivator family and the potential ramifications of this posttranslational modification for regulation of gene expression.
I. Introduction
A. Nuclear receptors (NRs) and coregulators
B. SRC family of coactivators: molecular structure and biological functions
II. Coactivator Phosphorylation as the Molecular Basis for Coactivator Specificity
III. Cellular Signaling, Coactivator Phosphorylation, and Cancer
IV. Concluding Remarks
I. Introduction
A. Nuclear receptors (NRs) and coregulators
STEROID RECEPTORS ARE part of the superfamily of NRs, which are transcription factors that regulate expression of numerous biologically important target genes; in the case of steroid receptors, transcriptional activity is regulated by steroid hormones (1). Collectively, the functions of NRs are required for a wide variety of important biological processes, including homeostasis, organogenesis, development, and reproduction. Structurally, NRs can be divided into several signature functional domains (1, 2). These include, from the amino to the carboxyl terminus, the first activation function (AF-1) domain, the DNA-binding domain, the hinge region, and the ligand-binding domain that also contains the second activation function (AF-2) domain.
Functionally, the full activity of NRs depends on a large number of cellular factors that do not bind DNA directly but are otherwise recruited to the promoters by NRs. These cellular proteins are collectively defined as coregulators. Depending on the effect of the coregulators on the outcome of gene expression, they can be broadly divided into coactivators that promote NR activation of transcription, and corepressors that suppress NR-dependent gene expression. In recent years, we have witnessed not only the identification of increasing numbers of coregulators, but also the achievement of a better understanding of the molecular basis by which these coregulators function. However, it is not the intention of this review to discuss in detail the role of coactivators or corepressors in NR function and, for this matter, readers are referred to several excellent recent reviews (3, 4). Instead, we will focus only on the p160 steroid receptor coactivator (SRC) family of coactivators, particularly on SRC-3, and discuss novel and interesting aspects of its diverse cellular biology.
B. SRC family of coactivators: molecular structure and biological functions
SRC-3 [also referred to as acetyltransferase/p300/cAMP response element binding protein (CREB)-binding protein (CBP)-interacting protein/receptor-associated coactivator 3/thyroid hormone receptor-activated molecule 1/amplified in breast cancer 1 (AIB1)] is one of the three members of the SRC (p160) family of coactivators. Structurally, all three SRCs (SRC-1/nuclear receptor coactivator-1, SRC-2/transcriptional intermediary factor 2/glucocorticoid receptor interacting protein 1/nuclear receptor coactivator-2, and SRC-3) contain a so-called basic helix-loop-helix-Per/ARNT/Sim domain at their amino termini, which is also the most conserved region among the SRCs (reviewed in Refs. 5 and 6). The basic helix-loop-helix-Per/ARNT/Sim domain was originally identified in Drosophila proteins, where it was shown to be involved in DNA binding and mediating protein-protein interactions (7). Because SRCs also contain this well-defined domain, it is reasonable to envision that cross-talk between different pathways involving SRCs might occur through interactions of this domain of SRCs with other proteins. In fact, members of the SRC family have been shown to interact with myogenin, MEF-2 C, and transcriptional enhancer factor (TEF) through this domain (8, 9). Moreover, the ability of SRCs to coactivate with other transcription factors, such as nuclear factor-B (NF-B), activator protein 1 (AP-1), signal transducers and activators of transcription (STAT), p53 and E2F1, suggests that SRCs are important components of multiple pathways (10, 11, 12, 13, 14, 15). In fact, different regions of SRCs were shown to be responsible for the interactions of p160s with these factors, further emphasizing the flexibility available to these coactivators to modulate the activity of various classes of transcription factors (10, 12, 16, 17, 18) (Fig. 1). These findings clearly suggest that SRCs may be more versatile coactivators, relative to their functional interactions with more diverse transcription factors, than was originally understood.
A centrally located region of the SRCs contains multiple LXXLL motifs (where L is leucine and X is any amino acid) that are responsible for ligand-dependent interaction with NRs (19, 20, 21). These motifs also are known as NR boxes. Further carboxy-terminal to the NR-interaction domain of SRCs is a region that was shown to contain the intrinsic transcriptional activation domain (19, 22). This domain also coincides with the region responsible for interaction of SRCs with the histone acetyltransferase activity (HAT)-containing p300/CBP cointegrators, and this interaction is shown to be critical for transcription activation mediated by SRCs (20, 23, 24). Additional functions of the carboxyl termini of SRCs are to bind other coactivators such as the coactivator-associated arginine methyltransferase-1 (CARM1) (25), and to provide intrinsic HAT activity in SRC-1 and SRC-3 (25, 26). The preponderance of intrinsic or associated enzymatic activities that can modify histone proteins suggests that chromatin modification by SRC coactivators, either directly or indirectly, is important for their transcriptional activity. Based on the presence of these important domains, it also has been suggested that the p160/SRC family of coactivators serve as adaptor molecules to recruit additional coactivators and basal transcription machinery to the promoter. In support of this, SRCs have been shown to synergistically activate NR activity in conjunction with p300/CBP and CARM1 in transient transfection assays (25, 27, 28, 29). Conversely, inhibition of p160 coactivator expression by small interfering RNAs or antisense oligonucleotides attenuates ligand-induced target gene expression (30, 31).
Recently, the biological functions of SRCs in vivo have been revealed by gene disruption experiments in mice. Mice lacking SRC-1 showed normal growth and fertility but displayed partial resistance to multiple steroid hormones (32). SRC-2–/– mice also exhibit normal growth, but have reduced fertility in both males and females due to gonadal failure (33). Additionally, SRC-2 null mice are leaner and contain less white adipose tissue but more active brown adipocytes (34). In stark contrast to SRC-1 and SRC-2 null mice, knockout of SRC-3 results in growth retardation and smaller body size (35, 36). SRC-3-null mice also display resistance to growth hormones and estrogen. Interestingly, although knockouts of each individual SRC did not result in embryonic lethality, likely due to some functional coactivator redundancy among family members, the mice clearly exhibited distinct phenotypic defects, suggesting the presence of unique functions for each of these family members. For a more detailed discussion of the phenotypes of the SRC null mice, readers are referred to a recent review by Xu and Li (6).
Despite substantial progress in our understanding of the importance of SRCs, many intriguing and physiologically relevant questions remain unanswered. In this review, we will explore posttranslational modifications of SRCs, particularly with emphasis on SRC-3 phosphorylation, as a novel type of regulation of the transcriptional activity of coactivators. We will discuss the involvement of phosphorylation in the determination of transcription factor specificity and the potential implications of phosphorylation in tumorgenesis and cancer progression.
II. Coactivator Phosphorylation as the Molecular Basis for Coactivator Specificity
During the last decade, it has been noted that the activities of several NRs, including estrogen receptor (ER), progesterone receptor, and androgen receptor, are regulated by posttranslational modifications (37, 38, 39). Prominent among these modifications is receptor phosphorylation elicited by kinase-mediated cellular signaling events. Generally speaking, increased receptor phosphorylation is associated with increased transcriptional activity, and available data indicate that the activity of receptors is subject to regulation by cellular signaling pathways. For example, mutation of the serine118 phosphorylation site of ER to an alanine yields a receptor with reduced transcriptional activity (40, 41), whereas induction of ER-dependent gene expression by either estradiol- or epidermal growth factor-signaling pathways is associated with increased phosphorylation at this site (42, 43). However, the significance of phosphorylation at serine118 of ER is not without debate. Recently, Font de Mora and Brown (44) reported that phosphorylation of serine118 is not required for epidermal growth factor-induced ER activation, but rather requires phosphorylation of SRC-3 (AIB1). The reason for this discrepancy surrounding serine118 phosphorylation is currently unknown; one possible explanation may involve the strong promoter and cell type specificities of the AF-1 domain (45). Additionally, it is conceivable that other phosphorylation sites on ER and other components of the transcriptional complex may also be required for the activation of ER. To avoid the potential pitfall of transfection and to unequivocally demonstrate its importance, the effects of serine118 phosphorylation on endogenous ER target genes in physiological relevant cell types and settings should be assessed. Despite the fact that the mechanism(s) by which phosphorylation affects transcriptional activity remains obscure, a large number of studies of various NRs support the conclusion that receptor function is subject to regulation by cellular signaling pathways.
In comparison, a realization that the activity and specificity of coactivators is subject to similar regulation has only recently occurred. Of note, SRCs, CBP, and peroxisomal proliferators-activated receptor--coactivator-1 can be modified by phosphorylation as well as other types of modification such as methylation, acetylation, and sumoylation (15, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54). Seven phosphorylation sites for SRC-1 and six for SRC-3 have been identified recently (50, 55). Subsequently, phosphorylation of SRCs was shown to be induced in response to a spectrum of environmental stimuli, including epidermal growth factor, steroid hormones and cytokines, and increased intracellular cAMP (15, 44, 50, 55, 56). Importantly, phosphorylation of SRCs induced by these agents was shown to be essential for their optimal activity. In the case of SRC-1, phosphorylation at threonine1179 and serine1185 induced by cAMP were shown to enhance both ligand-dependent and ligand-independent activity of progesterone receptor (56). Athough the actual underlying mechanism was not defined, phosphorylation was shown to be important for the physical or functional interaction of SRC-1 with p/CAF (p300/CBP associating factor) or CBP (56). Similarly, phosphorylation of SRC-3 induced by epidermal growth factor, steroid hormones, and cytokines also was shown to be important for its coactivator activity (44, 55). Interestingly, phosphorylation of SRC-3 was shown to selectively affect interactions with receptors, NF-B, and CBP, but not with CARM1 (55). Taken together, phosphorylation of SRCs seems to be involved in the regulation of protein-protein interactions; however, it is unclear and remains to be investigated whether phosphorylation affects other aspects of SRCs function, such as their intrinsic activation and HAT activities.
Taken together, available data suggest that SRCs are targets of distinct cellular signaling pathways that can regulate coactivator activities. This raised the interesting possibility that SRCs might function to integrate various distinct cellular signals, thereby permitting these signals to accurately and specifically affect a broad range of promoters for transcriptional activation. In support of this, SRCs coactivate an increasingly diverse number of transcription factors (see Section I.B.), and they can be phosphorylated by multiple kinases, such as MAPK and IB kinases (IKKs), that are involved in a variety of cellular signaling pathways. Nevertheless, the question remained as to how coactivators might distinguish one downstream class of transcription factors from another, so as to prevent global gene activation in response to phosphorylations induced by a specific pathway.
Interestingly, our recent study on SRC-3 demonstrated that not only is the activity of SRC-3 regulated by phosphorylation, but also the specificity of SRC-3 for different transcription factors can be determined by inducing distinct patterns of phosphorylation on the SRC-3 molecule (55). More specifically, phosphorylation at six amino acids of SRC-3 is shown to be induced by estrogen or androgen hormones, and all six sites are required for ER activation of target genes. However, phosphorylation of only five of these six amino acids is inducible by TNF-stimulated signaling pathways and required for coactivation of NF-B. In other words, a combination of phosphorylation events at specific sites on SRC-3 affords the coactivator the ability to participate specifically in the stimulation of transcription factor-dependent gene expression (55). It is also important to note that the initiating signal (e.g., estradiol) appropriately stimulates the phosphorylation of SRC-3 so that it is an efficient coactivator for the corresponding transcription factor (e.g., ER). This interesting finding suggests that an SRC-3 phosphorylation code exists for distinct regulatory responses to different environmental cues. Although the initial observation was made on SRC-3, it is likely that the activity of other coactivators could be regulated in a similar manner (Fig. 2).
Although all three SRCs contain both redundant and distinct functions, it is clear from experiments in mice that each individual SRC contains the capacity to regulate different biological functions. To date, the molecular basis underpinning these fundamental differences was unclear. Several recent notable differences were observed among SRCs that shed new light on this issue. For example, the IKK complex was copurified exclusively with SRC-3 but not SRC-1 and, accordingly, phosphorylation of SRC-3, but not SRC-1, is enhanced in response to TNF stimulation (15). This suggests that the activity of SRCs may be modulated by the signaling pathways that control the activity of the transcription factors with which they are associated. Second, comparison of the identified phosphoamino acids revealed very little conservation of these sequences among SRCs, both with respect to positions and type of phosphorylation sites (50, 55), arguing that phosphorylation is a significant determinant of the ultimate specificity of SRCs. Finally, among the steroid hormones tested, we found that only ligands for ER (estradiol) and androgen receptor (R1881), but not progesterone receptor (progesterone), can induce SRC-3 phosphorylation on the identified sites (R. C. Wu and B. W. O’Malley, unpublished results). Consistent with earlier findings (57), these results clearly demonstrated a biochemical degree of ligand, receptor, and coactivator specificity. Thus, we conclude that posttranslational phosphorylation provides a molecular basis that determines the ability of SRCs to distinguish among various NRs and other transcription factors to provide specific responses to distinct upstream signaling pathways (Fig. 2).
III. Cellular Signaling, Coactivator Phosphorylation, and Cancer
The gene for SRC-3 (also known as AIB1 for amplified in breast cancer-1) has been shown to be amplified in 10% of human breast tumors, and the same report indicated that elevated SRC-3 mRNA expression was found for 64% of tumors (58); this was the first indication that SRC-3 may confer some growth advantage. Subsequently, it was shown that SRC-3 overexpression enhances somatic cell growth in prostate cancer cells by activating the AKT signaling pathway (59). In addition, overexpression of SRC-3 in mice alone or in the presence of the Ras oncogene facilitates mammary gland tumorgenesis, and this is associated with increasing serum levels of IGF-I and AKT pathway activity (60, 61). Conversely, knockout of the SRC-3 gene reduced adult body size, due in part to lower circulating IGF-I levels and impairment of IGF-I signaling (36). Collectively, these studies demonstrate that SRC-3 alone is sufficient for the initiation of tumorgenesis, and SRC-3 also can function as a facilitator in the oncogenesis process. It is clearly an oncogene.
However, the levels of SRC-3 protein are not always the sole determinants of SRC-3 pathological function. As summarized above, SRC-3 is an integral and important component of cellular signaling pathways, suggesting a means by which its activity can be subject to other levels of regulation (15, 35, 44, 55). For example, MAPK and IKK signaling both augment SRC-3 activity by phosphorylating SRC-3. As these kinases are important regulators of cellular growth functions, it is evident that aberrant activation of SRC-3 by these kinases could have adverse effects. Therefore, to fully appreciate the function of SRC-3, it is paramount to account for the milieu of cellular signaling in context with SRC-3. However, it is of note that the mechanisms involved in the induction of SRC-3 phosphorylation appear to be complex in nature. First of all, an array of kinases was shown to be able to phosphorylate SRC-3 at least in vitro, and some of the same amino acids of SRC-3 are potential targets of different kinases. Although it broadens and supports the ever-expanding role of SRC-3 in diverse signaling events, it also raises an interesting question as to how different kinases are able to act in concert to phosphorylate and activate SRC-3 in response to a single type of stimulation. Second, it is unclear as to the location where phosphorylation of SRC-3 actually takes place, i.e., in the cytoplasm or in the nucleus where transcription occurs, or in both compartments. Given the fact that the NRs, SRC-3, and the kinases involved are present in both the cytoplasm and nucleus, and are able to shuttle back and forth between these two compartments, this issue proves to be difficult to answer definitively. Lastly, some of the kinases, such as MAPK, were shown to phosphorylate not only SRC-3, but the receptors as well. It is not clear whether phosphorylation of one will affect phosphorylation of the other, or whether there is a sequential order for the phosphorylation of these proteins. Despite all of these unanswered questions, it should not lessen our interest to better understand how and where the signaling cascade originates. Experiments using specific mutants of receptors and SRC-3 should be useful for answering these remaining questions.
As several studies have established SRC-3 as a bona fide oncogene, it also is noteworthy to point out that despite the significant progress made in treating endocrine-sensitive cancers, success is still severely hampered by the development of therapeutic resistance. Therefore, it is imperative to fully understand the underlying mechanisms responsible for the acquisition of this resistance. Perhaps the best studied example of this is the development of resistance to tamoxifen therapy in the progression of human breast cancer. It was proposed, based on work done in the mid-1990s (62, 63) that the relative expression of coactivators and corepressors in a given cell would be critical determinants of the biocharacter (e.g., agonist vs. antagonist activity) of selective receptor modulators such as the selective ER modulator, tamoxifen. Evidence accumulated since that time has supported that hypothesis, including demonstrations that coactivators promote tamoxifen agonist activity (62, 63, 64) and corepressors are important for the antagonistic activity of this widely used drug (65, 66). However, varied cellular signaling pathways (including phosphorylation, acetylation, sumoylation, and methylation) can exert distinct effects on NR and coregulator function and suggest that the intracellular environment also can play a significant role in determining the biological activity of selective receptor modulators such as tamoxifen (reviewed in Ref. 67). Of the recently delineated cellular pathways potentially contributing to this resistance, the cross-talk between NRs and the growth-/survival-promoting signaling pathways seems to hold much promise, particularly as it has become increasingly evident that the interaction between these different pathways occurs at multiple levels, involving both NRs and coactivators (42, 44, 55, 68). Thus, as another layer of complexity has been added to the NR-coactivator picture, the recent data suggest coactivators as viable and critical targets for therapeutic intervention.
As an interesting example, the oncogenic potential of SRC-3 was recently demonstrated to be closely associated with the phosphorylation state of SRC-3 in vitro (55), consistent with the ability of cellular signaling events to modulate SRC-3 activity. In a recent study of breast cancer patients, high SRC-3 (AIB1) expression in patients who had not received adjuvant tamoxifen therapy was associated with improved disease-free survival, especially for those patients who also had low expression levels of HER-2/neu receptor tyrosine kinase (69). However, in patients that had received tamoxifen treatment, those with overexpression of SRC-3 in conjunction with high HER-2/neu expression levels experienced a significantly reduced period of disease-free survival, suggesting that cross-talk between these two pathways is disadvantageous. In a follow-up in vitro study, overexpression of HER-2/neu was associated with 1) increased phosphorylation of SRC-3 in response to stimulating breast cancer cells with heregulin, estradiol or tamoxifen, and 2) increased tamoxifen-induced association of SRC-3 with a gene promoter and elevated tamoxifen stimulation of ER target gene expression (70). These findings add another dimension to our understanding of SRC-3 function and exemplify the importance of cellular signaling as an integral part of coactivator biology. Moreover, these considerations present an interesting scenario whereby aberrant signaling activation of SRC-3 contributes to tumorgenesis and/or development of resistance to hormone therapy in endocrine cancers, suggesting a platform for future experimentation and drug development.
IV. Concluding Remarks
As our understanding of the biology of NRs and coactivators increases, so does our appreciation of the complexity of their actions. Even with recent developments, a number of important questions remain to be addressed, and future efforts should be oriented toward providing a better understanding of a number of these issues.
First of all, the nature of receptor specificity and functional redundancy of each of the SRC members remains an interesting topic to many in the field. Although the spatial expression of SRC family members in different cell types contributes to selectivity, it remains to be determined what other factors contribute to their specificity. Here we hypothesize, as shown in the case of NRs and NF-B, that differential posttranslational modifications of SRC members help to determine their receptor specificity. A detailed examination of all types of modifications, including phosphorylation, acetylation, methylation, ubiquitination, neddylation, and sumoylation, will expand our knowledge and provide new insight into this matter. Second, although SRC-3 was demonstrated to be an oncogene, the exact role of SRC-3 in tumor initiation must be further characterized. Examination of additional signaling upstream of SRC-3 activation and subsequent downstream genetic events elicited by SRC-3 activation deserve equal attention. Finally, because the oncogenic potential of SRC-3 has been linked to its phosphorylation state, it is likely that aberrant phosphorylation and activation of SRC-3 will be linked to advanced stages of cancers. Therefore, whether assessment of a hyperphosphorylation state or phosphorylation of specific amino acids of SRC-3 can be used as a marker for tumor progression is a question begging further investigation, and it is possible that management of disease in the future will encompass not only therapies targeted to NRs, but also to the coregulators that are so critical for mediating their biological activity within the cell.
Acknowledgments
We apologize to our colleagues whose work could not be cited due to page limitation.
Footnotes
This work was supported by a postdoctoral fellowship from the Department of Defense (to R.-C.W.), National Institutes of Health (NIH) Grants DK53002 and DK64083 (to C.L.S.), and NIH Grants HD08818, HD07857, and NIDDK/NURSA-5U19DK62434-02 (to B.W.O.).
First Published Online April 6, 2005
Abbreviations: AF, Activation function; AIB1, amplified in breast cancer 1; CARM1, coactivator-associated arginine methyltransferase-1; CBP, cAMP response element-binding protein (CREB)-binding protein; ER, estrogen receptor; HAT, histone acetyltransferase; IKK, IB kinase; NF-B, nuclear factor-B; NR, nuclear receptor; SRC, steroid receptor coactivator.
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Abstract
The basic mechanisms underlying ligand-dependent transcriptional activation by nuclear receptors (NRs) require the sequential recruitment of various coactivators. Increasing numbers of coactivators have been identified in recent years, and both biochemical and genetic studies demonstrate that these coactivators are differentially used by transcription factors, including NRs, in a cell/tissue type- and promoter-specific manner. However, the molecular basis underlying this specificity remains largely unknown. Recently, NRs and coregulators were shown to be targets of posttranslational modifications activated by diverse cellular signaling pathways. It is argued that posttranslational modifications of these proteins provide the basis for a combinatorial code required for specific gene activation by NRs and coactivators, and that this code also enables coactivators to efficiently stimulate the activity of other classes of transcription factors. In this review, we will focus on coactivators and discuss the recent progress in understanding the role of phosphorylation of the steroid receptor coactivator family and the potential ramifications of this posttranslational modification for regulation of gene expression.
I. Introduction
A. Nuclear receptors (NRs) and coregulators
B. SRC family of coactivators: molecular structure and biological functions
II. Coactivator Phosphorylation as the Molecular Basis for Coactivator Specificity
III. Cellular Signaling, Coactivator Phosphorylation, and Cancer
IV. Concluding Remarks
I. Introduction
A. Nuclear receptors (NRs) and coregulators
STEROID RECEPTORS ARE part of the superfamily of NRs, which are transcription factors that regulate expression of numerous biologically important target genes; in the case of steroid receptors, transcriptional activity is regulated by steroid hormones (1). Collectively, the functions of NRs are required for a wide variety of important biological processes, including homeostasis, organogenesis, development, and reproduction. Structurally, NRs can be divided into several signature functional domains (1, 2). These include, from the amino to the carboxyl terminus, the first activation function (AF-1) domain, the DNA-binding domain, the hinge region, and the ligand-binding domain that also contains the second activation function (AF-2) domain.
Functionally, the full activity of NRs depends on a large number of cellular factors that do not bind DNA directly but are otherwise recruited to the promoters by NRs. These cellular proteins are collectively defined as coregulators. Depending on the effect of the coregulators on the outcome of gene expression, they can be broadly divided into coactivators that promote NR activation of transcription, and corepressors that suppress NR-dependent gene expression. In recent years, we have witnessed not only the identification of increasing numbers of coregulators, but also the achievement of a better understanding of the molecular basis by which these coregulators function. However, it is not the intention of this review to discuss in detail the role of coactivators or corepressors in NR function and, for this matter, readers are referred to several excellent recent reviews (3, 4). Instead, we will focus only on the p160 steroid receptor coactivator (SRC) family of coactivators, particularly on SRC-3, and discuss novel and interesting aspects of its diverse cellular biology.
B. SRC family of coactivators: molecular structure and biological functions
SRC-3 [also referred to as acetyltransferase/p300/cAMP response element binding protein (CREB)-binding protein (CBP)-interacting protein/receptor-associated coactivator 3/thyroid hormone receptor-activated molecule 1/amplified in breast cancer 1 (AIB1)] is one of the three members of the SRC (p160) family of coactivators. Structurally, all three SRCs (SRC-1/nuclear receptor coactivator-1, SRC-2/transcriptional intermediary factor 2/glucocorticoid receptor interacting protein 1/nuclear receptor coactivator-2, and SRC-3) contain a so-called basic helix-loop-helix-Per/ARNT/Sim domain at their amino termini, which is also the most conserved region among the SRCs (reviewed in Refs. 5 and 6). The basic helix-loop-helix-Per/ARNT/Sim domain was originally identified in Drosophila proteins, where it was shown to be involved in DNA binding and mediating protein-protein interactions (7). Because SRCs also contain this well-defined domain, it is reasonable to envision that cross-talk between different pathways involving SRCs might occur through interactions of this domain of SRCs with other proteins. In fact, members of the SRC family have been shown to interact with myogenin, MEF-2 C, and transcriptional enhancer factor (TEF) through this domain (8, 9). Moreover, the ability of SRCs to coactivate with other transcription factors, such as nuclear factor-B (NF-B), activator protein 1 (AP-1), signal transducers and activators of transcription (STAT), p53 and E2F1, suggests that SRCs are important components of multiple pathways (10, 11, 12, 13, 14, 15). In fact, different regions of SRCs were shown to be responsible for the interactions of p160s with these factors, further emphasizing the flexibility available to these coactivators to modulate the activity of various classes of transcription factors (10, 12, 16, 17, 18) (Fig. 1). These findings clearly suggest that SRCs may be more versatile coactivators, relative to their functional interactions with more diverse transcription factors, than was originally understood.
A centrally located region of the SRCs contains multiple LXXLL motifs (where L is leucine and X is any amino acid) that are responsible for ligand-dependent interaction with NRs (19, 20, 21). These motifs also are known as NR boxes. Further carboxy-terminal to the NR-interaction domain of SRCs is a region that was shown to contain the intrinsic transcriptional activation domain (19, 22). This domain also coincides with the region responsible for interaction of SRCs with the histone acetyltransferase activity (HAT)-containing p300/CBP cointegrators, and this interaction is shown to be critical for transcription activation mediated by SRCs (20, 23, 24). Additional functions of the carboxyl termini of SRCs are to bind other coactivators such as the coactivator-associated arginine methyltransferase-1 (CARM1) (25), and to provide intrinsic HAT activity in SRC-1 and SRC-3 (25, 26). The preponderance of intrinsic or associated enzymatic activities that can modify histone proteins suggests that chromatin modification by SRC coactivators, either directly or indirectly, is important for their transcriptional activity. Based on the presence of these important domains, it also has been suggested that the p160/SRC family of coactivators serve as adaptor molecules to recruit additional coactivators and basal transcription machinery to the promoter. In support of this, SRCs have been shown to synergistically activate NR activity in conjunction with p300/CBP and CARM1 in transient transfection assays (25, 27, 28, 29). Conversely, inhibition of p160 coactivator expression by small interfering RNAs or antisense oligonucleotides attenuates ligand-induced target gene expression (30, 31).
Recently, the biological functions of SRCs in vivo have been revealed by gene disruption experiments in mice. Mice lacking SRC-1 showed normal growth and fertility but displayed partial resistance to multiple steroid hormones (32). SRC-2–/– mice also exhibit normal growth, but have reduced fertility in both males and females due to gonadal failure (33). Additionally, SRC-2 null mice are leaner and contain less white adipose tissue but more active brown adipocytes (34). In stark contrast to SRC-1 and SRC-2 null mice, knockout of SRC-3 results in growth retardation and smaller body size (35, 36). SRC-3-null mice also display resistance to growth hormones and estrogen. Interestingly, although knockouts of each individual SRC did not result in embryonic lethality, likely due to some functional coactivator redundancy among family members, the mice clearly exhibited distinct phenotypic defects, suggesting the presence of unique functions for each of these family members. For a more detailed discussion of the phenotypes of the SRC null mice, readers are referred to a recent review by Xu and Li (6).
Despite substantial progress in our understanding of the importance of SRCs, many intriguing and physiologically relevant questions remain unanswered. In this review, we will explore posttranslational modifications of SRCs, particularly with emphasis on SRC-3 phosphorylation, as a novel type of regulation of the transcriptional activity of coactivators. We will discuss the involvement of phosphorylation in the determination of transcription factor specificity and the potential implications of phosphorylation in tumorgenesis and cancer progression.
II. Coactivator Phosphorylation as the Molecular Basis for Coactivator Specificity
During the last decade, it has been noted that the activities of several NRs, including estrogen receptor (ER), progesterone receptor, and androgen receptor, are regulated by posttranslational modifications (37, 38, 39). Prominent among these modifications is receptor phosphorylation elicited by kinase-mediated cellular signaling events. Generally speaking, increased receptor phosphorylation is associated with increased transcriptional activity, and available data indicate that the activity of receptors is subject to regulation by cellular signaling pathways. For example, mutation of the serine118 phosphorylation site of ER to an alanine yields a receptor with reduced transcriptional activity (40, 41), whereas induction of ER-dependent gene expression by either estradiol- or epidermal growth factor-signaling pathways is associated with increased phosphorylation at this site (42, 43). However, the significance of phosphorylation at serine118 of ER is not without debate. Recently, Font de Mora and Brown (44) reported that phosphorylation of serine118 is not required for epidermal growth factor-induced ER activation, but rather requires phosphorylation of SRC-3 (AIB1). The reason for this discrepancy surrounding serine118 phosphorylation is currently unknown; one possible explanation may involve the strong promoter and cell type specificities of the AF-1 domain (45). Additionally, it is conceivable that other phosphorylation sites on ER and other components of the transcriptional complex may also be required for the activation of ER. To avoid the potential pitfall of transfection and to unequivocally demonstrate its importance, the effects of serine118 phosphorylation on endogenous ER target genes in physiological relevant cell types and settings should be assessed. Despite the fact that the mechanism(s) by which phosphorylation affects transcriptional activity remains obscure, a large number of studies of various NRs support the conclusion that receptor function is subject to regulation by cellular signaling pathways.
In comparison, a realization that the activity and specificity of coactivators is subject to similar regulation has only recently occurred. Of note, SRCs, CBP, and peroxisomal proliferators-activated receptor--coactivator-1 can be modified by phosphorylation as well as other types of modification such as methylation, acetylation, and sumoylation (15, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54). Seven phosphorylation sites for SRC-1 and six for SRC-3 have been identified recently (50, 55). Subsequently, phosphorylation of SRCs was shown to be induced in response to a spectrum of environmental stimuli, including epidermal growth factor, steroid hormones and cytokines, and increased intracellular cAMP (15, 44, 50, 55, 56). Importantly, phosphorylation of SRCs induced by these agents was shown to be essential for their optimal activity. In the case of SRC-1, phosphorylation at threonine1179 and serine1185 induced by cAMP were shown to enhance both ligand-dependent and ligand-independent activity of progesterone receptor (56). Athough the actual underlying mechanism was not defined, phosphorylation was shown to be important for the physical or functional interaction of SRC-1 with p/CAF (p300/CBP associating factor) or CBP (56). Similarly, phosphorylation of SRC-3 induced by epidermal growth factor, steroid hormones, and cytokines also was shown to be important for its coactivator activity (44, 55). Interestingly, phosphorylation of SRC-3 was shown to selectively affect interactions with receptors, NF-B, and CBP, but not with CARM1 (55). Taken together, phosphorylation of SRCs seems to be involved in the regulation of protein-protein interactions; however, it is unclear and remains to be investigated whether phosphorylation affects other aspects of SRCs function, such as their intrinsic activation and HAT activities.
Taken together, available data suggest that SRCs are targets of distinct cellular signaling pathways that can regulate coactivator activities. This raised the interesting possibility that SRCs might function to integrate various distinct cellular signals, thereby permitting these signals to accurately and specifically affect a broad range of promoters for transcriptional activation. In support of this, SRCs coactivate an increasingly diverse number of transcription factors (see Section I.B.), and they can be phosphorylated by multiple kinases, such as MAPK and IB kinases (IKKs), that are involved in a variety of cellular signaling pathways. Nevertheless, the question remained as to how coactivators might distinguish one downstream class of transcription factors from another, so as to prevent global gene activation in response to phosphorylations induced by a specific pathway.
Interestingly, our recent study on SRC-3 demonstrated that not only is the activity of SRC-3 regulated by phosphorylation, but also the specificity of SRC-3 for different transcription factors can be determined by inducing distinct patterns of phosphorylation on the SRC-3 molecule (55). More specifically, phosphorylation at six amino acids of SRC-3 is shown to be induced by estrogen or androgen hormones, and all six sites are required for ER activation of target genes. However, phosphorylation of only five of these six amino acids is inducible by TNF-stimulated signaling pathways and required for coactivation of NF-B. In other words, a combination of phosphorylation events at specific sites on SRC-3 affords the coactivator the ability to participate specifically in the stimulation of transcription factor-dependent gene expression (55). It is also important to note that the initiating signal (e.g., estradiol) appropriately stimulates the phosphorylation of SRC-3 so that it is an efficient coactivator for the corresponding transcription factor (e.g., ER). This interesting finding suggests that an SRC-3 phosphorylation code exists for distinct regulatory responses to different environmental cues. Although the initial observation was made on SRC-3, it is likely that the activity of other coactivators could be regulated in a similar manner (Fig. 2).
Although all three SRCs contain both redundant and distinct functions, it is clear from experiments in mice that each individual SRC contains the capacity to regulate different biological functions. To date, the molecular basis underpinning these fundamental differences was unclear. Several recent notable differences were observed among SRCs that shed new light on this issue. For example, the IKK complex was copurified exclusively with SRC-3 but not SRC-1 and, accordingly, phosphorylation of SRC-3, but not SRC-1, is enhanced in response to TNF stimulation (15). This suggests that the activity of SRCs may be modulated by the signaling pathways that control the activity of the transcription factors with which they are associated. Second, comparison of the identified phosphoamino acids revealed very little conservation of these sequences among SRCs, both with respect to positions and type of phosphorylation sites (50, 55), arguing that phosphorylation is a significant determinant of the ultimate specificity of SRCs. Finally, among the steroid hormones tested, we found that only ligands for ER (estradiol) and androgen receptor (R1881), but not progesterone receptor (progesterone), can induce SRC-3 phosphorylation on the identified sites (R. C. Wu and B. W. O’Malley, unpublished results). Consistent with earlier findings (57), these results clearly demonstrated a biochemical degree of ligand, receptor, and coactivator specificity. Thus, we conclude that posttranslational phosphorylation provides a molecular basis that determines the ability of SRCs to distinguish among various NRs and other transcription factors to provide specific responses to distinct upstream signaling pathways (Fig. 2).
III. Cellular Signaling, Coactivator Phosphorylation, and Cancer
The gene for SRC-3 (also known as AIB1 for amplified in breast cancer-1) has been shown to be amplified in 10% of human breast tumors, and the same report indicated that elevated SRC-3 mRNA expression was found for 64% of tumors (58); this was the first indication that SRC-3 may confer some growth advantage. Subsequently, it was shown that SRC-3 overexpression enhances somatic cell growth in prostate cancer cells by activating the AKT signaling pathway (59). In addition, overexpression of SRC-3 in mice alone or in the presence of the Ras oncogene facilitates mammary gland tumorgenesis, and this is associated with increasing serum levels of IGF-I and AKT pathway activity (60, 61). Conversely, knockout of the SRC-3 gene reduced adult body size, due in part to lower circulating IGF-I levels and impairment of IGF-I signaling (36). Collectively, these studies demonstrate that SRC-3 alone is sufficient for the initiation of tumorgenesis, and SRC-3 also can function as a facilitator in the oncogenesis process. It is clearly an oncogene.
However, the levels of SRC-3 protein are not always the sole determinants of SRC-3 pathological function. As summarized above, SRC-3 is an integral and important component of cellular signaling pathways, suggesting a means by which its activity can be subject to other levels of regulation (15, 35, 44, 55). For example, MAPK and IKK signaling both augment SRC-3 activity by phosphorylating SRC-3. As these kinases are important regulators of cellular growth functions, it is evident that aberrant activation of SRC-3 by these kinases could have adverse effects. Therefore, to fully appreciate the function of SRC-3, it is paramount to account for the milieu of cellular signaling in context with SRC-3. However, it is of note that the mechanisms involved in the induction of SRC-3 phosphorylation appear to be complex in nature. First of all, an array of kinases was shown to be able to phosphorylate SRC-3 at least in vitro, and some of the same amino acids of SRC-3 are potential targets of different kinases. Although it broadens and supports the ever-expanding role of SRC-3 in diverse signaling events, it also raises an interesting question as to how different kinases are able to act in concert to phosphorylate and activate SRC-3 in response to a single type of stimulation. Second, it is unclear as to the location where phosphorylation of SRC-3 actually takes place, i.e., in the cytoplasm or in the nucleus where transcription occurs, or in both compartments. Given the fact that the NRs, SRC-3, and the kinases involved are present in both the cytoplasm and nucleus, and are able to shuttle back and forth between these two compartments, this issue proves to be difficult to answer definitively. Lastly, some of the kinases, such as MAPK, were shown to phosphorylate not only SRC-3, but the receptors as well. It is not clear whether phosphorylation of one will affect phosphorylation of the other, or whether there is a sequential order for the phosphorylation of these proteins. Despite all of these unanswered questions, it should not lessen our interest to better understand how and where the signaling cascade originates. Experiments using specific mutants of receptors and SRC-3 should be useful for answering these remaining questions.
As several studies have established SRC-3 as a bona fide oncogene, it also is noteworthy to point out that despite the significant progress made in treating endocrine-sensitive cancers, success is still severely hampered by the development of therapeutic resistance. Therefore, it is imperative to fully understand the underlying mechanisms responsible for the acquisition of this resistance. Perhaps the best studied example of this is the development of resistance to tamoxifen therapy in the progression of human breast cancer. It was proposed, based on work done in the mid-1990s (62, 63) that the relative expression of coactivators and corepressors in a given cell would be critical determinants of the biocharacter (e.g., agonist vs. antagonist activity) of selective receptor modulators such as the selective ER modulator, tamoxifen. Evidence accumulated since that time has supported that hypothesis, including demonstrations that coactivators promote tamoxifen agonist activity (62, 63, 64) and corepressors are important for the antagonistic activity of this widely used drug (65, 66). However, varied cellular signaling pathways (including phosphorylation, acetylation, sumoylation, and methylation) can exert distinct effects on NR and coregulator function and suggest that the intracellular environment also can play a significant role in determining the biological activity of selective receptor modulators such as tamoxifen (reviewed in Ref. 67). Of the recently delineated cellular pathways potentially contributing to this resistance, the cross-talk between NRs and the growth-/survival-promoting signaling pathways seems to hold much promise, particularly as it has become increasingly evident that the interaction between these different pathways occurs at multiple levels, involving both NRs and coactivators (42, 44, 55, 68). Thus, as another layer of complexity has been added to the NR-coactivator picture, the recent data suggest coactivators as viable and critical targets for therapeutic intervention.
As an interesting example, the oncogenic potential of SRC-3 was recently demonstrated to be closely associated with the phosphorylation state of SRC-3 in vitro (55), consistent with the ability of cellular signaling events to modulate SRC-3 activity. In a recent study of breast cancer patients, high SRC-3 (AIB1) expression in patients who had not received adjuvant tamoxifen therapy was associated with improved disease-free survival, especially for those patients who also had low expression levels of HER-2/neu receptor tyrosine kinase (69). However, in patients that had received tamoxifen treatment, those with overexpression of SRC-3 in conjunction with high HER-2/neu expression levels experienced a significantly reduced period of disease-free survival, suggesting that cross-talk between these two pathways is disadvantageous. In a follow-up in vitro study, overexpression of HER-2/neu was associated with 1) increased phosphorylation of SRC-3 in response to stimulating breast cancer cells with heregulin, estradiol or tamoxifen, and 2) increased tamoxifen-induced association of SRC-3 with a gene promoter and elevated tamoxifen stimulation of ER target gene expression (70). These findings add another dimension to our understanding of SRC-3 function and exemplify the importance of cellular signaling as an integral part of coactivator biology. Moreover, these considerations present an interesting scenario whereby aberrant signaling activation of SRC-3 contributes to tumorgenesis and/or development of resistance to hormone therapy in endocrine cancers, suggesting a platform for future experimentation and drug development.
IV. Concluding Remarks
As our understanding of the biology of NRs and coactivators increases, so does our appreciation of the complexity of their actions. Even with recent developments, a number of important questions remain to be addressed, and future efforts should be oriented toward providing a better understanding of a number of these issues.
First of all, the nature of receptor specificity and functional redundancy of each of the SRC members remains an interesting topic to many in the field. Although the spatial expression of SRC family members in different cell types contributes to selectivity, it remains to be determined what other factors contribute to their specificity. Here we hypothesize, as shown in the case of NRs and NF-B, that differential posttranslational modifications of SRC members help to determine their receptor specificity. A detailed examination of all types of modifications, including phosphorylation, acetylation, methylation, ubiquitination, neddylation, and sumoylation, will expand our knowledge and provide new insight into this matter. Second, although SRC-3 was demonstrated to be an oncogene, the exact role of SRC-3 in tumor initiation must be further characterized. Examination of additional signaling upstream of SRC-3 activation and subsequent downstream genetic events elicited by SRC-3 activation deserve equal attention. Finally, because the oncogenic potential of SRC-3 has been linked to its phosphorylation state, it is likely that aberrant phosphorylation and activation of SRC-3 will be linked to advanced stages of cancers. Therefore, whether assessment of a hyperphosphorylation state or phosphorylation of specific amino acids of SRC-3 can be used as a marker for tumor progression is a question begging further investigation, and it is possible that management of disease in the future will encompass not only therapies targeted to NRs, but also to the coregulators that are so critical for mediating their biological activity within the cell.
Acknowledgments
We apologize to our colleagues whose work could not be cited due to page limitation.
Footnotes
This work was supported by a postdoctoral fellowship from the Department of Defense (to R.-C.W.), National Institutes of Health (NIH) Grants DK53002 and DK64083 (to C.L.S.), and NIH Grants HD08818, HD07857, and NIDDK/NURSA-5U19DK62434-02 (to B.W.O.).
First Published Online April 6, 2005
Abbreviations: AF, Activation function; AIB1, amplified in breast cancer 1; CARM1, coactivator-associated arginine methyltransferase-1; CBP, cAMP response element-binding protein (CREB)-binding protein; ER, estrogen receptor; HAT, histone acetyltransferase; IKK, IB kinase; NF-B, nuclear factor-B; NR, nuclear receptor; SRC, steroid receptor coactivator.
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