The three homologous members of the p160 SRC family (SRC-1, SRC-2 and SRC-3) mediate the transcriptional functions of nuclear receptors and other transcription factors, and are the most studied of all transcriptional coactivators. Recent work has indicated that the SRC genes are subject to amplification and overexpression in various human cancers. Some of the molecular mechanisms responsible for SRC overexpression along with the mechanisms by which SRCs promote breast and prostate cancer cell proliferation and survival have been identified, as have the specific contributions of individual SRC family members in spontaneous breast and prostate carcinogenesis in genetically manipulated mouse models. These studies have identified new challenges for cancer research and therapy.

Although several lines of evidence have indicated that the activity of SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 could be regulated by phosphorylation, an important question remained as to how different signaling pathways can act through limiting concentrations of the same SRC-3 molecule to exert different physiological functions. Herein, we report the successful identification of six functional in vivo SRC-3 phosphorylation sites. Interestingly, all phosphorylation sites are required for coactivation of estrogen and androgen receptors, but not all sites are required for coactivation of NF-kappaB. Different combinations of site-specific phosphorylations of SRC-3 are required for induction of IL-6 gene expression by TNF-alpha as compared to oncogenic transformation of MEFs. Mechanisms of pathway selectivity involve protein-protein interactions of differentially phosphorylated SRC-3 with downstream transcriptional activators and coactivators. Our results uncovered an additional level of transcriptional regulation whereby specific modulations of SRC-3 phosphorylation allow this coactivator to function as a regulatable integrator for diverse signaling pathways in cells.

In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3. In this report, we describe the biochemical purification of SRC-1 and SRC-3 protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-3, but not SRC-1, with the IB kinase (IKK). IKK is known to be responsible for the degradation of IB and the subsequent activation of NF-B. Since NF-B plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-3-IKK complex. We demonstrated that SRC-3 was able to enhance NF-B-mediated gene expression in concert with IKK. In addition, we showed that SRC-3 was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-3 phosphorylation in vivo and translocation of SRC-3 from cytoplasm to nucleus in response to tumor necrosis factor alpha occurred in cells, suggesting control of subcellular localization of SRC-3 by phosphorylation. Finally, the hypothesis that SRC-3 is involved in NF-B-mediated gene expression is further supported by the reduced expression of interferon regulatory factor 1, a well-known NF-B target gene, in the spleens of SRC-3 null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-3 to be a regulated event that plays an important role but also substantiate the role of SRC-3 in multiple signaling pathways.The nuclear receptor (NR) superfamily is a large class of ligand-dependent transcription factors that play pivotal roles in a wide spectrum of biological processes such as development, reproduction, and homeostasis (32, 51). In the past few years, many nuclear receptor coactivators have been identified, including the steroid receptor coactivator (SRC) family. Members of the SRC family, which include SRC-1, SRC-2/GRIP1/ TIF2, and SRC-3/ACTR/AIB-1/pCIP/RAC3/TRAM-1, interact with nuclear receptors and enhance their transactivation in a ligand-dependent manner (3,11,21,25,27,29,38,49,50,52). Two members of the SRC family, SRC-1 and SRC-3, are also known to contain histone acetyltransferase activity (11,47), and all three members contain an intrinsic transcriptional activation function when tethered to the GAL4 DNA-binding domain (29, 37, 52). Additionally, it has recently been reported that SRC-1 and SRC-3 are phosphoproteins (18,45) and that the activity of SRC-3 is attenuated by acetylation (12). However, it is unclear how these posttranslational modifications might be regulated and what their actual roles are.Nuclear factor B (NF-B) is a family of signal-inducible transcription factors whose members, including p50, p52, p65 (RelA), c-Rel, and RelB, play an essential role in the regulation of genes involved in inflammatory responses and cell survival (4, 5, 39). All ...

SRC-3/AIB1 is an important growth coactivator whose activity should be tightly regulated since excess activation results in oncogenesis. Herein, we provide evidence that coordinated phosphorylation-dependent ubiquitination regulates SRC-3 coactivator activation and transcriptional specificity. We discovered a critical "actron/degron" element in SRC-3 that is required for this phosphorylation-dependent ubiquitination event and identified GSK3 and SCF(Fbw7alpha) as the respective responsible kinase and E3 ubiquitin ligase. Interestingly, despite that SCF(Fbw7alpha) enhances ubiquitination and promotes eventual transcription-coupled degradation of SRC-3 in a phosphorylation- and Fbw7alpha dosage-dependent manner, our results also uncovered a nonproteolytic "activation" code for SRC-3 ubiquitination induced by Fbw7alpha. We propose that ubiquitination of SRC-3 is a phospho-mediated biphasic event and that a transition from multi-(mono)ubiquitination (SRC-3 activation) to long-chain polyubiquitination (SRC-3 degradation) is processive during the transcriptional coactivation of select transcription factors and can serve as a "transcriptional time clock" to control both the activation and the functional lifetime of coactivators.

Autism, a pervasive neurodevelopmental disorder manifested by deficits in social behavior and interpersonal communication, and by stereotyped, repetitive behaviors, is inexplicably biased towards males by a ratio of ∼4∶1, with no clear understanding of whether or how the sex hormones may play a role in autism susceptibility. Here, we show that male and female hormones differentially regulate the expression of a novel autism candidate gene, retinoic acid-related orphan receptor-alpha (RORA) in a neuronal cell line, SH-SY5Y. In addition, we demonstrate that RORA transcriptionally regulates aromatase, an enzyme that converts testosterone to estrogen. We further show that aromatase protein is significantly reduced in the frontal cortex of autistic subjects relative to sex- and age-matched controls, and is strongly correlated with RORA protein levels in the brain. These results indicate that RORA has the potential to be under both negative and positive feedback regulation by male and female hormones, respectively, through one of its transcriptional targets, aromatase, and further suggest a mechanism for introducing sex bias in autism.

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.

The white adipocyte is at the center of dysfunctional regulatory pathways in various pathophysiological processes, including obesity, diabetes, inflammation, and cancer. Here, we show that the oncogenic steroid receptor coactivator-3 (SRC-3) is a critical regulator of white adipocyte development. Indeed, in SRC-3 ؊/؊ mouse embryonic fibroblasts, adipocyte differentiation was severely impaired, and reexpression of SRC-3 was able to restore it. The early stages of adipocyte differentiation are accompanied by an increase in nuclear levels of SRC-3, which accumulates to high levels specifically in the nucleus of differentiated fat cells. Moreover, SRC-3 ؊/؊ animals showed reduced body weight and adipose tissue mass with a significant decrease of the expression of peroxisome proliferator-activated receptor ␥2 (PPAR␥2), a master gene required for adipogenesis. At the molecular level, SRC-3 acts synergistically with the transcription factor CAAT͞enhancer-binding protein to control the gene expression of PPAR␥2. Collectively, these data suggest a crucial role for SRC-3 as an integrator of the complex transcriptional network controlling adipogenesis.adipogenesis ͉ peroxisome proliferator-activated receptor (PPAR) ͉ transcription ͉ coregulators ͉ metabolism

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