Tuberin nuclear localization can be regulated by phosphorylation of its carboxyl terminus.

Tuberin, the tuberous sclerosis 2 (TSC2) gene product, has been identified as a tumor suppressor protein genetically implicated in the pathology of tuberous sclerosis and the female-specific lung disease lymphangioleiomyomatosis. Tuberin and its predominant cytoplasmic binding partner hamartin have been shown to complex with a variety of intracellular signaling regulators and affect the processes of protein translation, cellular proliferation, cellular migration, and cellular transcription. In previous studies, we have presented evidence for tuberin binding to the calcium-dependent intracellular signaling protein calmodulin (CaM), overlap of tuberin CaM binding domain with a binding domain for estrogen receptor alpha, and the phosphorylation-associated nuclear localization of tuberin. In the study presented here, we expand our findings on the mechanism of tuberin nuclear localization to show that the CaM-estrogen receptor-alpha binding domain of tuberin can also serve as a tuberin nuclear localization sequence. Furthermore, we identify an Akt/p90 ribosomal S6 kinase-1 phosphorylation site within the carboxyl terminus of tuberin that can regulate tuberin nuclear localization and significantly affect the ability of tuberin to modulate estrogen genomic signaling events. These findings suggest a link between tuberin nuclear localization and a variety of intracellular signaling events that have direct implications with respect to the role of tuberin in the pathology of tuberous sclerosis and lymphangioleiomyomatosis.

Isolation and characterization of axolotl NPDC-1 and its effects on retinoic acid receptor signaling.

Retinoic acid, a key morphogen in early vertebrate development and tissue regeneration, mediates its effects through the binding of receptors that act as ligand-induced transcription factors. These binding events function to recruit an array of transcription co-regulatory proteins to specific gene promoters. One such co-regulatory protein, neuronal proliferation and differentiation control-1 (NPDC-1), is broadly expressed during mammalian development and functions as an in vitro repressor of retinoic acid receptor (RAR)-mediated transcription. To obtain comparative and developmental insights about NPDC-1 function, we cloned the axolotl (Ambystoma mexicanum) orthologue and measured transcript abundances among tissues sampled during the embryonic and juvenile phases of development, and also during spinal cord regeneration. Structurally, the axolotl orthologue of NPDC-1 retained sequence identity to mammalian sequences in all functional domains. Functionally, we observed that axolotl NPDC-1 mRNA expression peaked late in embryogenesis, with highest levels of expression occurring during the time of limb development, a process regulated by retinoic acid signaling. Also similar to what has been observed in mammals, axolotl NPDC-1 directly interacts with axolotl RAR, modulates axolotl RAR DNA binding, and represses cell proliferation and axolotl RAR-mediated gene transcription. These data justify axolotl as a model to further investigate NPDC-1 and its role in regulating retinoic acid signaling.

Cross-talk between tuberin, calmodulin, and estrogen signaling pathways.

Lymphangioleiomyomatosis (LAM) is a rare disease that occurs primarily in women and has been linked to both estrogen-mediated signaling events and mutations associated with the tuberous sclerosis complex 2 gene product tuberin. These two observations fostered the hypothesis that tuberin's impact on estrogen-mediated signaling might be through a direct interaction with the intracellular receptor for estrogen, estrogen receptor alpha (ERalpha). In the study presented here, tuberin was shown to co-immunoprecipitate and directly bind ERalpha through a domain localized within the carboxyl 73 amino acids of tuberin. This domain had previously been shown to serve as a binding domain for the intracellular calcium signaling molecule calmodulin (CaM). Competition binding studies identified a potential competitive relationship for binding of tuberin by ERalpha and CaM. Additionally, tuberin-ERalpha interactions were found to be modulated by the presence of tuberin's predominant intracellular binding partner hamartin, suggesting that tuberin-hamartin interactions negatively impact the ability of tuberin to modulate ERalpha-mediated gene transcription events. Cumulatively, data presented here support the hypothesis that interactions between tuberin, ERalpha, and CaM may play a critical role in the pathology of LAM disease.

Use of a modified yeast one-hybrid screen to identify BAF60a interactions with the Vitamin D receptor heterodimer.

A modified yeast one-hybrid screen was used to isolate proteins capable of interacting with the Vitamin D receptor (VDR) heterodimer complex while driving expression from a repressor Vitamin D response element (VDRE). Four of nine independent colonies recovered in the screen coded for full-length BAF60a, a component of the mammalian SWI/SNF complex. Deletion studies in yeast were unable to localize a unique region of BAF60a responsible for interaction with the heterodimer complex, as only the full-length protein would support reporter gene expression. Pull-down analyses revealed that BAF60a displayed strong interactions with either the unliganded or liganded heterodimer complex, but neither individual receptor component alone. Transient transfection analysis in opossum kidney (OK) cells indicated that BAF60a decreased basal transcriptional activity from the negative VDRE, but had no effect on hormone-induced repression. Transcriptional activity from an enhancer VDRE also exhibited decreased basal transcriptional activity, but also augmented hormone-dependent enhancer activity, resulting in an overall increased sensitivity to hormone. In summary, BAF60a has been identified as a factor that specifically interacts with the VDR heterodimer complex using a modified yeast one-hybrid selection strategy. This suggests that BAF60a may be a link between mammalian SWI/SNF-like chromatin remodeling complexes and the VDR heterodimer.

A high-throughput mammalian cell-based transient transfection assay.

In eukaryotic organisms gene expression is regulated through a variety of upstream transacting factors (transcription factors) whose primary function appears to be the targeting of coregulatory protein complexes, which interact with basal transcription machinery to define the relative rate of transcription for a specific gene. Understanding the regulatory forces mediating transcription factor activity has been the focus of both academic and industrial research efforts over the past 15 yr, and in this time frame a variety of methodologies have been developed for reconstituting and assaying transcription factor activities in mammalian cell environments. Presented here is a high-throughput version of one of these methodologies that can be readily adapted to the screening of a variety of transcription factors. This technology utilizes co-transfection of mammalian expression and luciferase reporter plasmids to reconstitute transcription events in a mammalian host cell. Included is a detailed protocol for the use of a 96-well plate format, along with a variety of cost-effective measures that can be implemented to facilitate the use of the technology in the average low budget academic laboratory.

Synthesis and functional analysis of novel bivalent estrogens.

The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERalpha) and beta (ERbeta), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERalpha)-mediated transcription events with efficacy and potency equal to or greater than that of ERalpha's cognate ligand, 17beta-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.

Abnormal growth of smooth muscle-like cells in lymphangioleiomyomatosis: Role for tumor suppressor TSC2.

The TSC1 and TSC2 proteins, which function as a TSC1/TSC2 tumor suppressor complex, are associated with lymphangioleiomyomatosis (LAM), a genetic disorder characterized by the abnormal growth of smooth muscle-like cells in the lungs. The precise molecular mechanisms that modulate LAM cell growth remain unknown. We demonstrate that TSC2 regulates LAM cell growth. Cells dissociated from LAM nodules from the lungs of five different patients with LAM have constitutively activated S6K1, hyperphosphorylated ribosomal protein S6, activated Erk, and increased DNA synthesis compared with normal cells from the same patients. These effects were augmented by PDGF stimulation. Akt activity was unchanged in LAM cells. Rapamycin, a specific S6K1 inhibitor, abolished increased LAM cell growth. The full-length TSC2 was necessary for inhibition of S6 hyperphosphorylation and DNA synthesis in LAM cells, as demonstrated by co-microinjection of the C-terminus, which contains the GTPase activating protein homology domain, and the N-terminus, which binds TSC1. Our data demonstrate that increased LAM cell growth is associated with constitutive S6K1 activation, which is extinguishable by TSC2 expression. Loss of TSC2 GAP activity or disruption of the TSC1/TSC2 complex dysregulates S6K1 activation, which leads to abnormal cell proliferation associated with LAM disease.

NPDC-1, a novel regulator of neuronal proliferation, is degraded by the ubiquitin/proteasome system through a PEST degradation motif.

Neural proliferation and differentiation control protein-1 (NPDC-1) is a protein expressed primarily in brain and lung and whose expression can be correlated with the regulation of cellular proliferation and differentiation. Embryonic differentiation in brain and lung has classically been linked to retinoid signaling, and we have recently characterized NPDC-1 as a regulator of retinoic acid-mediated events. Regulators of differentiation and development are themselves highly regulated and usually through multiple mechanisms. One such mechanism, protein degradation via the ubiquitin/proteasome degradation pathway, has been linked to the expression of a number of proteins involved in control of proliferation or differentiation, including cyclin D1 and E2F-1. The data presented here demonstrate that NPDC-1 is likewise degraded by the ubiquitin/proteasome system. MG-132, a proteasome inhibitor, stabilized the expression of NPDC-1 and allowed detection of ubiquitinated NPDC-1 in vivo. A PEST motif (rich in proline, glutamine, serine, and threonine) located in the carboxyl terminus of NPDC-1 was shown to target the protein for degradation. Deletion of the PEST motif increased NPDC-1 protein stability and NPDC-1 inhibitory effect on retinoic acid-mediated transcription. NPDC-1 was phosphorylated by several kinases, including extracellular signal-regulated kinase. Phosphorylation of NPDC-1 increased the in vitro rate of NPDC-1 ubiquitination. The MEK inhibitor, PD-98059, an inhibitor of extracellular signal-regulated activation, also inhibited the formation of ubiquitinated NPDC-1 in vivo. Together these results suggest that retinoic acid signaling can be modulated by the presence of NPDC-1 and that the protein level and activity of NPDC-1 can be regulated by phosphorylation-mediated proteasomal degradation.

A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation.

BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription. RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner. CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

A calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis.

Mutations in the tuberous sclerosis 2 (TSC2) gene product have been genetically linked to the pathology of both tuberous sclerosis (TSC) and the gender-specific lung disease, lymphangioleiomyomatosis (LAM). Both diseases are classified as disorders of cellular migration, proliferation, and differentiation. Earlier studies from our laboratory (1) linked TSC2 with steroid/nuclear receptor signaling. Studies presented here provide evidence for calmodulin (CaM) signaling in the propagation of this TSC2 activity. Far Western screening of a lambda phage human brain cDNA library to identify interacting proteins for the TSC2 gene product (tuberin) yielded multiple clones encoding human CaM. Direct binding with 32P-labeled tuberin demonstrated Ca2+-dependent binding to CaM-Sepharose which was lost upon deletion of the C-terminal 72 residues. The sequence (1740)WIARLRHIKRLRQRIC(1755) was identified as one capable of forming a basic amphipathic helix indicative of CaM binding domains in known calmodulin binding proteins. Studies with a synthetic peptide of this sequence demonstrated very tight Ca2+-dependent binding to CaM as judged by tryptophan fluorescence perturbation studies and phosphodiesterase activation by CaM. Deletion mutagenesis studies further suggested that this CaM binding domain is required for tuberin modulation of steroid receptor function and that mutations in this region may be involved in the pathology of TSC and LAM.

Pseudolaric acid analogs as a new class of peroxisome proliferator-activated receptor agonists.

Extracts of the root and trunk barks of the Chinese tree Pseudolarix kaempferi, which contain pseudolaric acids, are used in Chinese medicine for treatment of fungal infections. Pseudolaric acid B (PLAB) is the major constituent that exhibits anti-fungal activity. The nuclear peroxisome proliferator-activator receptors (PPAR) were proposed as a cellular target for the action of PLAB and its analogs. PLAB and two derivatives were tested for the activation of PPAR isoforms in two mammalian cell lines. CV-1 and H4IIEC3 cells were transfected with phorbol ester response element or PPAR response element reporter constructs, and CV-1 cells were co-transfected with the individual PPAR isoform expression plasmids. PLAB showed similar concentration-dependent effects for the activation of PPAR alpha, gamma and delta isoforms in CV-1 and H4IIEC3 cells. O-Deacetylation of PLAB (PLAC) or esterification of the free carboxy group of PLAB with beta-D-O-glucopyranoside (PLAG) markedly reduced or abolished the activation of these PPAR isoforms. In H4IIEC3 cells, PLAB increased the activation of endogenous PPARalpha and the phospholipase C signaling pathway; and stimulated peroxisomal fatty acyl-CoA oxidase activity. These effects of PLAB on the activation of endogenous PPARalpha and phospholipase C-dependent pathway were blocked by staurosporine. These results suggest that the action of PLAB on PPARalpha in H4IIEC3 cells is mediated by a protein kinase C dependent phosphorylation. Based upon these findings, the chemical class of biologically active diterpene acids related to PLAB may have promise for the treatment of metabolic and pathophysiological disorders that are regulated by these nuclear receptor isoforms.

Estrogen-induced smooth muscle cell growth is regulated by tuberin and associated with altered activation of platelet-derived growth factor receptor-beta and ERK-1/2.

The mechanisms that regulate the diverse responses to estrogen (E2) are unknown. Loss of function of the tuberous sclerosis 2 gene (TSC2), a tumor suppressor gene, has been associated with a growth-promoting effect of E2. We hypothesized that tuberin, the protein product of TSC2, binds to estrogen receptors (ER) and regulates the growth effect of E2. An in vivo association between full-length tuberin and ERalpha was observed in HEK 293 cells and ELT-3 smooth muscle cells. In contrast, poor association was observed between tuberin and ERbeta. Complex formation with ERalpha and the C-terminal end of tuberin was also observed in vivo and in vitro, indicating that binding between ERalpha and tuberin occurs at the C-terminal end of the tuberin molecule. We examined the effect of tuberin expression in ELT-3 smooth muscle cells on the growth response to E2. The growth-promoting effect of E2 in tuberin-null ELT-3 smooth muscle cells was ERalpha-specific, associated with up-regulation and activation of platelet-derived growth factor receptor-beta (PDGFRbeta) and activation of the signaling intermediate, extracellular signal-regulated kinase-1/-2 (ERK-1/2). In contrast, the expression of tuberin in ELT-3 smooth muscle cells resulted in significant abrogation of E2-stimulated growth. In parallel with this observation, the expression of tuberin in ELT-3 cells also resulted in significant inhibition of PDGFRbeta and ERK-1/2 activation in response to E2. These results demonstrate that tuberin binds specifically to ERalpha and inhibits E2-induced proliferation of ELT-3 cells. Furthermore, the opposing effects of tuberin on estrogen-induced activation of PDGFRbeta and ERK-1/-2 suggest a pivotal role for tuberin in directing the signaling events that dictate the growth response to E2.