HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/9/1202 04-3142fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by York, B. Articles by Noonan, D. J. Search for Related Content PubMed PubMed Citation Articles by York, B. Articles by Noonan, D. J.

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

Brian York^*, Dingyuan Lou^*, Reynold A. Panettieri, Jr, Vera P. Krymskaya, Thomas C. Vanaman^* and Daniel J. Noonan^*,1

^* Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA; and
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

¹ Correspondence: Department of Molecular and Cellular Biochemistry, University of Kentucky, 800 Rose St., Lexington, KY 40536-0298, USA. E-mail: dnoonan{at}pop.uky.edu

SPECIFIC AIMS

Lymphangioleiomyomatosis (LAM) is a devastating but rare female-specific disease. LAM in many ways is a subset of the more prevalent disease tuberous sclerosis (TSC). TSC, in spite of being a genetic disease (is associated with mutations in the TSC1 or TSC2 gene), has turned out to be an extremely complicated disease, associated with a variety of tissues and a variety of intracellular signaling pathways. The beauty of LAM disease is that it retains the genetic basis of TSC (is associated with mutations in the TSC2 gene), but occurs almost exclusively in women and manifests itself primarily as a proliferation of smooth muscle cells of the lung. Therefore, rather than dealing with 5 or more different types of tissues and untold number of signaling pathways, LAM permits a focus on the consequences of mutations in the TSC2 gene through the obligate estrogen signaling pathways and the obligate smooth muscle cell type. Our published work in this field has demonstrated that the TSC2 gene product, tuberin, could modulate both estrogen genomic and nongenomic signaling pathways. The specific aims of this study were to identify how tuberin might be modulating estrogen genomic and nongenomic signaling and whether this modulation could be associated with the pathology of LAM.

PRINCIPAL FINDINGS

1. Tuberin and ER participate in direct protein-protein interactions
Data published from our laboratory identified a novel association between tuberin and ER that localized binding of ER to the carboxyl 672 amino acids of tuberin. To assess the in vivo relevance of these in vitro associations, coimmunoprecipitations were performed. These studies clearly demonstrated that endogenously expressed tuberin and ER associate. A series of GST tagged deletion mutants were developed of tuberin’s carboxyl terminus and tested for their ability to directly bind recombinantly purified human ER. Data from this study further localized ER binding to the carboxyl 73 amino acids of tuberin, a domain our laboratory previously defined as one capable of binding the intracellular calcium signaling regulatory protein calmodulin (CaM).

Tuberin’s CaM binding domain has been shown to contain one of the most highly expressed LAM and TSC-associated mutations, a six amino acid in-frame deletion (1746-1752). To assess potential overlap in tuberin binding domains for ER and CaM, in vitro ER pull-down analyses were performed using GST-tagged recombinant tuberin proteins containing either a complete deletion of the CaM binding domain (TSC2CaM) or the six amino acid in-frame deletion within the CaM binding domain (mCBD). The TSC2CaM and mCBD proteins failed to bind ER, suggesting tuberin’s binding domain for ER overlaps with its binding domain for CaM.

2. ER is capable of disrupting tuberin-CaM interactions
The observation that ER binding localized to a similar domain as CaM binding prompted the investigation of ER-CaM competition for binding to tuberin. Competition binding analyses demonstrated ER bound tuberin in a calcium-independent manner and that this binding could not be disrupted upon addition of excess CaM/Ca²⁺. Additional competition analyses performed revealed that CaM-ER interactions could not be altered upon addition of excess tuberin. However, competition binding studies performed with ER prebound to tuberin’s C672 amino acids, followed by the addition of excess CaM, demonstrated that ER is capable of disrupting CaM’s ability to bind tuberin. These data suggest that ER’s binding affinity for tuberin may be marginally higher than CaM’s but that selective binding is most probably regulated by some yet to be defined mechanism.

3. Hamartin binding to tuberin effectively competes with ER but not CaM
Tuberin and hamartin have been genetically linked to the pathogenesis of TSC and shown to complex in a phosphorylation-specific manner, an event that has been demonstrated to play an important role in tuberin’s ability to modulate intracellular signaling pathways and tuberin’s ability to localize to the nucleus. We therefore investigated the role of tuberin-hamartin complexes in the binding of ER and CaM. Tuberin-hamartin interactions were demonstrated by immunoprecipitation analyses to inhibit ER’s ability to bind tuberin. Conversely, the tuberin-hamartin complex failed to inhibit CaM binding, suggesting the presence of a mechanism that regulates the accessibility and/or availability of binding partners in these complexes of proteins.

4. Tuberin inhibits ER-DNA interactions
ER genomic functions are centered on the receptor’s ability to bind estradiol, dimerize and to bind a DNA consensus sequence (ERE) in the promoter regions of genes housing this response element. Previous data from our laboratory identified tuberin’s ability to translocate to the nucleus and modulate steroid mediated transcription events. To investigate the functional consequences of tuberin-ER genomic interactions, we performed gel mobility shift assays (GMSAs) using an ERE-containing oligonucleotide sequence and recombinantly purified tuberin, CaM, and ER. Incorporation of the GST-TSC2-C672 protein in the GMSA reaction resulted in the complete loss of ER-specific complex formation (Fig. 1 ). Additional experiments helped to confirm tuberin’s direct involvement in this event and the importance of the carboxyl 73 amino acids of tuberin in mediating the disruption. Furthermore, as with the tuberin-ER binding studies, tuberin constructs containing mutations within the CaM binding domain or lacking the domain altogether failed to inhibit ER complex formation.

View larger version (37K): [in this window] [in a new window]   Figure 1. Oligonucleotides corresponding to an ERE consensus sequence were annealed, end-labeled with 32P-dCTP, and used to analyze tuberin’s effects on ER binding in gel mobility shift assays (GMSA). A) Labeled ERE was incubated with HepG2 nuclear extract (25 µg) and various recombinant proteins in the presence of 10 nM 17ß-estradiol. DNA:protein complexes were resolved on nondenaturing PAGE gels and analyzed by autoradiography. B) To test the dose-dependent effects of GST-TSC2-C672 on ER-mediated complex formation, GMSAs were repeated as described above but using increasing concentrations of GST-TSC2-C672 (1, 2.5, 5, and 10 µg, lanes 4–7). C) Recombinant GST-TSC2-C73, a 6 amino acid in-frame deletion of tuberin’s CaM binding domain (GST-TSC2-mCBD), and a 672 amino acid carboxyl terminal construct of tuberin lacking the last 73 amino acids (GST-TSC2CaM) (10 µg each) were analyzed in GMSAs for their ability to alter ER-mediated complex formation. ER, ERE, and free probe complexes are indicted by arrows.

5. Tuberin repression of ER-mediated gene transcription is rescued by hamartin
To further investigate the functional significance of tuberin-ER interactions, the expression of tuberin and hamartin was examined in ER-specific in vitro transcription assays. Tuberin expression was shown to repress ER-mediated gene transcription by 25 percent. Given the fact that only a small percentage of tuberin transiently localizes to the nucleus, this repression imposed by tuberin presents a significant mechanism by which tuberin may selectively regulate gene transcription events to regulate cell growth and proliferation. Hamartin, the well-documented cytoplasmic binding partner of tuberin, has been extensively characterized for its ability to regulate a variety of tuberin-associated intracellular signaling pathways. Here, we demonstrate that overexpression of hamartin along with tuberin rescues tuberin’s repression of ER-mediated transcription events. These data were further abrogated by the observation that a tuberin-hamartin complex inhibits tuberin-ER interactions. Collectively, these data provide the first evidence of how loss of a functional tuberin may relate to aberrations in estrogen signaling pathways in patients with LAM disease.

CONCLUSIONS AND SIGNIFICANCE

Our studies establish the following novel contributions:

1) We have characterized the interaction between tuberin-ER and identified the binding domain for ER on tuberin.

2) Competition analyses suggest ER binding to tuberin is preferential when compared with CaM; ER can bind tuberin in a calcium-independent manner.

3) Our studies provide a novel mechanism for tuberin’s ability to modulate ER transcription events, which was demonstrated to be facilitated by tuberin’s direct interaction with ER and regulated by tuberin-hamartin interactions.

The data presented here establish a functional relationship between tuberin, ER, hamartin, and CaM. These studies, along with published data demonstrating the functional interaction of CaM and ER and the colocalization of tuberin, CaM, and ER to cytoplasmic and nuclear compartments, suggest that tuberin and CaM may act coordinately to regulate ER genomic and nongenomic signaling events. They also suggest that mutations within the TSC2 gene that lead to the disruption of tuberin-hamartin complexes or the CaM/ER binding domain appear to abolish tuberin’s ability to regulate ER-mediated DNA complex formation and gene transcription. The observation that in-frame deletions in this domain and/or truncating mutations upstream of the domain predominate in LAM disease would suggest a mechanism wherein tuberin’s effect on differentiation and proliferation pathways in smooth muscle cells of the lung may be mediated through its ability to modulate ER-mediated gene expression events.

Further investigation is needed to fully understand the intricate mechanisms that regulate tuberin-ER-CaM interactions. Though there is still much to be discovered with respect to why LAM disease occurs almost exclusively in women, the studies presented here open a new window into a viable molecular explanation for this idiosyncrasy.

View larger version (36K): [in this window] [in a new window]   Figure 2. Model depicting the genomic and nongenomic signal pathways in which tuberin functions. Nuclear tuberin may function to regulate ER-mediated gene transcription by direct association with ER or by sequestering nuclear available CaM needed for efficient ER genomic activity. Alternatively, tuberin may also function to regulate ER’s nongenomic signaling events, which are capable of modulating global transcription mediated by ERK as well as translational control mediated by mTOR.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3142fje; doi: 10.1096/fj.04-3142fje

This article has been cited by other articles:

				K. H. Lu, W. Wu, B. Dave, B. M. Slomovitz, T. W. Burke, M. F. Munsell, R. R. Broaddus, and C. L. Walker Loss of Tuberous Sclerosis Complex-2 Function and Activation of Mammalian Target of Rapamycin Signaling in Endometrial Carcinoma Clin. Cancer Res., May 1, 2008; 14(9): 2543 - 2550. [Abstract] [Full Text] [PDF]

				S. C. Juvet, F. X. McCormack, D. J. Kwiatkowski, and G. P. Downey Molecular Pathogenesis of Lymphangioleiomyomatosis: Lessons Learned from Orphans Am. J. Respir. Cell Mol. Biol., April 1, 2007; 36(4): 398 - 408. [Abstract] [Full Text] [PDF]

				B. York, D. Lou, and D. J. Noonan Tuberin Nuclear Localization Can Be Regulated by Phosphorylation of Its Carboxyl Terminus Mol. Cancer Res., November 1, 2006; 4(11): 885 - 897. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/9/1202 04-3142fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by York, B. Articles by Noonan, D. J. Search for Related Content PubMed PubMed Citation Articles by York, B. Articles by Noonan, D. J.