Calmodulin Regulates the Transcriptional Activity of Estrogen Receptors

Abstract

The steroid hormone estrogen elicits biological effects in cells by binding to and activating the estrogen receptor (ER). Estrogen binding induces a conformational change in the receptor, inducing nuclear translocation and transcriptional activation of ER. The ubiquitous Ca 2؉ -binding protein calmodulin has been shown to interact directly with ER and enhance its stability. To further elucidate the functional sequelae of the association between calmodulin and ER, we examined the effect on ER transcriptional activation of specifically inhibiting calmodulin. The cell-permeable calmodulin antagonist CGS9343B prevented estrogen-induced transcriptional activation by ER, without altering basal transcription. The inhibition was dose-dependent and independent of the time of estrogen stimulation. To validate these findings, calmodulin function was also neutralized by targeted expression of a specific inhibitor peptide. By inserting localization signals, the inhibitor peptide was selectively targeted to different subcellular domains. Inactivation of calmodulin function in the nucleus virtually eliminated estrogen-stimulated ER transcriptional activation. By contrast, when membrane calmodulin was specifically neutralized, estrogen-stimulated transcriptional activation by ER was only slightly attenuated. Importantly, the inhibitor peptides did not significantly reduce the amount of ER in the cells. Together, these data demonstrate that calmodulin is a fundamental component of ER transcriptional activation.The classic steroid hormone estrogen promotes the proliferation of both normal and malignant breast epithelial cells and shortens the cell cycle. Estrogen mediates its biological effects in cells through the estrogen receptor (ER), 1 a member of the nuclear receptor family of ligand-dependent transcription factors (reviewed in Refs. 1 and 2). Analogous to other steroid hormone receptors, ER is an intracellular transcription factor composed of six domains. Estrogen binding to the C-terminal hormone-binding domain induces conformational changes in ER, thereby promoting its dimerization and nuclear localization. The DNA-binding domain of the activated ER binds to DNA sequences, termed estrogen response elements, found in the regulatory regions of target genes. Several factors, including coactivators, corepressors, and integrator proteins, are important in ER-mediated transcription (reviewed in Refs. 3 and 4). It is becoming apparent that transcriptional regulation requires the recruitment by ER of multiple, distinct proteins that cooperate to achieve the required response (3). These factors can alter the magnitude of cellular responses to estrogen.There are yet additional factors that modulate ER function. For example, ER interacts with members of the heat-shock protein family (1), and dissociation of heat-shock protein seems to be necessary for ER to activate transcription. One of the major roles of ligand binding is to change the nature of proteinprotein interactions between steroid receptors and other proteins (2). Convers...

Multiple proteins mediate IQGAP1-stimulated cell migration

“…If this hypothesis is correct, blocking the interaction of calmodulin with IQGAP1 at cell membranes should enhance the interaction of IQGAP1 with Cdc42 and augment IQGAP1-stimulated cell migration. Consistent with this model, a calmodulin-binding peptide that specifically and selectively inhibits calmodulin function at membranes [25] significantly increased cell migration promoted by IQGAP1. In contrast, when the peptide was targeted to the nucleus, which inhibits nuclear calmodulin function [25], IQGAP1-stimulated cell migration was not altered.…”

“…Consistent with this model, a calmodulin-binding peptide that specifically and selectively inhibits calmodulin function at membranes [25] significantly increased cell migration promoted by IQGAP1. In contrast, when the peptide was targeted to the nucleus, which inhibits nuclear calmodulin function [25], IQGAP1-stimulated cell migration was not altered. Collectively these data reveal that Ca 2+ /calmodulin signalling provides an additional level to fine tune the ability of IQGAP1 to increase cell migration.…”

“…A synthetic gene that encodes the calmodulin-binding sequence of myosin-light chain kinase [24,25] (kindly provided by Marcia Kaetzel and John Dedman, University of Cincinnati) was used. The construct contains four tandem calmodulin-binding peptide (CaMBP) repeats.…”

“…The CaMBP was tagged with a yellow-green variant of enhanced green fluorescent protein (EYFP) and inserted into plasmids that selectively target it to two subcellular domains, the nucleus or the plasma membrane [25]. The correct subcellular localization of these CaMBPs has been verified by confocal microscopy [25]. The EYFP-containing plasmid was used as control.…”

“…A peptide corresponding to the calmodulin-binding domain of myosin-light chain kinase specifically inhibits calmodulin function in cells [24,25]. By selectively targeting this construct to the nucleus or plasma membrane, we are able to inhibit calmodulin function in discrete subcellular domains [25]. To determine the effect on migration stimulated by IQGAP1, we used cells that stably overexpress IQGAP1.…”