Abstract
Cytosolic and nuclear Ca(2+) have been shown to differentially regulate transcription. However, the impact of spatially distinct Ca(2+) signals on mitogen-activated protein kinase-mediated gene expression remains unknown. Here we investigated the role of nuclear and cytosolic Ca(2+) signals in epidermal growth factor (EGF)-induced transactivation of the ternary complex factor Elk-1 using a GAL4-Elk-1 construct. EGF increased Ca(2+) in both the nucleus and cytosol of HepG2 or 293 cells. Pretreatment with the intracellular Ca(2+) chelator bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid significantly reduced EGF-induced transactivation of Elk-1, indicating that EGF-stimulated Elk-1 transcriptional activity is dependent on intracellular Ca(2+). To determine the relative contribution of nuclear and cytosolic Ca(2+) signals during EGF-mediated Elk-1 transactivation, Ca(2+) signals in either compartment were selectively impaired by targeted expression of the Ca(2+)-binding protein parvalbumin to either the nucleus or cytosol. Suppression of nuclear but not cytosolic Ca(2+) signals inhibited EGF-induced transactivation of Elk-1. However, suppression of nuclear Ca(2+) signals did not affect the ability of ERK either to become phosphorylated or to undergo translocation to the nucleus in response to EGF. Elk-1 phosphorylation and nuclear localization following EGF stimulation were also unaffected by suppressing nuclear Ca(2+) signals. These results suggest that nuclear Ca(2+) is required for EGF-mediated transcriptional activation of Elk-1 and that phosphorylation of Elk-1 alone is not sufficient to induce its transcriptional activation in response to EGF. Thus, subcellular targeting of parvalbumin reveals a distinct role for nuclear Ca(2+) signals in mitogen-activated protein kinase-mediated gene transcription.
Highlights
Cytosolic and nuclear Ca2؉ have been shown to differentially regulate transcription
These results suggest that nuclear Ca2؉ is required for epidermal growth factor (EGF)-mediated transcriptional activation of Elk-1 and that phosphorylation of Elk-1 alone is not sufficient to induce its transcriptional activation in response to EGF
Following phosphorylation by the mitogen-activated protein kinases (MAPK) kinases (MKKs), the extracellular signal-regulated kinases 1 and 2 (ERKs), c-Jun amino-terminal kinases, and p38 MAPKs each translocate to the nucleus [25] where they transactivate various transcription factors by serine and threonine phosphorylation [23, 24]
Summary
Can2ϩuc, nuclear free calcium; Cac2yϩt, cytosolic free calcium; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; EGF, epidermal growth factor; ERK, extracellular regulated kinase; PV, parvalbumin; NES, nuclear exclusion signal; NLS, nuclear localization signal; GFP, green fluorescent protein; AM, acetoxymethyl; HA, hemagglutinin; BAPTA, bis(2-aminophenyl)ethyleneglycol-N,N,NЈ,NЈ-tetraacetic acid. In contrast to the positive signaling role of Ca2ϩ in growth factor-mediated Elk-1 activation, Ca2ϩ has been proposed to play a negative role in. Growth factor-mediated changes in Ca2ϩ can lead to the regulation of Elk-1 via multiple pathways in both positive and negative manners. The distinct actions of Ca2ϩ when localized to either the nucleus or cytosol might provide some explanation for the complexity of Elk-1 regulation in growth factor signal transduction. In this study we have investigated the relative contribution of both Cac2yϩt and Can2ϩuc to the regulation of MAPK-mediated gene expression in response to stimulation with EGF by selective targeting of the Ca2ϩ chelator protein parvalbumin (PV). To either the cytosol or the nucleus Using this novel approach, we have demonstrated that Can2ϩuc but not Cac2yϩt is required for Elk-1 transcriptional activation in response to EGF stimulation
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have