Pusl Thomas, Wu Julie J, Zimmerman Tracy L, Zhang Lei, Ehrlich Barbara E, Berchtold Martin W, Hoek Joannes B, Karpen Saul J, Nathanson Michael H, Bennett Anton M
Department of Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
J Biol Chem. 2002 Jul 26;277(30):27517-27. doi: 10.1074/jbc.M203002200. Epub 2002 Apr 23.
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.
细胞溶质和细胞核中的Ca(2+)已被证明对转录具有不同的调节作用。然而，空间上不同的Ca(2+)信号对丝裂原活化蛋白激酶介导的基因表达的影响仍然未知。在这里，我们使用GAL4-Elk-1构建体研究了细胞核和细胞溶质Ca(2+)信号在表皮生长因子（EGF）诱导的三元复合因子Elk-1反式激活中的作用。EGF增加了HepG2或293细胞的细胞核和细胞溶质中的Ca(2+)。用细胞内Ca(2+)螯合剂双（2-氨基苯基）乙二醇-N,N,N',N'-四乙酸预处理显著降低了EGF诱导的Elk-1反式激活，表明EGF刺激的Elk-1转录活性依赖于细胞内Ca(2+)。为了确定在EGF介导的Elk-1反式激活过程中细胞核和细胞溶质Ca(2+)信号的相对贡献，通过将Ca(2+)结合蛋白小白蛋白靶向表达到细胞核或细胞溶质中，选择性地损害了任一隔室中的Ca(2+)信号。抑制细胞核而非细胞溶质中的Ca(2+)信号抑制了EGF诱导的Elk-1反式激活。然而，抑制细胞核Ca(2+)信号并不影响ERK响应EGF而磷酸化或转位到细胞核的能力。抑制细胞核Ca(2+)信号也不影响EGF刺激后Elk-1的磷酸化和核定位。这些结果表明，细胞核Ca(2+)是EGF介导的Elk-1转录激活所必需的，并且仅Elk-1的磷酸化不足以诱导其对EGF的转录激活。因此，小白蛋白的亚细胞靶向揭示了细胞核Ca(2+)信号在丝裂原活化蛋白激酶介导的基因转录中的独特作用。
