AP activity is increased by MAP kinase stimulation
AP-1 activity is increased by MAP kinase stimulation in different cell types (Whitmarsh and Davis, 1996), while MKP-1, the enzyme that catalyzes the dephosphorylation and inactivation of MAP kinases in the nucleus, inhibits AP-1 (Rössler et al., 2008, Rössler and Thiel, 2009, Mayer et al., 2011, Thiel et al., 2012, Thiel and Rössler, 2011, Thiel and Rössler, 2014, Thiel and Rössler, 2017). In this study, we showed that overexpression of MKP-1 attenuated B-Raf-induced AP-1 activation, indicating that MKP-1 functions as a shut-off device for the B-Raf-induced signaling cascade. In addition, we showed that calcineurin interferes with B-Raf-induced activation of AP-1, establishing a second negative feedback loop. Recently, it has been shown that pharmacological inhibition of calcineurin enhanced AP-1 gene expression in bone cells (Worton et al., 2014). We previously observed that calcineurin attenuated the activation of AP-1 in neuroblastoma cells expressing active M3 muscarinic exendin 4 receptors (Rössler et al., 2008). Together with this study, we conclude that calcineurin negatively regulates AP-1 activation in distinct signaling cascades. Mechanistically, calcineurin dephosphorylates the ternary complex factor (TCF) Elk-1 (Sugimoto et al., 1997, Tian and Karin, 1999), a protein that binds to the serum response element (SRE). The SRE, composed of the CArG box, the binding site of serum response factor, and an adjacent Ets binding motif that attracts ternary complex factors to the promoter is one of the best studied genetic landmark motifs of the c-Fos promoter. The TCF Elk-1 is phosphorylated by MAP kinases and regulates c-Fos gene transcription via the SRE. The data presented here showed that the transcriptional activation potential of Elk-1 was increased as a result of B-Raf stimulation. We propose that the stimulation of c-Fos expression via B-Raf-induced Elk-1 activation is attenuated by calcineurin via dephosphorylation of Elk-1, thus impairing SRE-mediated activation of c-Fos gene transcription. Microarray analysis has revealed that AP-1 plays an important role in the upregulation of transcription in insulinoma cells that had been stimulated with glucose and cpt-cAMP (Glauser et al., 2007). The phosphorylation and activation of c-Jun in insulinoma cells as a result of glucose stimulation has been shown (Maedler et al., 2008). In addition, stimulation of insulinoma cells with glucose was shown to trigger the activation of AP-1, which was attenuated by expressing an inhibitory A-Raf mutant (Müller et al., 2010), thus shedding light to the central role of Raf in glucose signaling in insulinoma cells. This study reinforces the view that B-Raf occupies center stage within the signaling pathway connecting glucose stimulation with the activation of AP-1. AP-1 functions in various cell types as convergence point for intracellular signaling cascades, regulating diverse cellular functions. The transcription factor c-Jun is often part of the AP-1 transcription factor complex, and experiments with a dominant-negative c-Jun mutant revealed, that B-Raf-induced activation of AP-1 relies on c-Jun. Homozygous transgenic mice with a disrupted c-Jun gene die at mid-gestation, precluding the analysis of c-Jun-deficient β-cells in adult animals (Johnson et al., 1993). c-Jun is a major substrate for the c-Jun N-terminal protein kinase (JNK), which regulates important functions in β-cells and has been correlated with insulin resistance, glucose intolerance, apoptosis, metabolic syndrome and type 2 diabetes (Ammendrup et al., 2000, Aguirre et al., 2000, Bonny et al., 2001, Kaneto et al., 2002, Weston and Davis, 2007, Ferdaoussi et al., 2008, Maedler et al., 2008, Lanuza-Masdeu et al., 2013). Thus, many biological functions of pancreatic β-cells are correlated with activated JNK and may be executed by the phosphorylation of c-Jun and the subsequent activation of the AP-1. As JNK is often correlated with β-cell apoptosis, c-Jun may execute the apoptotic program initiated by JNK via transcriptional activation of proapoptotic genes (Maedler et al., 2008). However, the fact that stimulation of β-cells with glucose, activation of L-type voltage-gated Ca2+ channels, and stimulation of B-Raf triggers an activation of AP-1, involving c-Jun, argues for an important role of AP-1 in genuine β-cell signaling cascades and functions. A strategy for elucidating the role of AP-1 in β-cells is the identification of the AP-1-regulated delayed response genes. As part of a search for these delayed response genes, we showed that B-Raf stimulation increased cyclin D1 promoter activity that was attenuated by mutating the AP-1 site of the cyclin D1 promoter. Cyclin D1 is essential for the postnatal growth of pancreatic β-cells and it has been shown that the cyclin D1 gene is part of a genuine signaling pathway in this cell type (Kushner et al., 2005, Kang et al., 2006).