ACAGT-007a, an anti-cancer compound that modulates ERK MAPK signaling, induces nuclear enrichment of phosphorylated ERK in T3M4 pancreatic cancer cells.

The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.

Real-time sensing of MAPK signaling in medulloblastoma cells reveals cellular evasion mechanism counteracting dasatinib blockade of ERK activation during invasion.

Aberrantly activated kinase signaling pathways drive invasion and dissemination in medulloblastoma (MB). A majority of tumor-promoting kinase signaling pathways feed into the mitogen-activated protein kinase (MAPK) extracellular regulated kinase (ERK1/2) pathway. The activation status of ERK1/2 during invasion of MB cells is not known and its implication in invasion control unclear. We established a synthetic kinase activation relocation sensor (SKARS) for the MAPK ERK1/2 pathway in MB cells for real-time measuring of drug response. We used 3D invasion assays and organotypic cerebellum slice culture to test drug effects in a physiologically relevant tissue environment. We found that hepatocyte growth factor (HGF), epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF) caused rapid nuclear ERK1/2 activation in MB cells, which persisted for several hours. Concomitant treatment with the BCR/ABL kinase inhibitor dasatinib completely repressed nuclear ERK1/2 activity induced by HGF and EGF but not by bFGF. Increased nuclear ERK1/2 activity correlated positively with speed of invasion. Dasatinib blocked ERK-associated invasion in the majority of cells, but we also observed fast-invading cells with low ERK1/2 activity. These ERK1/2-low, fast-moving cells displayed a rounded morphology, while ERK-high fast-moving cells displayed a mesenchymal morphology. Dasatinib effectively blocked EGF-induced proliferation while it only moderately repressed tissue invasion, indicating that a subset of cells may evade invasion repression by dasatinib through non-mesenchymal motility. Thus, growth factor-induced nuclear activation of ERK1/2 is associated with mesenchymal motility and proliferation in MB cells and can be blocked with the BCR/ABL kinase inhibitor dasatinib.

Spatial signal repression as an additional role of Sprouty2 protein variants.

Sprouty2 (Spry2) is a prominent member of a protein family with crucial functions in the modulation of signal transduction. One of its main actions is the repression of mitogen-activated protein kinase (MAPK) pathway in response to growth factor-induced signalling. A common single nucleotide polymorphism within the Spry2 gene creates two protein variants where a proline adjacent to the serine rich domain is converted to an additional serine. Both protein variants perform similar functions although their efficiency in fulfilling these tasks varies. In this report, we used biochemical fractionation methods as well as confocal microscopy to analyse quantitative and qualitative differences in the distribution of Spry2 variants. We found that Spry2 proteins localize not solely to the plasma membrane, but also to other membrane engulfed compartments like for example the Golgi apparatus. In these less dense organelles, predominantly slower migrating forms reside indicating that posttranslational modification contributes to the distribution profile of Spry2. However there is no significant difference in the distribution of the two variants. Additionally, we found that Spry2 could be found exclusively in membrane fractions irrespective of the mitogen availability and the phosphorylation status. Considering the interference of extracellular signal-regulated kinase (ERK) activation in the cytoplasm, both Spry2 variants inhibited the levels of phosphorylated ERK (pERK) significantly to a similar extent. In contrast, the induction profiles of pERK levels were completely different in the nuclei. Again, both Spry2 variants diminished the levels of pERK. While the proline variant lowered the activation throughout the observation period, the serine variant failed to interfere with immediate accumulation of nuclear pERK levels, but the signal duration was shortened. Since the extent of the pERK inhibition in the nuclei was drastically more pronounced than in the cytoplasm, we conclude that Spry2 - in addition to its known functions as a repressor of general ERK phosphorylation - functions as a spatial repressor of nucleic ERK activation. Accordingly, a dominant negative version of Spry2 was only able to enhance the pERK levels of serum-deprived cells in the cytosol, while in the nucleus the intensity of the pERK signal in response to serum addition was significantly increased.