VEGF Stimulates Activation of ERK5 in the Absence of C-Terminal Phosphorylation Preventing Nuclear Localization and Facilitating AKT Activation in Endothelial Cells

Extracellular-signal-regulated kinase 5 (ERK5) is critical for normal cardiovascular development. Previous studies have defined a canonical pathway for ERK5 activation, showing that ligand stimulation leads to MEK5 activation resulting in dual phosphorylation of ERK5 on Thr218/Tyr220 residues within the activation loop. ERK5 then undergoes a conformational change, facilitating phosphorylation on residues in the C-terminal domain and translocation to the nucleus where it regulates MEF2 transcriptional activity. Our previous research into the importance of ERK5 in endothelial cells highlighted its role in VEGF-mediated tubular morphogenesis and cell survival, suggesting that ERK5 played a unique role in endothelial cells. Our current data show that in contrast to EGF-stimulated HeLa cells, VEGF-mediated ERK5 activation in human dermal microvascular endothelial cells (HDMECs) does not result in C-terminal phosphorylation of ERK5 and translocation to the nucleus, but instead to a more plasma membrane/cytoplasmic localisation. Furthermore, the use of small-molecule inhibitors to MEK5 and ERK5 shows that instead of regulating MEF2 activity, VEGF-mediated ERK5 is important for regulating AKT activity. Our data define a novel pathway for ERK5 activation in endothelial cells leading to cell survival

Kinobead Profiling Reveals Reprogramming of BCR Signaling in Response to Therapy within Primary CLL Cells

Purpose: B-cell receptor (BCR) signaling is critical for the pathogenesis of chronic lymphocytic leukemia (CLL), promoting both malignant cell survival and disease progression. Although vital, understanding of the wider signaling network associated with malignant BCR stimulation is poor. This is relevant with respect to potential changes in response to therapy, particularly involving kinase inhibitors. In the current study, we describe a novel high-resolution approach to investigate BCR signaling in primary CLL cells and track the influence of therapy on signaling response. Experimental Design: A kinobead/mass spectrometry–based protocol was used to study BCR signaling in primary CLL cells. Longitudinal analysis of samples donated by clinical trial patients was used to investigate the impact of chemoimmunotherapy and ibrutinib on signaling following surface IgM engagement. Complementary Nanostring and immunoblotting analysis was used to verify our findings. Results: Our protocol isolated a unique, patient-specific signature of over 30 kinases from BCR-stimulated CLL cells. This signature was associated with 13 distinct Kyoto Encyclopedia of Genes and Genomes pathways and showed significant change in cells from treatment-naïve patients compared with those from patients who had previously undergone therapy. This change was validated by longitudinal analysis of clinical trials samples where BCR-induced kinome responses in CLL cells altered between baseline and disease progression in patients failing chemoimmunotherapy and between baseline and treatment in patients taking ibrutinib. Conclusions: These data comprise the first comprehensive proteomic investigation of the BCR signaling response within CLL cells and reveal unique evidence that these cells undergo adaptive reprogramming of this signaling in response to therapy

Power, Trust, and Institution: Tax Authority VS Taxpayer in Indonesia

This study estimates tax compliance in a relationship between power of authority and trust in authority in Indonesia, and how institution plays key role to shape tax reform. Using the aggregate data of individuals and firms per region tax office in all provinces in Indonesia in 10 years of taxing period (2008-2017) in time-fixed effect model, we found that soft and medium approach in power of authority gives significant effect in increasing tax compliance. However, other findings show that the higher taxpayer income, the lower the tax compliance. In addition, Java, Bali and Nusa Tenggara still give higher compliance than the other location in Indonesia. Tax reform needs institution as a rule to adapt in taxpayer behavior and understand the compliance risk

Performance evaluation of PV ventilated hybrid greenhouse dryer under no load condition

The PV ventilated hybrid greenhouse dryer was designed and fabricated for drying of food grains. The greenhouse dryer with 446.7 X 213.4 cm size and with central height of 259 cm was constructed using 50.8X25.4 mm MS pipe, 19X30.2 mm MS angles. Clear twin wall polycarbonate sheet with 6 mm thick was used insulate the greenhouse dryer structure. Performance evaluation was conducted under no load test condition. It was observed that, the average temperature inside the dryer was 7.6-13.40C and 6.3-13.20C (22-43%) higher and average relative humidity was 26-47% and 23-40% lower than ambient temperature and relative humidity during May and December months respectively. Exhaust air flow rate varied in the range of 38.93-81.95 m3/min and 28-63 m3/min during May and December respectively. Elevated greenhouse dryer air temperature and reduced relative humidity would reduce the drying time considerably

The ERK5 pathway in BRAFV600E melanoma cells plays a role in development of acquired resistance to dabrafenib but not vemurafenib.

Malignant melanoma, an aggressive skin cancer with a poor prognosis, frequently features BRAFV600E mutation resulting in activation of the MAPK pathway and melanocyte proliferation and survival. BRAFV600E inhibitors like vemurafenib and dabrafenib have enhanced patient survival, yet drug resistance remains a significant challenge. We investigated the role of the ERK5 pathway in BRAFV600E melanoma cells and cells with acquired resistance to PLX4720 (vemurafenib) and dabrafenib. In BRAFV600E melanoma, ERK5 inhibition minimally affected viability compared to ERK1/2 inhibition. In vemurafenib-resistant cells, ERK5 inhibition alone didn't impact viability or restore drug sensitivity to vemurafenib. However, in dabrafenib-resistant cells, ERK5 inhibition reduced viability and enhanced the anti-proliferative effect of MEK1/2 inhibition. Targeting the ERK5 pathway may represent a therapeutic opportunity in dabrafenib-resistant melanoma

Kinobead Profiling Reveals Reprogramming of BCR Signaling in Response to Therapy within Primary CLL Cells

PURPOSE: B-cell receptor (BCR) signaling is critical for the pathogenesis of chronic lymphocytic leukemia (CLL), promoting both malignant cell survival and disease progression. Although vital, understanding of the wider signaling network associated with malignant BCR stimulation is poor. This is relevant with respect to potential changes in response to therapy, particularly involving kinase inhibitors. In the current study, we describe a novel high-resolution approach to investigate BCR signaling in primary CLL cells and track the influence of therapy on signaling response. EXPERIMENTAL DESIGN: A kinobead/mass spectrometry–based protocol was used to study BCR signaling in primary CLL cells. Longitudinal analysis of samples donated by clinical trial patients was used to investigate the impact of chemoimmunotherapy and ibrutinib on signaling following surface IgM engagement. Complementary Nanostring and immunoblotting analysis was used to verify our findings. RESULTS: Our protocol isolated a unique, patient-specific signature of over 30 kinases from BCR-stimulated CLL cells. This signature was associated with 13 distinct Kyoto Encyclopedia of Genes and Genomes pathways and showed significant change in cells from treatment-naïve patients compared with those from patients who had previously undergone therapy. This change was validated by longitudinal analysis of clinical trials samples where BCR-induced kinome responses in CLL cells altered between baseline and disease progression in patients failing chemoimmunotherapy and between baseline and treatment in patients taking ibrutinib. CONCLUSIONS: These data comprise the first comprehensive proteomic investigation of the BCR signaling response within CLL cells and reveal unique evidence that these cells undergo adaptive reprogramming of this signaling in response to therapy