Sec6 modulates PP2A phosphatase activity through alteration of the binding affinity of PP2A in A and C subunits

Dephosphorylation of the activated the regulator of a-fetoprotein (Raf)-MAP/ERK kinase (MEK)-the extracellular signal-regulated protein kinase (ERK)-p90 ribosomal protein 6 kinase (RSK) cascade plays an essential role in regulating the magnitude and duration of kinase activation and the nature of the physiological response. The protein phosphatase 2A (PP2A), which is one of the major serine/threonine phosphatases, plays a pivotal role in various signaling pathways including cell cycle, metabolism, migration, and cell death. The impairment of PP2A activity is associated with various diseases including neurodegenerative disorders, autoimmune diseases, type II diabetes, and tumors. However, little is known about how cells control the assembly of PP2A subunits and stabilize PP2A phosphatase activity. In the present study, we demonstrate that Sec6 regulates PP2A phosphatase activity by modulating the binding affinity of the A and C subunits of PP2A, thereby modulating the phosphorylation of p90RSK at Thr359 and Ser380, glycogen synthase kinase 3b (GSK3b) at Ser9, and the expression of zinc finger E-box binding homeobox 1 (ZEB1), vimentin, and zonula occludens 3 (ZO-3).

NF106: A Neurofibromatosis Clinical Trials Consortium Phase II Trial of the MEK Inhibitor Mirdametinib (PD-0325901) in Adolescents and Adults With NF1-Related Plexiform Neurofibromas

PURPOSE Patients with neurofibromatosis type 1 (NF1) frequently develop plexiform neurofibromas (PNs), which can cause significant morbidity. We performed a phase II trial of the MAPK/ERK kinase inhibitor, mirdametinib (PD-0325901), in patients with NF1 and inoperable PNs. The primary objective was response rate based on volumetric magnetic resonance imaging analysis. METHODS Inclusion criteria included age ≥ 16 years and a PN that was either progressive or causing significant morbidity. First-dose pharmacokinetics were performed. Patients completed patient-reported outcome measures. Patients received mirdametinib by mouth twice a day at 2 mg/m2/dose (maximum dose = 4 mg twice a day) in a 3-week on/1-week off sequence. Each course was 4 weeks in duration. Evaluations were performed after four courses for the first year and then after every six courses. Patients could receive a maximum of 24 total courses. RESULTS Nineteen patients were enrolled, and all 19 received mirdametinib. The median age was 24 years (range, 16-39 years); the median baseline tumor volume was 363.8 mL (range, 3.9-5,161 mL). Eight of the 19 patients (42%) achieved a partial response of the target PN by course 12, and 10 (53%) had stable disease. One patient (5%) developed progressive disease at course 8. Significant and durable decreases were observed in pain ratings. CONCLUSION To our knowledge, this analysis represents the first characterization of the activity and pharmacokinetics of mirdametinib in patients with NF1 and PNs and is the first published response study for MAPK/ERK kinase inhibitors in adults with NF1 and PNs. Mirdametinib given at 2 mg/m2/dose (maximum dose, 4 mg) twice daily in a 3-week on/1-week off sequence resulted in a 42% partial response rate with preliminary evidence of reduction in pain.

Modulation of Differentiation of Embryonic Stem Cells by Polypyrrole: The Impact on Neurogenesis

The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.

Simultaneous readout of multiple FRET pairs using photochromism

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral band-width required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for ERK kinase activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.

The Regenerative Potential of Donkey and Human Milk on the Redox-Sensitive and Proliferative Signaling Pathways of Skin Fibroblasts

The influence of milk bioactive peptides on skin regenerative potential and rejuvenation is very often limited because of allergic reactions. The current study is aimed at exploring the influence of donkey colostrum and mature milk, human colostrum and mature milk, and β-casein and β-casomorphine-7, on the growth and inflammatory response of the culture of cultured skin fibroblasts exposed to these conditions for twenty-four hours. Their effects on the growth-regulatory kinases and redox-sensitive, proinflammatory transcriptional factor NF-κB were detected by using specific primary antibodies against NF-κB p65, Akt-1, phospho-Akt-1, Erk-1, phospho-Erk-1, JNK, phospho-JNK, phospho-STAT-1, and CD26, while logarithmic integrated fluorescence intensity patterns were recorded by flow cytometry. The downregulation of NF-κB p65 was observed after the exposure of skin fibroblasts to donkey milk and human colostrum, while β-casein and β-casomorphine-7 exerted the opposite effect, which suggests that noncasein bioactive peptides of donkey and human milk may be responsible for anti-inflammatory properties. The exposure to all milk species examined and β-casein leads to the activation of growth-regulatory kinases (Akt1/2/3 kinase, Erk kinase, JNK kinase, and Stat-1 kinase), especially for the p-Erk pathway, which suggests that essential amino acids of casein may be responsible for Erk-induced cell cycle activation and proliferation. The opposite effect was observed when cells were exposed to β-casomorphine-7, which may affect the skin fibroblast survival and their proliferative and regenerative potential. Donkey milk did not significantly change the CD26 antigen expression. In conclusion, our results suggest that among cell signaling molecules, the most sensitive but nonspecific downstream effector is p-Erk kinase, which may point to donkey milk usefulness in wound healing, regenerative, and aesthetic dermatology. The noncasein bioactive peptides of donkey milk may be responsible for the anti-inflammatory property of donkey milk and colostrum, which may indicate the usefulness in the treatment of inflammatory skin diseases.

The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells

Identifying additional mitogen-activated protein kinase (MAPK) pathway regulators is invaluable in aiding our understanding of the complex signaling networks that regulate cellular processes, including cell proliferation and survival. Here, using in vitro kinase assays and by expressing WT or kinase-dead MAPK kinase kinase 19 (MAP3K19) in the HEK293T cell line and assessing activation of the extracellular signal–regulated kinase (ERK) and JUN N-terminal kinase (JNK) signaling pathways, we defined MAP3K19 as a novel regulator of MAPK signaling. We also observed that overexpression of WT MAP3K19 activates both the ERK and JNK pathways in a panel of cancer cell lines. Furthermore, MAP3K19 sustained ERK pathway activation in the presence of inhibitors targeting the RAF proto-oncogene Ser/Thr protein kinase (RAF) and MAPK/ERK kinase, indicating that MAP3K19 activates ERK via a RAF-independent mechanism. Findings from in vitro and in-cell kinase assays demonstrate that MAP3K19 is a kinase that directly phosphorylates both MAPK/ERK kinase (MEK) and MAPK kinase 7 (MKK7). Results from an short-hairpin RNA screen indicated that MAP3K19 is essential for maintaining survival in KRAS-mutant cancers; therefore, we depleted or inhibited MAP3K19 in KRAS-mutant cancer cell lines and observed that this reduces viability and decreases ERK and JNK pathway activation. In summary, our results reveal that MAP3K19 directly activates the ERK and JNK cascades and highlight a role for this kinase in maintaining survival of KRAS-mutant lung cancer cells.

p21 inhibitor UC2288 ameliorates MPTP induced Parkinson’s disease progression through inhibition of oxidative stress and neuroinflammation

BackgroundParkinson's disease (PD) is a neurodegenerative disease characterized by the early prominent death of dopaminergic neurons and a decrease of dopamine levels. Dopamine depletion leads to several motor dysfunctions, including resting tremor, muscular rigidity, bradykinesia and postural instability. Our previous study determined that knockout of parkin, a gene of PD degrade p21, suppresses neurogenesis which is critical for a neurodegenerative disease. MethodsThus, we investigated the effect of UC2288, an inhibitor of p21, for its therapeutic effect on PD. We found that UC2288 attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral impairment in Rota-rod and Pole test as well as dopamine depletion.ResultsMoreover, UC2288 recovered the number of TH positive cells, but decreased the number of GFAP and Iba-1 positive cells accompanied the decrease of BAX and cleaved caspase3 as well as iNOS and COX-2 expression. In cultured neurons, UC2288 recovered MPP+-induced neuronal cell death in a concentration dependent manner. We also found that UC2288 decreased the p21 reactive cell number, oxidative neuronal damages, cytokines product in vivo and cultured neurons. In a mechanism study, we found that UC2288 significantly decreased the activation of ERK and p38 kinase pathway in the mitogen-activated protein kinase (MAPK) pathway. In addition, 1-10 μM concentration of ERK kinase inhibitor U0126 recovered MPP+-induced neuronal cell death. However, ERK kinase inhibitor U0126 further decreased cell viability with the increase of H2O2.ConclusionThese results indicated that the administration of UC2288 exerted neuroprotective effects on the death of dopaminergic neurons through the suppression of oxidative stress and neuroinflammation via ERK pathway inhibition.