BRAFWT BRAF in melanoma cells was either not detected or was about 10 to 50 times less active than its counterpart mutant. IN, where the activity of t Is measurably inhibits PLX4032 BRAF kinase wild-type as well. Anything similar studies have shown no detectable activity T shown with RAF1 MK-2866 in YULAC BRAFV600E cells and YUMAC BRAFV600K. In contrast, a wide range of RAF1 Kinaseaktivit T in four melanoma cells was observed in all BRAFWT F Cases independently-Dependent increased again Ht after treatment with PLX4032. In addition, five times resulted in lower levels of Raf1 YUFIC BRAFWT melanoma cells by siRNA in a Hnlichen suppression both PLX4032 induced ERK activation compared to untreated cells, support lends to the conclusion that RAF1 the main kinase, activating the caused by ERK. We have considered several known ways in which PLX4032 could activate RAF1.
We concluded that the bet Account a discharge path, such as a receptor tyrosine kinase, PD0325901 independently by two-Dependent methods. First failed Herk mmlichen Western blotting with anti-phospholipid antique Rpern that detect specific Raf1 by an increase in the phosphorylation or RAF1 RAF1. It was a sustained increase in RAF1 phosphorylated ERK1 2 phosphorylation in cells treated with the drug BRAFWT. However, the phosphorylation of these sites is a feedback mechanism to mitigate ERK1 2 activity t and can therefore not explained Ren RAF1 activation. Second, transiently expressed RAF1 R89L mutant. Not bind Ras GTP, a key step in the stimulation of the receptor is mediated by PLX4032 was equally Ma S activated, expressed as the wild-type RAF1 ectopically We therefore concluded that PLX4032 is not possible to change a channel upstream Rts kinase RAF1 escape.
Another important mechanism for activating RAF1 homodimerization or heterodimerization with wild-type or mutated BRAF enzyme ver changed. However, we were not able Immunpr Zipitation the endogenous association with BRAF or RAF1 MYC transfected fa Transient and demonstrate labeled FLAG RAF1 PLX4032 in melanoma cells under conditions where the drug has been activated by RAF1 treated. Differential activation of downstream targets ERK1 We also examined the activation of ERK and downstream targets Ver Changes in gene expression, which shed more light on the PLX4032 and cellular responses provide k Can gauge for monitoring the treatment k Nnten. Rpern Western blot with phospho-specific antibody revealed P90rsk marked activation of ERK1 this 2 effector cells in melanoma YUDOSO BRAFWT if it has been suppressed in V600E YULACBRAF.
The nuclear transcription factor CREB to the SSIG, a known target p90rsk downstream was also activated. Real-time RT-PCR showed that early response genes are known, were FOS and JunB in 30 minutes in melanoma cells YUDOSO BRAFWT in response to PLX4032, an effect that is activated up to 8 retain h, then the FOS was down-regulated within 30 min YULAC BRAFV600E cells, in agreement with the kinetics of ERK1 2 activation and functional inactivation of wild-type and mutant cells. We explored the spectrum of genes affected by hybridization NimbleGen all tables of gene expression of the genome, the comparison with non-treated melanoma cells YUDOSO BRAFWT PLX4032 is treated. The results showed a strong up-regulation of 28 genes in response to PLX4032, including normal early response genes EGR1 EGR3.