| Signaling by the Ras/Raf/MEK/ERK pathway was activated
in A431 cells by stimulation of the epidermal growth factor
receptor (EGFR) with EGF. Cells were also treated with
drug compounds (red arrows) that inhibit the signaling
cascade at two different points. PD168393 is a known EGFR
inhibitor and should affect not only EGFR auto-phosphorylation
but also downstream targets such as STAT3 and ERK1/2. In
contrast, U0126 is an inhibitor of MEK1/2. This drug should
inhibit signaling by MEK1/2 and cause a decrease in ERK1/2
phosphorylation, without affecting phosphorylation of EGFR
or STAT3. |
 |
| Effects of pathway inhibitors on EGFR, Stat3, and ERK
phosphorylation*. A431 cells were cultured and treated
with serial dilutions of drug, then stimulated with EGF.
IC50 curves for each drug were determined in a single microwell
plate using duplicate samples. |
 |
| A) The MEK inhibitor U0126 displayed the
expected specificity and caused a dramatic decrease in
ERK phosphorylation, but did not affect phosphorylation
of EGFR or Stat3. |
 |
| B) The EGFR inhibitor PD168393 decreased the phosphorylation
observed for all three target proteins in the pathway – the
receptor as well as its downstream effectors. However,
the concentration of drug required to achieve 50% inhibition
of phosphorylation (IC50) was almost 10-fold higher for
ERK (~100 nM) than for EGFR and Stat3 (~11.1 nM). This
may be due to EGFR-independent signaling pathways that
also signal through ERK and were not inhibited by the drug
tested. |
