Figure 9: Positive feedback in the PYK2–STAT3–c-MET axis.

(a) PYK2 depletion or its overexpression affects the steady-state level of c-Met protein as well as the phosphorylation of STAT3 in MDA-MB-468 and BT-549 cells as determined by WB analysis. (b) PYK2 affects EGF-induced upregulation of c-Met mRNA in MDA-MB-468 cells as determined by RT–PCR (left panel) and by real-time PCR (qRT–PCR; graph, right panel). Overexpression (OX). (c) The c-Met inhibitor PHA-66752 (10 μM) reduces the phosphorylation of STAT3 and PYK2 in response to EGF (24 h) and also of the mRNA levels of EMT transcription factors as determined by WB analysis (c) and by RT–PCR (d, left panel) or qRT–PCR (d, graph), respectively. The intensity of the RT–PCR bands was assessed by densitometry (Image J). Mean values of qRT–PCR±s.d. were calculated and presented in the graph as fold of control EGF-untreated values. (e) A scheme depicting the positive feedback loop between EGFR/c-Met, PYK2 and STAT3. EGFR and c-Met, which can trans-phosphorylate each other, activate PYK2 (PY402), which in turn, enhances STAT3 phosphorylation (pY705). Phospho-STAT3(pY705) translocates to the nucleus, binds to PYK2 prompter and enhances its transcription. pSTAT3 also enhances the expression of c-Met, possibly indirectly, and of Twist, which is required for EMT. The uncropped western blotting images are shown in Supplementary Fig 13.