Internalization of Met requires the co-receptor CD44v6 and its link to ERM proteins.

Receptor Tyrosine Kinases (RTKs) are involved in many cellular processes and play a major role in the control of cell fate. For these reasons, RTK activation is maintained under tight control. Met is an essential RTK that induces proliferation, differentiation, migration, survival and branching morphogenesis. Deregulation of Met by overexpression, amplification or lack of effective degradation leads to cancer and metastasis. We have shown that Met relies on CD44v6 for its activation and for signaling in several cancer cell lines and also in primary cells. In this paper, we show that internalization of Met is dependent on CD44v6 and the binding of Ezrin to the CD44v6 cytoplasmic domain. Both CD44v6 and Met are co-internalized upon Hepatocyte Growth Factor induction suggesting that Met-induced signaling from the endosomes relies on its collaboration with CD44v6 and the link to the cytoskeleton provided by ERM proteins.

Labeling of oxidizable proteins with a photoactivatable analog of the antitumor agent DMXAA: evidence for redox signaling in its mode of action.

The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-alpha production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA.