375 ng/ml, approximately half of the TNF�� release of the LPS-treated U937 cells. Applying their supernatants to HCT116 tumor cells, we observed moreover a minor induction of p-p38 at 6 hours but a further reinforcement after 24 hours by Western blot analysis (Figure 4C). Identification of p-p38 as DAPK Binding-Protein To determine whether DAPK is interacting with p-p38, we first performed co-immunoprecipitation with an anti-DAPK antibody followed by immunoblotting with anti- p-p38 antibody or vice versa in HCT116 tumor cells subjected to TNF�� (Figure 4D). Here, p-p38 was identified as a DAPK-interacting protein. In a second step, we analyzed the localization of both proteins in HCT116 tumor cells with or without TNF��. We selected the 6 hours time point for co-immunofluorescence studies because p-p38 and DAPK were simultaneously induced here.
In the absence of TNF��, DAPK was found in the cytoplasm of colorectal tumor cells, whereas p-p38 was not detectable in the cells. On TNF�� exposure, both proteins were found to be co-localized in the cytoplasm of HCT116 tumor cells (Figure 4E). Altogether, these results further strengthen our assumption that p-p38 is a new DAPK binding partner in HCT116 tumor cells on TNF�� exposure. Triggering DAPK-Mediated Apoptosis by p-p38 Having identified the physical interaction between DAPK and p-p38, we then aimed to demonstrate the direct induction of DAPK catalytic activity by p-p38. For this, we used p38-siRNA to reduce the endogenous expression levels of p38 in HCT116 tumor cells on TNF�� exposure. siRNA transfection resulted in 70% reduction of p38 protein levels.
As expected p-p38 protein that is recruited from the 30% remaining p38 pool was not longer detectable at 6 hours (Figure 5A). In parallel p38 activity reached the low control level (Figure 5B). Furthermore, siRNA-mediated loss in p-p38 did not influence the DAPK protein level in cell lysates at 24 hours (Figure 5B), but resulted in a significant repression of TNF��-induced DAPK catalytic activity (Figure 5B). This loss of catalytic activity was accompanied by a significant reduction in TNF��-induced apoptosis in HCT116 tumor cells (Figure 5C) at 48 hours. This was not surprising, because it is well known that the catalytic activity of DAPK is absolutely required for all biological functions of this kinase.
The same results were obtained after treatment of tumor cells with the p38 inhibitor SB203580 (Figure 5, D and E). Figure 5 TNF��-induced and DAPK-mediated apoptosis is triggered by p-p38. A: Dacomitinib Lysates of HCT116 cells knocked down for p38 and subsequently subjected to TNF�� for 48 hours were analyzed by Western blotting using anti-p38, anti-p-p38, or anti-��-actin … Altogether, these results suggest that p-p38 may be necessary for DAPK-activation and DAPK-mediated apoptosis in HCT116 tumor cells on TNF�� exposure.