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    • It has been shown that DAPK induces cell death

    2020-07-02

    It has been shown that DAPK induces cell death through an increase in DAPK catalytic activity and subsequent phosphorylation of target proteins (Shang et al., 2005, Yamamoto et al., 2002), but its multidomain structure enables also a close physical interaction with other proteins (Bialik and Kimchi, 2006). To obtain a mechanistic insight into the role of DAPK in LIMK/cofilin signaling, we studied the effect of DAPK inhibition, siRNA knockdown and overexpression on the phosphorylation status of LIMK and cofilin under TNF treatment. After DAPK inhibition with potent and selective DAPK inhibitor (Gandesiri et al., 2012, Okamoto et al., 2010), the LIMK/p-cofilinSer3 signaling was diminished. Furthermore, the levels of p-cofilinSer3 and p-LIMKThr508 correlated with the DAPK expression status as shown by DAPK siRNA experiments. DAPK-overexpression led to increased p-cofilinSer3 levels, surprisingly however without any detectable change in p-LIMKThr508 levels. Because the cofilinSer3-residue is not located within a consensus DAPK motif, we speculate that DAPK-overexpression might (directly or indirectly) block the activity of cofilin phosphatases. Another explanation could be a possible interplay between cofilin serine3 residue and another phosphorylation site. Inspection of the human cofilin sequence revealed the presence of a DAPK–phosphorylation consensus motif (Lin et al., 2010) around serine24 (RksS), but after mutating serine24 to Ala/Glu, we could not detect any influence on cofilinSer3 phosphorylation and TNF-induced apoptosis (unpublished data). Moreover, DAPK-phosphorylation consensus motifs (Lin et al., 2010) are present in Cdc42 (Kuo et al., 2006), and can also be detected in the amino PHA 543613 hydrochloride sequences of ROCK1, Rho A, and PAK1 (LIMK up-stream kinases). Although these proteins were not identified in our peptide array, we cannot exclude their involvement in our model. There also exist DAPK motifs in the LIMK protein sequence, but the 8 interaction residues proposed in the 3D model do not include any phosphorylatable amino acid. Another potential connection between DAPK and cofilin may be provided by 14-3-3 proteins, which are known to bind to DAPK (Henshall et al., 2003) and to stabilize phosphorylated cofilin (Gohla and Bokoch, 2002). Interestingly, the 3D structural model suggests no direct interaction between DAPK and cofilin. The evidence of cofilin in the DAPK immunoprecipitation instead suggests a strong LIMK/cofilin interaction. The energy analysis on the complexes of LIMK/cofilin and of DAPK/LIMK/cofilin could explain that DAPK provides further stability to the complex. Thus, we propose that both the kinase activity and scaffolding functions of DAPK are important in TNF-induced apoptosis. The significance of cofilin activation/inactivation during apoptosis is only poorly understood. To date, there are only few reports showing a role of cofilin in inducing cell death under oxidative stress (Campos et al., 2009, Hsieh et al., 2010, Klamt et al., 2009, Rehklau et al., 2012). Studying the dephosphorylated, active form of cofilin1, Chua et al., 2003, reported its mitochondrial translocation preceding the release of cytochrome c, indicating a role in the initiation phase of apoptosis. Other studies described a participation of active cofilin in growth-stimulating processes (Samstag et al., 1991, Samstag et al., 1994, Samstag et al., 1996, Saito et al., 1994). Interestingly, treatment of endothelial cells with a cytokine cocktail containing IL-1, IL-6 and TNF increased the phosphorylation of cofilin together with the formation of actin stress fibers (Campos et al., 2009). Similarly, inhibition of p-cofilinSer3 dephosphorylation by the serine phosphatase inhibitor okadaic acid was accompanied by apoptosis (Samstag et al., 1996). By varying the cofilin phosphorylation using p-cofilinSer3 mutants, we demonstrated that the expression of the pseudophosphorylated (cofilinGlu3 and cofilinAsp3) and nonphosphorylatable mutant cofilinAla3 did not significantly alter TNF-induced apoptosis in comparison with cofilin wt expression. Thus, we suggest that p-cofilinSer3 is an indicator of TNF-induced apoptosis and does not further trigger the apoptotic signal. In this regard, p-cofilinSer3 levels were found to be increased in TNF-induced apoptotic cells with condensed chromatin, pronounced membrane blebs and Annexin V up-regulation. In detached cells, we see a massive accumulation of inactive p-cofilinSer3 which has lost the actin filament disrupting activity. According to Wiggan et al. (2012), cofilin silencing could induce a crucial disturbance of normal homeostatic actin-myosin cytoarchitecture, resulting in bleb formation. The high levels of activated caspases might mediate the cleavage of further cytoskeletal proteins such as myosin light chain, actin, vimentin, and paxillin (Byun et al., 2001, Mashima et al., 1999, Moretti et al., 2002, Shim et al., 2001). The reinforced caspase 8, 9, and 3 activation could be explained as a cell death induced by loss of cell adhesion (anoikis) as described for ceramide-induced cell death (Widau et al., 2010). Although there is a role of DAPK in anoikis this form of cell death might be not associated with the described DAPK/LIMK/cofilin complex under TNF stimulus. The loss of pLIMKThr508 and DAPK in detached cells suggests that the multiprotein complex consisting of pLIMKThr508, cofilin, and DAPK is abrogated in detached apoptotic cells and its signaling is interrupted. Obviously TNF-treated cells have committed to caspase-dependent apoptosis prior to detachment from the culture plate. Here upon TNF a striking redistribution of LIMK, DAPK, and cofilin to the perinuclear compartment was observed. Whereas assembly of these three proteins was not associated with condensation of the chromatin, adherent apoptotic cells were positively marked by p-cofilinSer3 and p-LIMKThr508 co-localization. In summary, we present a novel pro-apoptotic multi-protein complex including DAPK, LIMK and cofilin that is reinforced following TNF-treatment. 3D structural modeling suggests that DAPK can act as a scaffold to integrate the LIMK1/cofilin1 complex. Furthermore, we suggest that p-cofilinSer3 is a marker but not a trigger of TNF-induced apoptosis in HCT116 cells, and is mainly responsible for the cytoskeletal changes that apoptotic cells undergo.