Introduction Tumor necrosis factor TNF is a pro
Tumor necrosis factor (TNF) is a pro-inflammatory mediator with the capacity to induce apoptosis (Benderska et al., 2012). Recent reports have shown that TNF might trigger cell death, at least in part, by directly affecting the reorganization of the p38 pathway cytoskeleton (Campos et al., 2009). The most apparent consequences of TNF-induced apoptosis are drastic changes in cell morphology – such as the rounding-up of the cell and blebbing of the plasma membrane (Mathew et al., 2009, Scanlon et al., 1989). Nevertheless, the responsible cytoskeletal proteins and their signaling pathways are not well understood.
A potential candidate kinase that might connect TNF signaling to actin reorganization during cell death is the death-associated protein kinase (DAPK), a calmodulin-regulated and actin filament-associated serine/threonine kinase (Deiss et al., 1995). DAPK is known to exert tumor-suppressive functions, and this has been attributed to its apoptosis-promoting activity (Deiss et al., 1995, Inbal et al., 1997, Inbal et al., 2000, Kögel et al., 2003, Raveh and Kimchi, 2001). DAPK exerts this pro-apoptotic effect by inside-out inactivation of integrin β1, thereby suppressing the matrix survival signal and activating a p53-dependent apoptosis pathway (Wang et al., 2002). It also participates in TNF- and Fas-induced apoptosis (Cohen et al., 1997). However, despite of a broad involvement in different cell death systems, very little is known about DAPK\'s role in cytoskeletal reorganization.
DAPK\'s localization to specific cytoskeletal components might bring it into close proximity with its physiological substrates. One known cytoskeletal-associated substrate of DAPK is the myosin-II light chain (MLC), which has previously been shown to be phosphorylated by DAPK, thus stabilizing actin stress fibers (Cohen et al., 1997, Kuo et al., 2003). Moreover, paxillin, a component of focal adhesions, was found to be localized in close proximity to the tips of the DAPK-positive filaments, indicating that stress fibers containing DAPK extend to focal contacts (Bialik and Kimchi, 2004, Bialik and Kimchi, 2006). Recently we have shown that activation of the p38/DAPK complex induces caspase3-dependent apoptotic cell death under TNF stimulation (Bajbouj et al., 2009). The signaling molecules involved in the TNF-mediated downstream pathway between DAPK and caspase3 have not yet been identified. We performed a phospho-peptide array to test for cytoskeleton-associated proteins that might interact with DAPK under TNF stimulation. We found that cofilin1 showed a marked increase in phosphorylation at serine3 residue in response to TNF. Cofilin1 is a key regulator of actin filament dynamics and reorganization by stimulating depolymerisation and severing of actin filaments (Bamburg et al., 1999, Bernstein and Bamburg, 2010, Moon and Drubin, 1995, Pollard and Borisy, 2003). The activity of cofilin is reversibly regulated by phosphorylation and dephosphorylation at serine3, with the phosphorylated form being inactive. LIM kinase1 (LIMK1) phosphorylates cofilin and thereby inhibits its actin filament-disrupting activity (Arber et al., 1998, Yang et al., 1998). The inactive serine3-phosphorylated cofilin (p-cofilinSer3) is dephosphorylated and reactivated by phosphatases of the Slingshot family and by Chronophin (Gohla et al., 2005, Niwa et al., 2002). LIMK1-mediated cofilin phosphorylation is critically involved in a variety of physiological and pathological processes, including directional migration, chemotaxis, cell polarity and tumor invasion (Aizawa et al., 2001, Kobayashi et al., 2006, Mouneimne et al., 2006, Nishita et al., 2005, Yoshioka et al., 2003). Its role in apoptosis, however, is not well investigated.
Materials and methods
Discussion The serine/threonine kinase DAPK plays an important role in a wide range of different cell death modes (Lin et al., 2010). To date, only a limited number of proteins that interact with DAPK have been identified. Recently, we have described a novel interaction between the MAPkinase p38 and DAPK in TNF-induced apoptosis of colorectal cancer cells (Bajbouj et al., 2009). To better understand how DAPK mediates caspase3-dependent apoptotic cell death, we performed a peptide array screen to identify phosphorylated targets under TNF treatment. The peptide array showed pronounced cofilin phosphorylation at serine3. In the Western blot analysis we confirmed a phosphorylation of its up-stream kinase LIMK at threonine508 in response to TNF. Although there was a consistent LIMK activation and subsequent cofilin inactivation pattern after TNF treatment, the broad variation in the time frame between 24 and 48h cannot be fully explained. Because LIMK was shown to be involved in cell cycle progression (Manetti, 2012), we might speculate that this variation results from cells in different phases of the cell cycle. Due to high cytotoxicity of the combination of lipofectamin with starvation this experimental proof was technically impossible. In a set of experiments that are discussed below, we then identified DAPK as an interaction partner of LIMK. Our data suggest that DAPK serves as a scaffold of the LIMK/cofilin complex.