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    • The structural analysis of the ASK activation segment which

    2023-01-28

    The structural analysis of the ASK1 activation segment, which was not phosphorylated in the crystal structure, showed interactions mimicking those found in activated kinases. In addition, the isolated unphosphorylated ASK1-CD is active and able to autophosphorylate itself at three sites, Thr813, Thr838 and Thr842 (Fig. 1C) (Bunkoczi et al., 2007). Although Thr838 is located in the activation segment and its autophosphorylation is crucial for ROS-induced ASK1 activation (Tobiume et al., 2002), Thr842 is close to the catalytic Asp803. Moreover, the comparison between ASK1-CD and the protein kinases DAPK3 (Graves et al., 2005) and CHK2 (Lee and Chung, 2001) suggests that Thr842 may also play a regulatory role. The third site, Thr813, is far from the catalytic site and its role in ASK1 regulation is unknown. Interestingly, ASK1-CD mutants with all three threonines replaced by Ala were still catalytically active, thus indicating that their colony stimulating factor 1 receptor may be required for recruiting ASK1 binding partners within the signalosome instead of directly regulating its catalytic activity (Bunkoczi et al., 2007).
    Interaction with 14-3-3 14-3-3 proteins, a family of highly conserved dimeric proteins (Fig. 1F) ubiquitously expressed in all eukaryotic cells, interact with and regulate the function of several hundreds of partner proteins by recognizing phosphoserine- (pS) or phosphothreonine (pT)-containing motifs (Fu et al., 2000, Liu et al., 1995, Muslin et al., 1996, Xiao et al., 1995). Through these binding interactions, 14-3-3 proteins act as allosteric regulators and/or molecular scaffolds that constrain the conformation of the binding partner; if the target protein is an enzyme, this can affect its catalytic activity (Obsil and Obsilova, 2011, Yaffe, 2002). The role of the 14-3-3 protein in ASK1 regulation was first suggested by observations, that, under non-stress conditions, ASK1 specifically binds 14-3-3 proteins through a phosphoserine (pSer966)-containing motif located approximately 25 residues downstream of the C terminus of ASK1-CD (Fig. 1A) and that mutating this residue to alanine dramatically accelerated ASK1-induced cell death (Zhang et al., 1999). It was subsequently shown that the oxidative stress-induced dephosphorylation of pSer966 followed by 14-3-3 dissociation is accompanied by an increase in the autokinase activity of ASK1 and that the 14-3-3 binding-defective mutant ASK1 Ser966Ala shows increased autokinase and transkinase activities (Goldman et al., 2004). In addition, studies have also shown that ASK1-interacting protein (AIP1) binding to the ASK1 sequence surrounding the 14-3-3 binding site is another contributing factor to 14-3-3 dissociation from ASK1 (Zhang et al., 2003). Conversely, Klotho-dependent phosphorylation of the 14-3-3 protein at the dimerization interface, which promotes 14-3-3 monomer formation, presumably stabilizes the interaction between 14-3-3 and ASK1, thereby enhancing ASK1 inhibition (Brobey et al., 2015). The close proximity of the 14-3-3 binding motif to ASK1-CD suggests that the 14-3-3 protein modulates the structure of ASK1-CD and/or the accessibility of its active site, as shown for other enzymes regulated in a 14-3-3-dependent manner (Kopecka et al., 2014, Obsil et al., 2001). Indeed, structural characterization of the complex between ASK1-CD phosphorylated at Ser966 (pASK1-CD) and the 14-3-3ΞΆ protein indicated that the 14-3-3 protein may inhibit ASK1 through several mechanisms, including structural modulation of its active site, steric blocking of Thr838 phosphorylation and/or blocking of interactions between ASK1 and its substrates (Petrvalska et al., 2016). However, this study also showed that the interaction between the dimeric pASK1-CD and the 14-3-3 protein was transient, and the resulting complex was dynamic and conformationally heterogeneous, most likely due to the lack of a large and well-defined contact interface between 14-3-3 and ASK1-CD. The observed instability of the pASK1-CD:14-3-3 complex could have been caused by the absence of another protein, e.g. ASK2, because evidence shows that ASK1, ASK2 and 14-3-3 form a ternary complex in which the interaction between ASK2 and 14-3-3 determines the extent of ASK1 binding to 14-3-3 (Cockrell et al., 2010, Federspiel et al., 2016).