Kiki Chu

Identification of ROCK1 kinase as a critical regulator of Beclin1-mediated autophagy during metabolic stress

The Ser/Thr Rho kinase 1 (ROCK1) is known to play major roles in a wide range of cellular activities, including those involved in tumor metastasis and apoptosis. Here we identify an indispensable function of ROCK1 in metabolic stress-induced autophagy. Applying a proteomics approach, we characterize Beclin1, a proximal component of the PI(3)kinase class III lipid-kinase complex that induces autophagy, as an interacting partner of ROCK1. Upon nutrient deprivation, activated ROCK1 promotes autophagy by binding and phosphorylating Beclin1 at Thr119. This results in the specific dissociation of the Beclin1-Bcl-2 complex, without affecting the Beclin1-UVRAG interaction. Conversely, inhibition of ROCK1 activity increases Beclin1-Bcl-2 association, thus reducing nutritional stress-mediated autophagy. Genetic knockout of ROCK1 function in mice also leads to impaired autophagy as evidenced by reduced autophagosome formation. These results show that ROCK1 acts as a prominent upstream regulator of Beclin1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy.

Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53

Tumor suppressor p53 linked to immune function We thought we knew all we needed to about the tumor suppressor p53. However, Yoon et al. now describe a previously unrecognized function of p53 (see the Perspective by Zitvogel and Kroemer). p53 induces expression of the gene encoding DD1α, a receptor-like transmembrane protein of the immunoglobulin superfamily. In conditions of stress, p53 activation can lead to cell death. p53-induced expression of DD1α also promotes the clearance of dead cells by promoting engulfment by macrophages. Furthermore, expression of DD1α on T cells inhibits T cell function. Thus, p53 offers protection from inflammatory disease caused by the accumulation of apoptotic cells, and its suppression of T cells might help cancer cells to escape immune detection. Science, this issue 10.1126/science.1261669; see also p. 476 p53 promotes clearance of dead cells and proper immune function. [Also see Perspective by Zitvogel and Kroemer] INTRODUCTION Programmed cell death occurs throughout life in all tissues of the body, and more than a billion cells die every day as part of normal processes. Thus, rapid and efficient clearance of cell corpses is a vital prerequisite for homeostatic maintenance of tissue health. Failure to clear dying cells can lead to the accumulation of autoantigens in tissues that foster diseases, such as chronic inflammation, autoimmunity, and developmental abnormalities. In the normal immune system, phagocytic engulfment of apoptotic cells is accompanied by induction of a certain degree of immune tolerance in order to prevent self-antigen recognition. Over the past few decades, enormous efforts have been made toward understanding various mechanisms of tumor suppressor p53–mediated apoptosis. However, the involvement of p53 in postapoptosis has yet to be addressed. RATIONALE One of the most intriguing, yet enigmatic, questions in studying homeostatic control of efficient dead cell clearance and proper immune tolerance is how these two essential activities are interrelated: The complexity of these processes is demonstrated by the many receptors and signaling pathways involved in the engulfment of apoptotic cells and stringent discrimination of self antigens from nonself antigens. Thus, there must be key connection(s) linking the balance between immune homeostasis and inflammation. In addition to the antitumor functions of p53, p53 has been implicated in immune responses and inflammatory diseases, with various roles in the immune system becoming apparent. We identified a postapoptotic target gene of p53, Death Domain1α (DD1α), that is responsive to genotoxic stresses and expressed in immune cells. DD1α appears to function as an immunoregulator of T cell tolerance. We hypothesized that p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, DD1α. We determined that DD1α functions as an engulfment ligand or receptor that is involved in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages. We also addressed whether DD1α deficiency caused any defects in dead cell clearance in vivo. RESULTS DD1α has similarity with several members of the immunoglobulin superfamily with the extracellular immunoglobulin V (IgV) domain, such as TIM family proteins and an immune checkpoint regulator, PD-L1. We found that the p53 induction and maintenance of DD1α expression in apoptotic cells and its subsequent functional intercellular homophilic interaction between apoptotic cells and macrophages are required for engulfment of apoptotic cells. DD1α-deficient mice showed less reduction in organ size and cell number after ionizing radiation (IR), owing to defective dead cell clearance. DD1α-null mice are viable and indistinguishable in appearance from wild-type littermates at an early age. However, at a later age, DD1α deficiency resulted in the development of autoimmune phenotypes and prominent formation of immune infiltrates in the skin, lung, and kidney, which indicated an immune dysregulation and breakdown of self-tolerance in DD1α-null mice. We demonstrated that DD1α also plays an important role as an intercellular homophilic receptor on T cells, which suggests that DD1α is a key-connecting molecule linking postapoptotic processes to immune surveillance. We found that DD1α deficiency in T cells impaired DD1α-mediated inhibitory activity of T cell proliferation. These data indicate that potential homophilic DD1α interactions are important for the DD1α-mediated T cell inhibitory role. Therefore, the results indicate a role for p53 in regulating expression of immune checkpoint regulators, including PD-1, PD-L1, and DD1α. CONCLUSION We found that the tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target DD1α, which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α functions as an engulfment ligand that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages. DD1α-deficient mice showed in vivo defects in clearing dying cells that led to damage to multiple organs indicative of immune dysfunction. p53-induced expression of DD1α is a vital phase for the phagocytic engulfment process of dead cells and then facilitates the stepwise priming of immune surveillance. As a downstream target of the tumor suppressor p53, DD1α activation may extend the repertoire of p53 activities to “guardian of the immune integrity.” p53-dependent accumulation of DD1α and its involvement in dead cell clearance and immune tolerance. DD1α functions as an engulfment ligand that participates in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and phagocytes. p53 induction of DD1α is a critical step in ensuring proper clearance of cell corpses to warrant the efficient generation of precise immune responses, leading to immune tolerance. The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.

Structural Mechanisms Determining Inhibition of the Collagen Receptor Ddr1 by Selective and Multi-Targeted Type II Kinase Inhibitors

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9 nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.

CDIP1-BAP31 Complex Transduces Apoptotic Signals from Endoplasmic Reticulum to Mitochondria under Endoplasmic Reticulum Stress

Resolved endoplasmic reticulum (ER) stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31) as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid) and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.

Revisiting calcein AM: Alternative tool for identifying dye-effluxing cancer stem cells?

Commentary to: Visualization and enrichment of live putative cancer stem cell populations following p53 inactivation or Bax deletion using non-toxic fluorescent dyes Joshua E. Allen, Lori S. Hart, David T. Dicker, Wenge Wang and Wafik S. El-Deiry

Abstract 4605: CDIP-BAP31 complex bridges endoplasmic reticulum and mitochondria during ER-stress mediated apoptosis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Resolved ER stress response is fundamentally essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. In addition, imbalances in homeostasis would result in improper cell functioning that may become an important pathogenic factor in a number of prevalent diseases, including neurodegenerative diseases, metabolic disorders, and cancers. However, the molecular mechanisms relating ER stress to apoptosis still remain largely unfamiliar. Here, we show that a proapoptotic p53 target, CDIP, acts as a key signal transducer of ER stress response. Applying a biochemical/proteomics approach, we characterize BAP31, B-cell receptor-associated protein 31 that is an integral protein of ER and known to crosstalk with mitochondrial proteins during the apoptosis process, as an interacting partner of CDIP. Upon ER stress, CDIP is induced and enhances an association with BAP31 at the ER membrane. We found that the CDIP-BAP31 complex formation is essential for ER stress-mediated apoptotic process. CDIP-/- or CDIP knock-down cells are strongly resistant to ER-stress-induced apoptosis. Moreover, we show that CDIP up-regulation and binding to BAP31 is required for BAP31 cleavage via caspase-8 activation in response to ER stress and also responsible for BAP31-Bcl-2 association, then resulting in Bcl-2 sequestration from BAX. The recruitment of Bcl-2 with BAP31-CDIP complex triggers BAX oligomerization/activation that causes its translocation to the mitochondria, followed by cyctochrome c release. Genetic knockout of CDIP in mice also leads to impaired response to ER-stress-mediated apoptosis, likely through reduced BAX activation. Together, our data demonstrate that the CDIP-BAP31→Bcl-2/BAX regulatory circuit represents a novel mechanism for establishing an ER-mitochondrial cross-talk for ER stress-mediated apoptosis signaling. Citation Format: Takushi Namba, Fang Tian, Kiki Chu, So-Young Hwang, Sam W. Lee. CDIP-BAP31 complex bridges endoplasmic reticulum and mitochondria during ER-stress mediated apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4605. doi:10.1158/1538-7445.AM2013-4605