Filamin (280-kDa Actin-binding Protein) Is a Caspase Substrate and Is Also Cleaved Directly by the Cytotoxic T Lymphocyte Protease Granzyme B during Apoptosis

Browne, Kylie A.; Johnstone, Ricky W.; Jans, David A.; Trapani, Joseph A.

doi:10.1074/jbc.c000622200

Cited by 82 publications

(47 citation statements)

References 44 publications

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“…Therefore, apoptotic response to radiation is unlikely to be the cause of enhanced toxicity in M2 cells. This observation is consistent with a recent report that a defect of filamin-A did not enhance the apoptotic response in M2 cells but rather that filamin-A might be a caspase substrate and is cleaved directly by protease granzyme B during apoptosis (35).…”

Section: Filamin-a and G 2 Arrestsupporting

confidence: 82%

Recovery from DNA Damage-induced G2 Arrest Requires Actin-binding Protein Filamin-A/Actin-binding Protein 280

Meng

Yuan

Maestas

et al. 2004

Journal of Biological Chemistry

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Filamin-A (filamin-1) is an actin-binding protein involved in the organization of actin networks. Our previous study shows that filamin-A interacts with BRCA2, and lack of filamin-A expression results in increased cellular sensitivity to several DNA damaging agents in melanoma cells (Yuan, Y., and Shen, Z. (2001) J. Biol. Chem. 276, 48318 -48324), suggesting a role of filamin-A in DNA damage response. In this report, we demonstrated that deficiency of filamin-A results in an 8-h delay in the recovery from G 2 arrest in response to ionizing radiation. However, filamin-A deficiency does not affect the initial activation of the G 2 /M checkpoint. We also found that filamin-A deficiency results in sustained activation of Chk1 and Chk2 after irradiation. This in turn causes a delay in the dephosphorylation of phospho-Cdc2, which is inhibitory to the G 2 /M transition. In addition, filamin-A-deficient M2 cells undergo mitotic catastrophe-related nuclear fragmentation after they are released from the G 2 arrest. Together, these data suggest a functional role of filamin-A in the recovery from G 2 arrest and subsequent mitotic cell death after DNA damage.In response to DNA damage, cell cycle checkpoints are activated to delay or block the progression of the cell cycle. This may prevent damaged cells from progressing into the next phase of the cell cycle, thus facilitating the maintenance of genomic stability (1-4). When DNA damage is repaired, cells recover from cell cycle arrest and resume their normal cell cycle progression. Studies in yeast suggest that the transition from cell cycle arrest to cell cycle progression is not simply a passive response to the removal of DNA damage. It appears to be an active process in which cells can adapt to prolonged cell cycle arrest even when the DNA damage has not been completely repaired (5-8). However, the mechanism that regulates the recovery of cell cycle arrest is poorly understood, especially in mammalian cells.Our previous study has shown that an actin-binding protein filamin-A (filamin-1, FLNa, 1 or ABP-280) interacts with BRCA2, and deficiency of filamin-A renders melanoma cells more sensitive to ␥-rays, bleomycin, and UV irradiation (9). In this study, we further investigated the role of filamin-A in cell cycle regulation using a filamin-A-deficient melanoma cell line (M2) and a C8161 melanoma cell line in which filamin-A expression was knocked down by RNA interference. We found that filamin-A deficiency correlated with an approximate 8-h delay in the recovery from G 2 arrest after irradiation. However, filamin-A deficiency had little effect on the initiation of G 2 arrest, indicating intact activation of the G 2 /M checkpoint. The delayed G 2 recovery correlated with delayed dephosphorylation of the phospho-Cdc2 protein and sustained activation of the Chk1 and Chk2 kinases. Furthermore, we found that deficiency of filamin-A renders cells more prone to mitotic cell death. These data provide the first evidence that filamin-A plays a significant role in the recovery from rad...

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Section: Filamin-a and G 2 Arrestsupporting

confidence: 82%

Recovery from DNA Damage-induced G2 Arrest Requires Actin-binding Protein Filamin-A/Actin-binding Protein 280

Meng

Yuan

Maestas

et al. 2004

Journal of Biological Chemistry

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“…Hypoxia is known to increase cytosolic calcium (26) and has been shown to up-regulate calpain protease activity in several cell types, such as pulmonary arterial endothelial cells (27). The ability of calpain to cleave FLNA is also regulated by phosphorylation (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Several kinases, including PKA, Pak1, and RSK, as well as phosphatases such as calcineurin, have been proposed to regulate FLNA phosphorylation (10).…”

Section: Discussionmentioning

confidence: 99%

Hypoxia-induced and calpain-dependent cleavage of filamin A regulates the hypoxic response

Zheng¹,

Zhou

Rouhi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The cellular response to hypoxia is regulated by hypoxia-inducible factor-1α and -2α (HIF-1α and -2α). We have discovered that filamin A (FLNA), a large cytoskeletal actin-binding protein, physically interacts with HIF-1α and promotes tumor growth and angiogenesis. Hypoxia induces a calpain-dependent cleavage of FLNA to generate a naturally occurring C-terminal fragment that accumulates in the cell nucleus. This fragment interacts with the N-terminal portion of HIF-1α spanning amino acid residues 1-390 but not with HIF-2α. In hypoxia this fragment facilitates the nuclear localization of HIF-1α, is recruited to HIF-1α target gene promoters, and enhances HIF-1α function, resulting in up-regulation of HIF-1α target gene expression in a hypoxia-dependent fashion. These results unravel an important mechanism that selectively regulates the nuclear accumulation and function of HIF-1α and potentiates angiogenesis and tumor progression.cancer | subcellular localization | signal transduction

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“…In addition, about 5% of the GrBa transfectants appeared as a volume extension, often with multiple nuclei (Fig. 1B), which suggested some cytoskeletal changes caused by GrBa (27). As assayed by immunofluorescent staining with an anti-␣-tubulin antibody, GrBa-expressing giant cells displayed abnormal cytoskeleton with evidence of leakage of nuclear materials into the cytoplasm as well as multiple spindle poles (Fig.…”

Section: Ectopic Expression Of Grba Contributes To Effective Cellmentioning

confidence: 99%

Secreted Antibody/Granzyme B Fusion Protein Stimulates Selective Killing of HER2-overexpressing Tumor Cells

Zhao

Zhang

Jia

et al. 2004

Journal of Biological Chemistry

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HER2/ErbB2, an epidermal growth factor receptor-related tyrosine kinase, is overexpressed on the surface of a variety of tumors of epithelial origin, such as breast tumors, ovarian carcinomas, and gastric tumors, and therefore constitutes an ideal therapeutic target (1-3). Among the many advances in the development of HER2-targeted cancer therapy over the past decade (4 -7), antibody-based strategies (e.g. herceptin (trastuzumab) (8 -10), intracellular antibody (11, 12), and fusion proteins for targeted delivery of various classes of therapeutic agents (13-17)) are especially notable. Our previous studies exploited the in vitro and in vivo cytotoxicity of immunotoxins by generating a new class of antigen-specific killer cells (18,19), but this approach may well be limited in its clinical applications because of antigenicity of heterogeneous toxin protein. Hence, our attention turned to human endogenous proteins, especially those acting as executioners during apoptosis, which in principle should be optimal candidates for triggering the death of tumor cells in an intrinsic physiological manner.Granzymes are a family of granular serine proteinases produced by cytotoxic T lymphocytes and natural killer cells, with a triad of key residues, histidine, aspartate, and serine, conserved at the catalytic site (20, 21). One member of the family, granzyme B (GrB), 1 plays an essential role in granule-mediated killing. GrB is originally synthesized as zymogen and activated by removal of the amino-terminal signal peptide and an acidic dipeptide after lymphocyte receptor recognition (21). The active GrB (GrBa) is then released from leukocyte granules by exocytosis, entering target cells to cause extensive proteolysis at specific amino acid residues (22, 23). GrB-mediated apoptosis involves the caspase-dependent pathway, direct nuclear entry, and caspase-independent pathways (24 -28). The potential of GrB therapy to induce multiple apoptotic pathways makes this approach to tumor cell killing especially attractive.In our most recent previous study, chimeric immunocasp-3 protein was shown to have a potent selective antitumor effect both in vitro and in vivo (29). This result led us to another targeted pro-apoptotic fusion protein, designated immunoGrB, which consisted of a single-chain anti-HER2 antibody, e23sFv (30, 31), a Pseudomonas exotoxin A (PE) translocation domain (32-35), and active GrB (GrBa) (21). The recombinant immunoGrB molecule was predicted to bind to tumor cells overexpressing HER2 and be internalized, such that endosomal cleavage would give rise to the active granzyme B domain that would consequently translocate into the cytosol to induce tumor cell death (18,19,29). Here we evaluate the antitumor activity of immunoGrB and immunoGrB-secreting lymphocytes in both in vitro and in vivo models. EXPERIMENTAL PROCEDURESConstruction of Expression Plasmids-The human GrB gene was obtained by reverse-transcription PCR from total RNA derived from healthy human peripheral blood mononuclear cells (PBMCs) following stimulatio...

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Filamin (280-kDa Actin-binding Protein) Is a Caspase Substrate and Is Also Cleaved Directly by the Cytotoxic T Lymphocyte Protease Granzyme B during Apoptosis

Cited by 82 publications

References 44 publications

Recovery from DNA Damage-induced G2 Arrest Requires Actin-binding Protein Filamin-A/Actin-binding Protein 280

Recovery from DNA Damage-induced G2 Arrest Requires Actin-binding Protein Filamin-A/Actin-binding Protein 280

Hypoxia-induced and calpain-dependent cleavage of filamin A regulates the hypoxic response

Secreted Antibody/Granzyme B Fusion Protein Stimulates Selective Killing of HER2-overexpressing Tumor Cells

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