Identification of a Critical Tyrosine Residue in Caspase 8 That Promotes Cell Migration

Barbero, Simone; Barilá, Daniela; Mielgo, Ainhoa; Stagni, Venturina; Clair, Kiran; Stupack, Dwayne G.

doi:10.1074/jbc.m800549200

Cited by 78 publications

(120 citation statements)

References 16 publications

(27 reference statements)

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“…These data differ from caspase-3, a related enzyme that has been reported to alter transport via a proteolysis-dependent mechanism (37). Nonetheless, recent evidence supports non-apoptotic functions for caspase 8 that are independent of catalytic activity, with several studies supporting a role for caspase 8 in cell migration and invasion (13)(14)(15)(16)22). In fact, caspase 8 was found to be phosphorylated on tyrosine 380 in these studies.…”

Section: Discussionmentioning

confidence: 38%

“…Interestingly, the SH2 domains of p85␣ were recently shown to interact with a phosphorylated tyrosine (Tyr-380) located in a peptide loop between the small and large subunits of the catalytic domain of caspase 8. Interactions between SH2 domains and this tyrosine do not require caspase 8 catalytic activity (13,16). Moreover, p85␣ acts as a Rab-GAP, promoting Rab5 hydrolysis of GTP (8).…”

Section: Caspase 8 Catalytic Activity Is Not Required To Influence Rab5mentioning

confidence: 99%

“…Localization-Proteolytic activity is required for the induction of apoptosis by caspase 8 but is dispensable for other activities, such as the promotion of cell migration (13,14,16). Both apoptosis and cell migration are dependent upon endocytic processes, and it was therefore unclear whether proteolytic activity was required for caspase 8 to regulate the Rab5-mediated trafficking.…”

Section: Caspase 8 Catalytic Activity Is Not Required To Influence Rab5mentioning

confidence: 99%

“…Human caspase 8 was available in the pcDNA3.1(ϩ) and pEGFP-N2 vectors, as previously described (13,19). The caspase 8 mutants C360A and Y380F were previously reported and cloned into pEGFP-N2 vector (13). Bovine FLAG-tagged wild type p85␣, p85␣/R274A (mutant defective for Rab-GAP activity), and p85␣/R368A/R649A (double mutant ⌬R, defective in both SH2 domains via point mutations) were previously described (8).…”

mentioning

confidence: 99%

“…The PCR product was digested and ligated into pGEX-6P1 (GE Healthcare) by using the restriction sites SalI/NotI. Human caspase 8 was available in the pcDNA3.1(ϩ) and pEGFP-N2 vectors, as previously described (13,19). The caspase 8 mutants C360A and Y380F were previously reported and cloned into pEGFP-N2 vector (13).…”

mentioning

confidence: 99%

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Caspase 8 Promotes Peripheral Localization and Activation of Rab5

Torres

Mielgo

Barilá

et al. 2008

Journal of Biological Chemistry

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Caspase 8 is a cysteine protease that initiates apoptotic signaling via the extrinsic pathway in a manner dependent upon association with early endosomes. Previously, we identified caspase 8 as an effector of migration, promoting motility in a manner dependent upon phosphorylation on Tyr-380 by Src family kinases and its subsequent association with Src homology 2 domain-containing proteins. Here we demonstrate the regulation of the small GTPase Rab5, which mediates early endosome formation, homotypic fusion, and maturation by caspase 8. Regulation requires the Tyr-380 phosphorylation site but not caspase proteolytic activity. Tyr-380 is essential for interaction with the Src homology 2 domains of p85␣, a multifunctional adaptor for phosphatidylinositol 3-kinase, that possesses Rab-GAP activity. Interaction between caspase 8 and p85␣ promotes Rab5 GTP loading, alters endosomal trafficking, and results in the accumulation of Rab5-positive endosomes at the edge of the cell. Conversely, caspase 8-dependent GTP loading of Rab5 is overcome by increased expression of p85␣ in a Rab-GAPdependent manner. Thus, we demonstrate a novel function for caspase 8 as a modulator of p85␣ Rab-GAP activity and endosomal trafficking.Rab5 is a small GTPase involved in clathrin-coated vesicle formation, vesicle-early endosome, and early endosome homotypic fusion as well as endosome maturation (for review, see Refs. 1 and 2). Rab5 cycling between the GDP-(inactive) and GTP-bound (active) forms is a process tightly controlled by GTPase-activating proteins (GAPs), 2 guanine nucleotide-exchange factors, and GDP dissociation inhibitors. This strict control is critical to the correct "activation" of Rab5 in time and space (1). GTP-bound Rab5 binds many effectors, including EEA1 (3), Rabaptin5 (4), Rabenosyn5 (5), and phosphatidylinositol 3-kinases (6), thus accounting for its influence on endosome tethering, fusion, and transport (2). The amount of GTP-loaded Rab5 acts as a rate-limiting step influencing the extent of endosome docking and fusion (7). Characterization of GAPs and guanine nucleotide-exchange factors continues to provide new insights on how membrane trafficking is regulated. Recently we characterized the p85␣ subunit of phosphatidylinositol 3-kinase as a Rab-GAP, binding Rab5 via its BH domain, providing a "timing" mechanism for GTP-bound Rab5 (8). Mutation in the BH domain (R274A) impairs Rab-GAP activity, altering the trafficking and degradation of tyrosine kinase receptors (9).Caspase 8 is a cysteine protease that initiates apoptotic signaling via the extrinsic pathway in a manner dependent upon association with early endosomes (10 -12). However, increasing evidence has revealed unexpected non-apoptotic functions of caspase 8, including enhancement of cell adhesion and motility (13-17). Importantly, after phosphorylation (13, 21) caspase 8 was shown to influence cell adhesion and migration via an interaction with p85␣ in a Rac-dependent manner (16).Interestingly, Rab5 was recently shown to regulate cell motility, acting in co...

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Section: Discussionmentioning

confidence: 38%

Section: Caspase 8 Catalytic Activity Is Not Required To Influence Rab5mentioning

confidence: 99%

Section: Caspase 8 Catalytic Activity Is Not Required To Influence Rab5mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Caspase 8 Promotes Peripheral Localization and Activation of Rab5

Torres

Mielgo

Barilá

et al. 2008

Journal of Biological Chemistry

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Caspase‐8 Variant G Regulates Rheumatoid Arthritis Fibroblast‐Like Synoviocyte Aggressive Behavior

Ansalone

Ainsworth

Nygaard

et al. 2021

ACR Open Rheumatology

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Objective Fibroblast‐like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase‐8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs. Methods RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5‐8. Caspase‐8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase‐8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase‐8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase‐8B and G were determined. Results Caspase‐8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase‐8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase‐8) and induced by PDGF. The crystal structures of caspase‐8 variant G and B were identical except for a unique unstructured 59 amino acid N‐terminal domain in variant G. Selective knockdown of caspase‐8G was solely responsible for the effects of caspase‐8 on calpain activity and cell invasion in FLS. Conclusion Blocking caspase‐8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase‐8 inhibition.

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Novel role of ASC as a regulator of metastatic phenotype

et al. 2016

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Disorders of cytoskeletal remodeling and signal transduction are frequently involved in cancer progression. In particular, apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (ASC) has been reported a proapoptotic molecule that is epigenetically silenced in several human cancers. ASC is a well‐characterized adaptor protein involved in the formation of multiprotein oligomers, called inflammasomes, and plays a crucial role in the activation and secretion of interleukin‐1β and interleukin‐18 in innate immune cells. However, the function of ASC in the regulation of tumor progression remains elusive. The present investigation examined the involvement of ASC in cancer progression and the acquisition of metastatic ability. To determine the effect of ASC depletion in in vitro and in vivo model systems, ASC was stably knocked down in B16 murine melanoma cell lines using retroviral transduction of shRNA. ASC suppression increased the motility of B16BL6 cells in scratch assays and augmented invasiveness as assessed by a Matrigel‐coated transwell system. Invadopodia formation and Src phosphorylation level were markedly enhanced in ASC‐knockdown cells as well. Since caspase‐8 has been reported to enhance cellular migration by Tyr380 phosphorylation via Src, we examined Tyr380 phosphorylation of caspase‐8 in ASC‐knockdown cells and found it to be elevated in ASC‐knockdown cells but attenuated by z‐VAD‐fmk or z‐IETD‐fmk. Moreover, ASC ablation increased pulmonary metastasis in mice after intravenous injection of B16BL6 cells. Our cumulative findings indicate that ASC suppresses cancer metastasis and progression via the modulation of cytoskeletal remodeling and the Src‐caspase‐8 signaling pathway.

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Identification of a Critical Tyrosine Residue in Caspase 8 That Promotes Cell Migration

Cited by 78 publications

References 16 publications

Caspase 8 Promotes Peripheral Localization and Activation of Rab5

Caspase 8 Promotes Peripheral Localization and Activation of Rab5

Caspase‐8 Variant G Regulates Rheumatoid Arthritis Fibroblast‐Like Synoviocyte Aggressive Behavior

Novel role of ASC as a regulator of metastatic phenotype

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