Caspase 8 Promotes Peripheral Localization and Activation of Rab5

Abstract

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...

Mutant PIK3CA licenses TRAIL and CD95L to induce non-apoptotic caspase-8-mediated ROCK activation

“…So, in NB7 neuroblastoma cells with hyperactive Src or stimulated with EGF, caspase-8 becomes phosphorylated on Y380 enabling the protein to bind the p85 subunit of PI3K and to activate the PI3K effector Rac, which is of pivotal relevance for cell migration. 29,30 In this model of cell migration, the enzymatic activity of caspase-8 is again dispensable. 29,30 In the same cellular model, a non-catalytic function of caspase-8 in Src-dependent cell adhesion and activation of extracellular-regulated kinase 1/2 has also been shown, but the relation of these events to p85/Rac-mediated cell migration is still unclear.…”

“…29,30 In this model of cell migration, the enzymatic activity of caspase-8 is again dispensable. 29,30 In the same cellular model, a non-catalytic function of caspase-8 in Src-dependent cell adhesion and activation of extracellular-regulated kinase 1/2 has also been shown, but the relation of these events to p85/Rac-mediated cell migration is still unclear. 31 In addition, caspase-8 has also been found to be recruited to the focal adhesion complex in NB7 cells in which it enhances cell migration by stimulating talin cleavage by calpains in a caspase-activity/apoptosisindependent manner.…”

Rab5A is associated with axillary lymph node metastasis in breast cancer patients

“…(32) Caspase-8 was shown to influence cell adhesion and migration through an interaction with PI3K in a Rac-dependent manner, and Rab5 can be activated by caspase-8. (33,34) Some intimate relationships between Rab5 and integrin trafficking are thought to coordinate signaling among endocytic, cytoskeletal, and apoptotic pathways during cell migration. (34,35) Our finding, that cell migration increased under the stimulation of EGF in MDA-MB-231 breast cancer cells, is consistent with these observations.…”

Src-inducible association of CrkL with procaspase-8 promotes cell migration

“…6,7 However, additional roles have been shown by our lab and others, including influencing cellular differentiation, altering endosome trafficking, and enhancing motility and attachment to the extracellular matrix. [8][9][10][11][12][13][14] Molecular toggling between apoptotic and migratory roles can switch caspase-8 from anti-metastatic to pro-metastatic function, 2,3,13 Procaspase-8, the zymogen form of the apoptosis-initiator caspase-8, undergoes phosphorylation following integrinmediated cell attachment to an extracellular matrix substrate. Concordant with cell attachment to fibronectin, a population of procaspase-8 becomes associated with a peripheral insoluble compartment that includes focal complexes and lamellar microfilaments.…”