Antiapoptotic signaling generated by caspase-induced cleavage of RasGAP

Activation of caspases 3 and 9 is thought to commit a cell irreversibly to apoptosis. There are, however, several documented situations (e.g., during erythroblast differentiation) in which caspases are activated and caspase substrates are cleaved with no associated apoptotic response. Why the cleavage of caspase substrates leads to cell death in certain cases but not in others is unclear. One possibility is that some caspase substrates generate antiapoptotic signals when cleaved. Here we show that RasGAP is one such protein. Caspases cleave RasGAP into a C-terminal fragment (fragment C) and an N-terminal fragment (fragment N). Fragment C expressed alone induces apoptosis, but this effect could be totally blocked by fragment N. Fragment N could also block apoptosis induced by low levels of caspase 9. As caspase activity increases, fragment N is further cleaved into fragments N1 and N2. Apoptosis induced by high levels of caspase 9 or by cisplatin was strongly potentiated by fragment N1 or N2 but not by fragment N. The present study supports a model in which RasGAP functions as a sensor of caspase activity to determine whether or not a cell should survive. When caspases are mildly activated, the partial cleavage of RasGAP protects cells from apoptosis. When caspase activity reaches levels that allow completion of RasGAP cleavage, the resulting RasGAP fragments turn into potent proapoptotic molecules.     

Impaired Akt activity down-modulation, caspase-3 activation, and apoptosis in cells expressing a caspase-resistant mutant of RasGAP at position 157

RasGAP bears two caspase-3 cleavage sites that are used sequentially as caspase activity increases in cells. When caspase-3 is mildly activated, RasGAP is first cleaved at position 455. This leads to the production of an N-terminal fragment, called fragment N, that activates the Ras-PI3K-Akt pathway and that promotes cell survival. At higher caspase activity, RasGAP is further cleaved at position 157 generating two small N-terminal fragments named N1 and N2. We have now determined the contribution of this second cleavage event in the regulation of apoptosis using cells in which the wild-type RasGAP gene has been replaced by a cDNA encoding a RasGAP mutant that cannot be cleaved at position 157. Our results show that cleavage of fragment N at position 157 leads to a marked reduction in Akt activity. This is accompanied by efficient processing of caspase-3 that favors cell death in response to various apoptotic stimuli. In nontumorigenic cells, fragments N1 and N2 do not modulate apoptosis. Therefore, the role of the second caspase-mediated cleavage of RasGAP is to allow the inactivation of the antiapoptotic function of fragment N so that caspases are no longer hampered in their ability to kill cells.     

A subset of caspase substrates functions as the Jekyll and Hyde of apoptosis

Cleavage of caspase substrates is believed to be the commitment point that will lead a cell towards apoptosis. While the cleavage of some caspase substrates participates directly in the dismantling of the cell, others regulate the extent of caspase activation. In this communication, we discuss some recent findings indicating that two caspase substrates, MEKK1 and RasGAP, change their functions from anti- to pro-apoptotic as caspase activity increases. MEKK1 is a MAPK kinase kinase regulating the JNK MAPK pathway. As a full-length protein, MEKK1 generates protective signals (e.g. in cardiomyocytes), but potentiates apoptosis when cleaved by caspases. This switch is mediated by a translocation of the kinase activity from insoluble to soluble cellular structures. RasGAP is a regulator of Ras GTPase family members. As a full-length protein, RasGAP does not modulate apoptosis. However, low caspase activity readily induces the cleavage of RasGAP into an N-terminal fragment that generates potent anti-apoptotic signals. At higher caspase activity, the N-terminal fragment is further cleaved into two fragments that strongly potentiate apoptosis. RasGAP can, thus, be viewed as an apoptostat because it allows the cells to determine when caspases have been mildly activated to fulfill functions other than apoptosis or when caspases are strongly activated to mediate apoptosis.     

Partial cleavage of RasGAP by caspases is required for cell survival in mild stress conditions

Tight control of apoptosis is required for proper development and maintenance of homeostasis in multicellular organisms. Cells can protect themselves from potentially lethal stimuli by expressing antiapoptotic factors, such as inhibitors of apoptosis, FLICE (caspase 8)-inhibitory proteins, and members of the Bcl2 family. Here, we describe a mechanism that allows cells to survive once executioner caspases have been activated. This mechanism relies on the partial cleavage of RasGAP by caspase 3 into an amino-terminal fragment called fragment N. Generation of this fragment leads to the activation of the antiapoptotic Akt kinase, preventing further amplification of caspase activity. Partial cleavage of RasGAP is required for cell survival under stress conditions because cells expressing an uncleavable RasGAP mutant cannot activate Akt, cannot prevent amplification of caspase 3 activity, and eventually undergo apoptosis. Executioner caspases therefore control the extent of their own activation by a feedback regulatory mechanism initiated by the partial cleavage of RasGAP that is crucial for cell survival under adverse conditions.     

The role of endogenous and exogenous RasGAP-derived fragment N in protecting cardiomyocytes from peroxynitrite-induced apoptosis

Peroxynitrite (PN) is a potent nitrating and oxidizing agent generated during various pathological situations affecting the heart. The negative effects of PN result, at least in part, from its ability to activate caspases and apoptosis. RasGAP is a ubiquitously expressed protein that is cleaved sequentially by caspase-3. At low caspase-3 activity, RasGAP is cleaved into an N-terminal fragment, called fragment N, that protects cells by activating the Ras/PI3K/Akt pathway. At high caspase-3 activity, fragment N is further cleaved and this abrogates its capacity to stimulate the antiapoptotic Akt kinase. Fragment N formation is crucial for the survival of cells exposed to a variety of stresses. Here we investigate the pattern of RasGAP cleavage upon PN stimulation and the capacity of fragment N to protect cardiomyocytes. PN did not lead to sequential cleavage of RasGAP. Indeed, PN did not allow accumulation of fragment N because it induced its rapid cleavage into smaller fragments. No situations were found in cells treated with PN in which the presence of fragment N was associated with survival. However, expression of a caspase-resistant form of fragment N in cardiomyocytes protected them from PN-induced apoptosis. Our results indicate that the antiapoptotic pathway activated by fragment N is effective at inhibiting PN-induced apoptosis (as seen when cardiomyocytes express a capase-3-resistant form of fragment N) but because fragment N is too transiently generated in response to PN, no survival response is effectively produced. This may explain the marked deleterious consequences of PN generation in various organs, including the heart.     

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G₂/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G₂/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G₂/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.     

Expression of an uncleavable N-terminal RasGAP fragment in insulin-secreting cells increases their resistance toward apoptotic stimuli without affecting their glucose-induced insulin secretion

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.     

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.     

Cleavage and inactivation of antiapoptotic Akt/PKB by caspases during apoptosis

The oncogene Akt/PKB/RAC-PK is a serine/threonine kinase that mediates survival signals and has protective effects against apoptosis induced by a variety of stimuli. The kinase activity of Akt has been demonstrated to be critical in transmitting survival signals. We found that Akt protein was down-regulated during apoptosis. The down-regulation was blocked by a caspase inhibitor, indicating that Akt was cleaved by caspases during apoptosis. The Akt protein incubation with active caspases in vitro revealed that it was cleaved at three sites to produce 40- and 44-kDa fragments. The two cleavage sites were between the NH(2)-terminal pleckstrin homology domain (PH domain) and the kinase domain (TVAD(108 downward arrow)G and EEMD(119 downward arrow)F) and in the COOH-terminal regulatory domain (SETD(434 downward arrow)T). The loss of COOH-terminal domain of the Akt protein reduced its kinase activity and the overexpression of NH(2)-terminal and COOH-terminal-deleted Akt fragment increased the sensitivity to apoptosis-inducing stimuli. These results indicate that caspase-dependent cleavage of anti-apoptotic Akt turns off the survival signals, resulting in the acceleration of apoptotic cell death.     

Role of mTOR,Bad, and Survivin in RasGAP Fragment N-Mediated Cell Protection

Partial cleavage of p120 RasGAP by caspase-3 in stressed cells generates an N-terminal fragment, called fragment N, which activates an anti-apoptotic Akt-dependent survival response. Akt regulates several effectors but which of these mediate fragment N-dependent cell protection has not been defined yet. Here we have investigated the role of mTORC1, Bad, and survivin in the capacity of fragment N to protect cells from apoptosis. Neither rapamycin, an inhibitor of mTORC1, nor silencing of raptor, a subunit of the mTORC1 complex, altered the ability of fragment N from inhibiting cisplatin- and Fas ligand-induced death. Cells lacking Bad, despite displaying a stronger resistance to apoptosis, were still protected by fragment N against cisplatin-induced death. Fragment N was also able to protect cells from Fas ligand-induced death in conditions where Bad plays no role in apoptosis regulation. Fragment N expression in cells did neither modulate survivin mRNA nor its protein expression. Moreover, the expression of cytoplasmic survivin, known to exert anti-apoptotic actions in cells, still occurred in UV-B-irradiated epidermis of mouse expressing a caspase-3-resistant RasGAP mutant that cannot produce fragment N. Additionally, survivin function in cell cycle progression was not affected by fragment N. These results indicate that, taken individually, mTOR, Bad, or Survivin are not required for fragment N to protect cells from cell death. We conclude that downstream targets of Akt other than mTORC1, Bad, or survivin mediate fragment N-induced protection or that several Akt effectors can compensate for each other to induce the pro-survival fragment N-dependent response.     

Cleavage of zetaPKC but not lambda/iotaPKC by caspase-3 during UV-induced apoptosis

The stimulation of caspases is a critical event in apoptotic cell death. Several kinases critically involved in cell proliferation pathways have been shown to be cleaved by caspase-mediated mechanisms. Thus, the degradation of delta protein kinase C (PKC) and MEKK-1 by caspase-3 generates activated fragments corresponding to their catalytic domains, consistent with the observations that both enzymes are important for apoptosis. In contrast, other kinases reported to have anti-apoptotic properties, such as Raf-1 and Akt, are inactivated by proteolytic degradation by the caspase system. Since the atypical PKCs have been shown to play critical roles in cell survival, in the study reported here we have addressed the potential degradation of these PKCs by the caspase system in UV-irradiated HeLa cells. Herein we show that although zetaPKC and lambda/iotaPKC are both inhibited in UV-treated cells, only zetaPKC but not lambda/iotaPKC is cleaved by a caspase-mediated process. This cleavage generates a fragment that corresponds to its catalytic domain that is enzymatically inactive. The sequence where caspase-3 cleaves zetaPKC was mapped, and a mutant resistant to degradation was shown to protect cells from apoptosis more efficiently than the wild-type enzyme.     

The cleavage of Akt/protein kinase B by death receptor signaling is an important event in detachment-induced apoptosis.

Epithelial cells undergo death receptor-dependent apoptosis when detached from matrix, a process termed anoikis. Activation of Akt/protein kinase B (PKB) by matrix attachment protects cells from anoikis. In this study, we establish a link between anoikis and Akt/PKB-mediated survival by demonstrating that Akt/PKB is cleaved by caspases in matrix-detached epithelial cells by a mechanism that involves death receptors. Reduced levels of Akt/PKB protein were observed in detached Madin-Darby canine kidney cells relative to cells attached to collagen. Equivalent levels of Akt/PKB, however, were detected in matrix-adherent and detached cells after inhibition of caspase activity or expression of an Akt/PKB mutant (D108+119A) that is resistant to caspase cleavage. The contribution of death domain-containing proteins to Akt/PKB cleavage was evidenced by the ability of dominant negative Fas-associated death domain to restore normal levels of Akt/PKB in matrix-detached cells. Importantly, expression of a cleavage-resistant Akt/PKB mutant protected matrix-detached cells from apoptosis. These studies suggest that members of the death receptor family promote the caspase-mediated cleavage of Akt/PKB and that this event contributes to anoikis.     

SHP-2 regulates the phosphatidylinositide 3'-kinase/Akt pathway and suppresses caspase 3-mediated apoptosis

The Src homology domain 2 (SH2)-containing tyrosine phosphatase SHP-2 has been implicated in the regulation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway. The ability of SHP-2 to regulate the PI3K/Akt pathway is suggested to result in the positive effect of SHP-2 on cell survival. Whether SHP-2 regulates insulin-like growth factor-1 (IGF-1)-dependent activation of Akt at the level of PI3K has yet to be established. Furthermore, the identification of the down-stream apoptotic target engaged by SHP-2 in cell survival also has yet to be determined. Here, we show that overexpression of a catalytically inactive mutant of SHP-2 inhibited insulin-like growth factor-1 (IGF-1)-dependent PI3K and Akt activation. Consistent with the observation that SHP-2 participates in pro-survival signaling fibroblasts expressing a deletion within exon 3 of SHP-2, which results in a truncation of the amino-terminus SH2 domain (SHP-2(Ex3-/-)), were hypersensitive to etoposide-induced cell death. SHP-2(Ex3-/-) fibroblasts exhibited enhanced levels of etoposide-induced caspase 3 activity as compared to wild-type fibroblasts and the enhanced level of caspase 3 activity was suppressed by a caspase 3-specific inhibitor. Re-introduction of wild-type SHP-2 into the SHP-2(Ex3-/-) fibroblasts rescued the hypersensitivity to etoposide-induced caspase 3 activation. The effects of abrogating SHP-2 function on cell survival were not specific to the loss of the amino-terminus SH2 domain of SHP-2 since RNAi-mediated knock-down of SHP-2 also reduced cell survival. Taken together, these data indicate that the catalytic activity of SHP-2 is required to regulate the PI3K/Akt pathway and thus likely participates in anti-apoptotic signaling by suppressing caspase 3-mediated apoptosis.     

Ras GTPase-activating protein binds to Akt and is required for its activation

RasGAP (Ras GTPase-activating protein) is a negative regulator as well as a downstream effector of Ras. To identify partners of RasGAP we used it as the bait in a yeast two-hybrid screen. This resulted in discovering its interaction with Akt. Overexpression of RasGAP or a mutant lacking the GTPase-activating domain (nGAP) enhanced phosphorylation and activity of Akt, which was dependent on the upstream integrin-linked kinase. Also, nGAP protected the cells against staurosporin-induced apoptosis through an Akt-dependent pathway. To determine the role of RasGAP in receptor-mediated activation of Akt, we used short hairpin RNA interference to knock out endogenous RasGAP expression. Although this procedure resulted in enhanced Ras activity, it inhibited Akt phosphorylation. Thus, we propose that Ras-GAP interacts with Akt and is necessary for its activation, possibly via integrin-linked kinase-mediated phosphorylation of Ser-473. The data suggest that this effect is independent of Ras activity.     

Regulation of apoptosis by phosphatidylinositol 4,5-bisphosphate inhibition of caspases, and caspase inactivation of phosphatidylinositol phosphate 5-kinases

Phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the apoptotic machinery. We now report that another phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) is a direct inhibitor of initiator caspases 8 and 9, and their common effector caspase 3. PIP2 inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apoptosome system. It inhibited purified caspase 3 and 8 activity, at physiologically attainable PIP2 levels in mixed lipid vesicles. Caspase 3 binding to PIP2 was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase alpha (PIP5KIalpha), which synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KIalpha was accompanied by decreases in the generation of activated caspases and of caspase 3-cleaved PARP. Protection was not mediated through PIP3 or Akt activation. An anti-apoptotic role for PIP(2) is further substantiated by our finding that PIP5KIalpha was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KIalpha in vitro. Mutation of the P(4) position (D279A) of the PIP5KIalpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KIalpha mutant was more effective in suppressing apoptosis than the wild-type kinase. These results show that PIP2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KIalpha inactivation contributes to the progression of apoptosis. This novel feedforward amplification mechanism for maintaining the balance between life and death of a cell works through phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.     

A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.     

Mechanisms for the magnolol-induced cell death of CGTH W-2 thyroid carcinoma cells

Magnolol, a substance purified from the bark of Magnolia officialis, inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The aim of this study was to study the effects of magnolol on CGTH W-2 thyroid carcinoma cells. After 24 h treatment with 80 microM magnolol in serum-containing medium, about 50% of the cells exhibited apoptotic features and 20% necrotic features. Cytochrome-c staining was diffused in the cytoplasm of the apoptotic cells, but restricted to the mitochondria in control cells. Western blot analyses showed an increase in levels of activated caspases (caspase-3 and -7) and of cleaved poly (ADP-ribose) polymerase (PARP) by magnolol. Concomitantly, immunostaining for apoptosis inducing factor (AIF) showed a time-dependent translocation from the mitochondria to the nucleus. Inhibition of either PARP or caspase activity blocked magnolol-induced apoptosis, supporting the involvement of the caspases and PARP. In addition, magnolol activated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. These data suggest that magnolol promoted apoptosis probably by alleviating the inhibitory effect of Akt on caspase 9. Furthermore, inhibition of PARP activity, but not of caspase activity, completely prevented magnolol-induced necrosis, suggesting the notion that it might be caused by depletion of intracellular ATP levels due to PARP activation. These results show that magnolol initiates apoptosis via the cytochrome-c/caspase 3/PARP/AIF and PTEN/Akt/caspase 9/PARP pathways and necrosis via PARP activation.     

Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1

RasGAP is a multifunctional protein that controls Ras activity and that is found in chromosomal passenger complexes. It also negatively or positively regulates apoptosis depending on the extent of its cleavage by caspase-3. RasGAP has been reported to bind to G3BP1 (RasGAP SH3-domain-binding protein 1), a protein regulating mRNA stability and stress granule formation. The region of RasGAP (amino acids 317–326) thought to bind to G3BP1 corresponds exactly to the sequence within fragment N2, a caspase-3-generated fragment of RasGAP, that mediates sensitization of tumor cells to genotoxins. While assessing the contribution of G3BP1 in the anti-cancer function of a cell-permeable peptide containing the 317–326 sequence of RasGAP (TAT-RasGAP_317–326), we found that, in conditions where G3BP1 and RasGAP bind to known partners, no interaction between G3BP1 and RasGAP could be detected. TAT-RasGAP_317–326 did not modulate binding of G3BP1 to USP10, stress granule formation or c-myc mRNA levels. Finally, TAT-RasGAP_317–326 was able to sensitize G3BP1 knock-out cells to cisplatin-induced apoptosis. Collectively these results indicate that G3BP1 and its putative RasGAP binding region have no functional influence on each other. Importantly, our data provide arguments against G3BP1 being a genuine RasGAP-binding partner. Hence, G3BP1-mediated signaling may not involve RasGAP.     

Vagus Nerve Stimulation Attenuates Cerebral Ischemia and Reperfusion Injury via Endogenous Cholinergic Pathway in Rat

Inflammation and apoptosis play critical roles in the acute progression of ischemic injury pathology. Emerging evidence indicates that vagus nerve stimulation (VNS) following focal cerebral ischemia and reperfusion (I/R) may be neuroprotective by limiting infarct size. However, the underlying molecular mechanisms remain unclear. In this study, we investigated whether the protective effects of VNS in acute cerebral I/R injury were associated with anti-inflammatory and anti-apoptotic processes. Male Sprague-Dawley (SD) rats underwent VNS at 30 min after focal cerebral I/R surgery. Twenty-four h after reperfusion, neurological deficit scores, infarct volume, and neuronal apoptosis were evaluated. In addition, the levels of pro-inflammatory cytokines were detected using enzyme-linked immune sorbent assay (ELISA), and immunofluorescence staining for the endogenous “cholinergic anti-inflammatory pathway” was also performed. The protein expression of a7 nicotinic acetylcholine receptor (a7nAchR), phosphorylated Akt (p-Akt), and cleaved caspase 3 in ischemic penumbra were determined with Western blot analysis. I/R rats treated with VNS (I/R+VNS) had significantly better neurological deficit scores, reduced cerebral infarct volume, and decreased number of TdT mediated dUTP nick end labeling (TUNEL) positive cells. Furthermore, in the ischemic penumbra of the I/R+VNS group, the levels of pro-inflammatory cytokines and cleaved caspase 3 protein were significantly decreased, and the levels of a7nAchR and phosphorylated Akt were significantly increased relative to the I/R alone group. These results indicate that VNS is neuroprotective in acute cerebral I/R injury by suppressing inflammation and apoptosis via activation of cholinergic and a7nAchR/Akt pathways.     

Anti-apoptotic Role of Caspase-cleaved GAB1 Adaptor Protein in Hepatocyte Growth Factor/Scatter Factor-MET Receptor Protein Signaling

The GRB2-associated binder 1 (GAB1) docking/scaffold protein is a key mediator of the MET-tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). Activated MET promotes recruitment and tyrosine phosphorylation of GAB1, which in turn recruits multiple proteins and mediates MET signaling leading to cell survival, motility, and morphogenesis. We previously reported that, without its ligand, MET is a functional caspase target during apoptosis, allowing the generation of a p40-MET fragment that amplifies apoptosis. In this study we established that GAB1 is also a functional caspase target by evidencing a caspase-cleaved p35-GAB1 fragment that contains the MET binding domain. GAB1 is cleaved by caspases before MET, and the resulting p35-GAB1 fragment is phosphorylated by MET upon HGF/SF binding and can interact with a subset of GAB1 partners, PI3K, and GRB2 but not with SHP2. This p35-GAB1 fragment favors cell survival by maintaining HGF/SF-induced MET activation of AKT and by hindering p40-MET pro-apoptotic function. These data demonstrate an anti-apoptotic role of caspase-cleaved GAB1 in HGF/SF-MET signaling.