Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated with Phosphorylation of Raptor by mTOR

mTORC1 contains multiple proteins and plays a central role in cell growth and metabolism. Raptor (regulatory-associated protein of mammalian target of rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential for mTORC1 activity and critical for the regulation of mTORC1 activity in response to insulin signaling and nutrient and energy sufficiency. Herein we demonstrate that mTOR phosphorylates raptor in vitro and in vivo. The phosphorylated residues were identified by using phosphopeptide mapping and mutagenesis. The phosphorylation of raptor is stimulated by insulin and inhibited by rapamycin. Importantly, the site-directed mutation of raptor at one phosphorylation site, Ser⁸⁶³, reduced mTORC1 activity both in vitro and in vivo. Moreover, the Ser⁸⁶³ mutant prevented small GTP-binding protein Rheb from enhancing the phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate that mTOR-mediated raptor phosphorylation plays an important role on activation of mTORC1.Mammalian target of rapamycin (mTOR)² has been shown to function as a critical controller in cellular growth, survival, metabolism, and development (1). mTOR, a highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase, structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each of which catalyzes the phosphorylation of different substrates (1). The best characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also known as PHAS) and p70 S6 kinase (S6K) (1), whereas mTORC2 phosphorylates the hydrophobic and turn motifs of protein kinase B (Akt/protein kinase B) (2) and protein kinase C (3, 4). mTORC1 constitutively consists of mTOR, raptor, and mLst8/GβL (1), whereas the proline-rich Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that disassociates after growth factor stimulation (5, 6). Raptor is essential for mTORC1 activity by providing a substrate binding function (7) but also plays a regulatory role on mTORC1 with stimuli of growth factors and nutrients (8). In response to insulin, raptor binding to substrates is elevated through the release of the competitive inhibitor PRAS40 from mTORC1 (9, 10) because PRAS40 and the substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus sequence, the TOR signaling (TOS) motif (10–14). In response to amino acid sufficiency, raptor directly interacts with a heterodimer of Rag GTPases and promotes mTORC1 localization to the Rheb-containing vesicular compartment (15).mTORC1 integrates signaling pathways from growth factors, nutrients, energy, and stress, all of which generally converge on the tuberous sclerosis complex (TSC1-TSC2) through the phosphorylation of TSC2 (1). Growth factors inhibit the GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via the PI3K/Akt pathway (16, 17), whereas energy depletion activates TSC2 GTPase-activating protein activity by stimulating AMP-activated protein kinase (AMPK) (18). Rheb binds directly to mTOR, albeit with very low affinity (19), and upon charging with GTP, Rheb functions as an mTORC1 activator (6). mTORC1 complexes isolated from growth factor-stimulated cells show increased kinase activity yet do not contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding to mTOR leads to an increase in mTORC1 activity toward substrates, and what the role of raptor is in this activation is currently unknown. More recently, the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative regulation mechanism independent of TSC2 in response to energy supply (20). RSK-mediated raptor phosphorylation enhances mTORC1 activity and provides a mechanism whereby stress may activate mTORC1 independent of the PI3K/Akt pathway (21). Therefore, the phosphorylation status of raptor can be critical for the regulation of mTORC1 activity.In this study, we investigated phosphorylation sites in raptor catalyzed by mTOR. Using two-dimensional phosphopeptide mapping, we found that Ser⁸⁶³ and Ser⁸⁵⁹ in raptor were phosphorylated by mTOR both in vivo and in vitro. mTORC1 activity in vitro and in vivo is associated with the phosphorylation of Ser⁸⁶³ in raptor.     

Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects ∼1% of the population worldwide. The causes of sporadic cases are unknown, although mitochondrial or oxidative toxins such as 1-methyl-4-phenylpyridinium, 6-hydroxydopamine (6-OHDA),³ and rotenone reproduce features of the disease in animal and cell culture models (1). Abnormalities in mitochondrial respiration and increased oxidative stress are observed in cells and tissues from parkinsonian patients (2, 3), which also exhibit increased mitochondrial autophagy (4). Furthermore, mutations in parkinsonian genes affect oxidative stress response pathways and mitochondrial homeostasis (5). Thus, disruption of mitochondrial homeostasis represents a major factor implicated in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD encodes for PTEN-induced kinase 1 (PINK1) (6, 7). PINK1 is a cytosolic and mitochondrially localized 581-amino acid serine/threonine kinase that possesses an N-terminal mitochondrial targeting sequence (6, 8). The primary sequence also includes a putative transmembrane domain important for orientation of the PINK1 domain (8), a conserved kinase domain homologous to calcium calmodulin kinases, and a C-terminal domain that regulates autophosphorylation activity (9, 10). Overexpression of wild-type PINK1, but not its PD-associated mutants, protects against several toxic insults in neuronal cells (6, 11, 12). Mitochondrial targeting is necessary for some (13) but not all of the neuroprotective effects of PINK1 (14), implicating involvement of cytoplasmic targets that modulate mitochondrial pathobiology (8). PINK1 catalytic activity is necessary for its neuroprotective role, because a kinase-deficient K219M substitution in the ATP binding pocket of PINK1 abrogates its ability to protect neurons (14). Although PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated mutations differentially destabilize the protein, resulting in loss of neuroprotective activities (13, 15).Recent studies indicate that PINK1 and Parkin interact genetically (3, 16-18) to prevent oxidative stress (19, 20) and regulate mitochondrial morphology (21). Primary cells derived from PINK1 mutant patients exhibit mitochondrial fragmentation with disorganized cristae, recapitulated by RNA interference studies in HeLa cells (3).Mitochondria are degraded by macroautophagy, a process involving sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs) for delivery to lysosomes (22, 23). Interestingly, mitochondrial fission accompanies autophagic neurodegeneration elicited by the PD neurotoxin 6-OHDA (24, 25). Moreover, mitochondrial fragmentation and increased autophagy are observed in neurodegenerative diseases including Alzheimer and Parkinson diseases (4, 26-28). Although inclusion of mitochondria in autophagosomes was once believed to be a random process, as observed during starvation, studies involving hypoxia, mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic substrates in facultative anaerobes support the concept of selective mitochondrial autophagy (mitophagy) (29, 30). In particular, mitochondrially localized kinases may play an important role in models involving oxidative mitochondrial injury (25, 31, 32).Autophagy is involved in the clearance of protein aggregates (33-35) and normal regulation of axonal-synaptic morphology (36). Chronic disruption of lysosomal function results in accumulation of subtly impaired mitochondria with decreased calcium buffering capacity (37), implicating an important role for autophagy in mitochondrial homeostasis (37, 38). Recently, Parkin, which complements the effects of PINK1 deficiency on mitochondrial morphology (3), was found to promote autophagy of depolarized mitochondria (39). Conversely, Beclin 1-independent autophagy/mitophagy contributes to cell death elicited by the PD toxins 1-methyl-4-phenylpyridinium and 6-OHDA (25, 28, 31, 32), causing neurite retraction in cells expressing a PD-linked mutation in leucine-rich repeat kinase 2 (40). Whereas properly regulated autophagy plays a homeostatic and neuroprotective role, excessive or incomplete autophagy creates a condition of “autophagic stress” that can contribute to neurodegeneration (28).As mitochondrial fragmentation (3) and increased mitochondrial autophagy (4) have been described in human cells or tissues of PD patients, we investigated whether or not the engineered loss of PINK1 function could recapitulate these observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous PINK1 gave rise to mitochondrial fragmentation and increased autophagy and mitophagy, whereas stable or transient overexpression of PINK1 had the opposite effect. Autophagy/mitophagy was dependent upon increased mitochondrial oxidant production and activation of fission. The data indicate that PINK1 is important for the maintenance of mitochondrial networks, suggesting that coordinated regulation of mitochondrial dynamics and autophagy limits cell death associated with loss of PINK1 function.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45: MULTIPROTEIN COMPLEXES RETAIN ACTIVE PHOSPHORYLATED ERK AND RSK AND PROTECT THEM FROM DEPHOSPHORYLATION*

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities (Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82 ,1838 -1850). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signal-regulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase (ERK)² mitogen-activated protein kinase (MAPK) signaling pathway has been implicated in diverse cellular physiological processes including proliferation, survival, growth, differentiation, and motility (1-4) and is also exploited by a variety of viruses such as Kaposi sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, coxsackie, vaccinia, coronavirus, and influenza virus (5-17). The MAPK kinases relay the extracellular signaling through sequential phosphorylation to an array of cytoplasmic and nuclear substrates to elicit specific responses (1, 2, 18). Phosphorylation of MAPK is reversible. The kinetics of deactivation or duration of signaling dictates diverse biological outcomes (19, 20). For example, sustained but not transient activation of ERK signaling induces the differentiation of PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells (20-22). During viral infection, a unique biphasic ERK activation has been observed for some viruses (an early transient activation triggered by viral binding or entry and a late sustained activation correlated with viral gene expression), but the responsible viral factors and underlying mechanism for the sustained ERK activation remain largely unknown (5, 8, 13, 23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie at the terminus of the ERK pathway (1, 24-26). In mammals, four isoforms are known, RSK1 to RSK4. Each one has two catalytically functional kinase domains, the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) as well as a linker region between the two. The NTKD is responsible for phosphorylation of exogenous substrates, and the CTKD and linker region regulate RSK activation (1, 24, 25). In quiescent cells ERK binds to the docking site in the C terminus of RSK (27-29). Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase (MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD activation loop. The activated CTKD then phosphorylates Ser-380 in the linker region, creating a docking site for 3-phosphoinositide-dependent protein kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates Ser-221 of RSK in the activation loop and activates the NTKD. The activated NTKD autophosphorylates the serine residue near the ERK docking site, causing a transient dissociation of active ERK from RSK (25, 26, 28). The stimulation of quiescent cells by a mitogen such as epidermal growth factor or a phorbol ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually results in a transient RSK activation that lasts less than 30 min. RSKs have been implicated in regulating cell survival, growth, and proliferation. Mutation or aberrant expression of RSK has been implicated in several human diseases including Coffin-Lowry syndrome and prostate and breast cancers (1, 24, 25, 30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman disease (33, 34). Infection and reactivation of KSHV activate multiple MAPK pathways (6, 12, 35). Noticeably, the ERK/RSK activation is sustained late during KSHV primary infection and reactivation from latency (5, 6, 12, 23), but the mechanism of the sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45, an immediate early and also virion tegument protein of KSHV, interacts with RSK1 and RSK2 and strongly stimulates their kinase activities (23). We also demonstrated that the activation of RSK plays an essential role in KSHV lytic replication (23). In the present study we determined the mechanism of ORF45-induced sustained ERK/RSK activation. We found that ORF45 increases the association of RSK with ERK and protects them from dephosphorylation, causing sustained activation of both ERK and RSK.     

Role of JNK1-dependent Bcl-2 Phosphorylation in Ceramide-induced Macroautophagy

Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C₂-ceramide and C₆-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr⁶⁹, Ser⁷⁰, and Ser⁸⁷, located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress.Macroautophagy (referred to below as “autophagy”) is a vacuolar, lysosomal degradation pathway for cytoplasmic constituents that is conserved in eukaryotic cells (1–3). Autophagy is initiated by the formation of a multimembrane-bound autophagosome that engulfs cytoplasmic proteins and organelles. The last stage in the process results in fusion with the lysosomal compartments, where the autophagic cargo undergoes degradation. Basal autophagy is important in controlling the quality of the cytoplasm by removing damaged organelles and protein aggregates. Inhibition of basal autophagy in the brain is deleterious, and leads to neurodegeneration in mouse models (4, 5). Stimulation of autophagy during periods of nutrient starvation is a physiological response present at birth and has been shown to provide energy in various tissues of newborn pups (6). In cultured cells, starvation-induced autophagy is an autonomous cell survival mechanism, which provides nutrients to maintain a metabolic rate and level of ATP compatible with cell survival (7). In addition, starvation-induced autophagy blocks the induction of apoptosis (8). In other contexts, such as drug treatment and a hypoxic environment, autophagy has also been shown to be cytoprotective in cancer cells (9, 10). However, autophagy is also part of cell death pathways in certain situations (11). Autophagy can be a player in apoptosis-independent type-2 cell death (type-1 cell death is apoptosis), also known as autophagic cell death. This situation has been shown to occur when the apoptotic machinery is crippled in mammalian cells (12, 13). Autophagy can also be part of the apoptotic program, for instance in tumor necrosis factor-α-induced cell death when NF-κB is inhibited (14), or in human immunodeficiency virus envelope-mediated cell death in bystander naive CD4 T cells (15). Moreover autophagy has recently been shown to be required for the externalization of phosphatidylserine, the eat-me signal for phagocytic cells, at the surface of apoptotic cells (16).The complex relationship between autophagy and apoptosis reflects the intertwined regulation of these processes (17, 18). Many signaling pathways involved in the regulation of autophagy also regulate apoptosis. This intertwining has recently been shown to occur at the level of the molecular machinery of autophagy. In fact the anti-apoptotic protein Bcl-2 has been shown to inhibit starvation-induced autophagy by interacting with the autophagy protein Beclin 1 (19). Beclin 1 is one of the Atg proteins conserved from yeast to humans (it is the mammalian orthologue of yeast Atg6) and is involved in autophagosome formation (20). Beclin 1 is a platform protein that interacts with several different partners, including hVps34 (class III phosphatidylinositol 3-kinase), which is responsible for the synthesis of phosphatidylinositol 3-phosphate. The production of this lipid is important for events associated with the nucleation of the isolation membrane before it elongates and closes to form autophagosomes in response to other Atg proteins, including the Atg12 and LC3² (microtubule-associated protein light chain 3 is the mammalian orthologue of the yeast Atg8) ubiquitin-like conjugation systems (3, 21). Various partners associated with the Beclin 1 complex modulate the activity of hVps34. For instance, Bcl-2 inhibits the activity of this enzyme, whereas UVRAG, Ambra-1, and Bif-1 all up-regulate it (22, 23).In view of the intertwining between autophagy and apoptosis, it is noteworthy that Beclin 1 belongs to the BH3-only family of proteins (24–26). However, and unlike most of the proteins in this family, Beclin 1 is not able to trigger apoptosis when its expression is forced in cells (27). A BH3-mimetic drug, ABT-737, is able to dissociate the Beclin 1-Bcl-2 complex, and to trigger autophagy by mirroring the effect of starvation (25).The sphingolipids constitute a family of bioactive lipids (28–32) of which several members, such as ceramide and sphingosine 1-phosphate, are signaling molecules. These molecules constitute a “sphingolipid rheostat” that determines the fate of the cell, because in many settings ceramide is pro-apoptotic and sphingosine 1-phosphate mitigates this apoptotic effect (31, 32). However, ceramide is also engaged in a wide variety of other cell processes, such as the formation of exosomes (33), differentiation, cell proliferation, and senescence (34). Recently we showed that both ceramide and sphingosine 1-phosphate are able to stimulate autophagy (35, 36). It has also been shown that ceramide triggers autophagy in a large panel of mammalian cells (37–39). However, elucidation of the mechanism by which ceramide stimulates autophagy is still in its infancy. We have previously demonstrated that ceramide induces autophagy in breast and colon cancer cells by inhibiting the Class I phosphatidylinositol 3-phosphate/mTOR signaling pathway, which plays a central role in inhibiting autophagy (36). Inhibition of mTOR is another hallmark of starvation-induced autophagy (17). This finding led us to investigate the effect of ceramide on the Beclin 1-Bcl-2 complex. The results presented here show that ceramide is more potent than starvation in dissociating the Beclin 1-Bcl-2 complex (see Ref. 40). This dissociation is dependent on three phosphorylation sites (Thr⁶⁹, Ser⁷⁰, and Ser⁸⁷) located in a non-structural loop of Bcl-2. Ceramide induces the c-Jun N-terminal kinase 1-dependent phosphorylation of Bcl-2. Expression of a dominant negative form of JNK1 blocks Bcl-2 phosphorylation, and thus the induction of autophagy by ceramide. These findings help to explain how autophagy is regulated by a major lipid second messenger.     

Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

ATP-binding cassette (ABC) transporters transduce the free energy of ATP hydrolysis to power the mechanical work of substrate translocation across cell membranes. MsbA is an ABC transporter implicated in trafficking lipid A across the inner membrane of Escherichia coli. It has sequence similarity and overlapping substrate specificity with multidrug ABC transporters that export cytotoxic molecules in humans and prokaryotes. Despite rapid advances in structure determination of ABC efflux transporters, little is known regarding the location of substrate-binding sites in the transmembrane segment and the translocation pathway across the membrane. In this study, we have mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster at the cytoplasmic end of helices 3 and 6 at a site accessible from the membrane/water interface and extending into an aqueous chamber formed at the interface between the two transmembrane domains. Binding of a nonhydrolyzable ATP analog inverts the transporter to an outward-facing conformation and relieves DNR quenching by spin labels suggesting DNR exclusion from proximity to the spin labels. The simplest model consistent with our data has DNR entering near an elbow helix parallel to the water/membrane interface, partitioning into the open chamber, and then translocating toward the periplasm upon ATP binding.ATP-binding cassette (ABC)² transporters transduce the energy of ATP hydrolysis to power the movement of a wide range of substrates across the cell membranes (1, 2). They constitute the largest family of prokaryotic transporters, import essential cell nutrients, flip lipids, and export toxic molecules (3). Forty eight human ABC transporters have been identified, including ABCB1, or P-glycoprotein, which is implicated in cross-resistance to drugs and cytotoxic molecules (4, 5). Inherited mutations in these proteins are linked to diseases such as cystic fibrosis, persistent hypoglycemia of infancy, and immune deficiency (6).The functional unit of an ABC transporter consists of four modules. Two highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze ATP to supply the active energy for transport (7). ABCs drive the mechanical work of proteins with diverse functions ranging from membrane transport to DNA repair (3, 5). Substrate specificity is determined by two transmembrane domains (TMDs) that also provide the translocation pathway across the bilayer (7). Bacterial ABC exporters are expressed as monomers, each consisting of one NBD and one TMD, that dimerize to form the active transporter (3). The number of transmembrane helices and their organization differ significantly between ABC importers and exporters reflecting the divergent structural and chemical nature of their substrates (1, 8, 9). Furthermore, ABC exporters bind substrates directly from the cytoplasm or bilayer inner leaflet and release them to the periplasm or bilayer outer leaflet (10, 11). In contrast, bacterial importers have their substrates delivered to the TMD by a dedicated high affinity substrate-binding protein (12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on the inner membrane to its final destination in the outer membrane requires the ABC transporter MsbA (13). Although MsbA has not been directly shown to transport lipid A, suppression of MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits bacterial growth strongly suggesting a role in translocation (14-16). In addition to this role in lipid A transport, MsbA shares sequence similarity with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus lactis, and Sav1866 of Staphylococcus aureus (16-19). ABCB1, a prototype of the ABC family, is a plasma membrane protein whose overexpression provides resistance to chemotherapeutic agents in cancer cells (1). LmrA and MsbA have overlapping substrate specificity with ABCB1 suggesting that both proteins can function as drug exporters (18, 20). Indeed, cells expressing MsbA confer resistance to erythromycin and ethidium bromide (21). MsbA can be photolabeled with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342 ({"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342) across membrane vesicles in an energy-dependent manner (21).The structural mechanics of ABC exporters was revealed from comparison of the MsbA crystal structures in the apo- and nucleotide-bound states as well as from analysis by spin labeling EPR spectroscopy in liposomes (17, 19, 22, 23). The energy harnessed from ATP binding and hydrolysis drives a cycle of NBD association and dissociation that is transmitted to induce reorientation of the TMD from an inward- to outward-facing conformation (17, 19, 22). Large amplitude motion closes the cytoplasmic end of a chamber found at the interface between the two TMDs and opens it to the periplasm (23). These rearrangements lead to significant changes in chamber hydration, which may drive substrate translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile, wasting the energy of ATP hydrolysis without substrate extrusion (7). Consistent with this model, ATP binding reduces ABCB1 substrate affinity, potentially through binding site occlusion (24-26). Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP hydrolysis increasing transport efficiency (1, 27, 28). However, there is a paucity of information regarding the location of substrate binding, the transport pathway, and the structural basis of substrate recognition by ABC exporters. In vitro studies of MsbA substrate specificity identify a broad range of substrates that stimulate ATPase activity (29). In addition to the putative physiological substrates lipid A and lipopolysaccharide (LPS), the ABCB1 substrates Ilmofosine, {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342, and verapamil differentially enhance ATP hydrolysis of MsbA (29, 30). Intrinsic MsbA tryptophan (Trp) fluorescence quenching by these putative substrate molecules provides further support of interaction (29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model of substrate binding to ABC efflux exporters. This so-called “hydrophobic cleaner model” describes substrates binding from the inner leaflet of the bilayer and then translocating through the TMD (10, 31, 32). These studies also identified a large number of residues involved in substrate binding and selectivity (33). When these crucial residues are mapped onto the crystal structures of MsbA, a subset of homologous residues clusters to helices 3 and 6 lining the putative substrate pathway (34). Consistent with a role in substrate binding and specificity, simultaneous replacement of two serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport of ethidium and taxol, although {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 and erythromycin interactions remain unaffected (34).The tendency of lipophilic substrates to partition into membranes confounds direct analysis of substrate interactions with ABC exporters (35, 36). Such partitioning may promote dynamic collisions with exposed Trp residues and nonspecific cross-linking in photo-affinity labeling experiments. In this study, we utilize a site-specific quenching approach to identify residues in the vicinity of the daunorubicin (DNR)-binding site (37). Although the data on DNR stimulation of ATP hydrolysis is inconclusive (20, 29, 30), the quenching of MsbA Trp fluorescence suggests a specific interaction. Spin labels were introduced along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent quenching of DNR fluorescence. Residues that quench DNR cluster along the cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR. Furthermore, many of these residues are not lipid-exposed but face the putative substrate chamber formed between the two TMDs. These residues are proximal to two Trps, which likely explains the previously reported quenching (29). Our results suggest DNR partitions to the membrane and then binds MsbA in a manner consistent with the hydrophobic cleaner model. Interpretation in the context of the crystal structures of MsbA identifies a putative translocation pathway through the transmembrane segment.     

Overexpression of E-cadherin on Melanoma Cells Inhibits Chemokine-promoted Invasion Involving p190RhoGAP/p120ctn-dependent Inactivation of RhoA

Melanoma cells express the chemokine receptor CXCR4 that confers high invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial stages of the disease show reduction or loss of E-cadherin expression, but recovery of its expression is frequently found at advanced phases. We overexpressed E-cadherin in the highly invasive BRO lung metastatic cell melanoma cell line to investigate whether it could influence CXCL12-promoted cell invasion. Overexpression of E-cadherin led to defective invasion of melanoma cells across Matrigel and type I collagen in response to CXCL12. A decrease in individual cell migration directionality toward the chemokine and reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent inhibition of RhoA activation was responsible for the impairment in chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore, we show that p190RhoGAP and p120ctn associated predominantly on the plasma membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association. These results suggest that melanoma cells at advanced stages of the disease could have reduced metastatic potency in response to chemotactic stimuli compared with cells lacking E-cadherin, and the results indicate that p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca²⁺-dependent adhesion molecules that mediate cell-cell contacts and are expressed in most solid tissues providing a tight control of morphogenesis (1, 2). Classical cadherins, such as epithelial (E) cadherin, are found in adherens junctions, forming core protein complexes with β-catenin, α-catenin, and p120 catenin (p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin, whereas α-catenin associates with the complex through its binding to β-catenin, providing a link with the actin cytoskeleton (1, 2). E-cadherin is frequently lost or down-regulated in many human tumors, coincident with morphological epithelial to mesenchymal transition and acquisition of invasiveness (3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis starts, it is responsible for 80% of deaths from skin cancers (7). Melanocytes express E-cadherin (8-10), but melanoma cells at early radial growth phase show a large reduction in the expression of this cadherin, and surprisingly, expression has been reported to be partially recovered by vertical growth phase and metastatic melanoma cells (9, 11, 12).Trafficking of cancer cells from primary tumor sites to intravasation into blood circulation and later to extravasation to colonize distant organs requires tightly regulated directional cues and cell migration and invasion that are mediated by chemokines, growth factors, and adhesion molecules (13). Solid tumor cells express chemokine receptors that provide guidance of these cells to organs where their chemokine ligands are expressed, constituting a homing model resembling the one used by immune cells to exert their immune surveillance functions (14). Most solid cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called SDF-1), which is expressed in lungs, bone marrow, and liver (15). Expression of CXCR4 in human melanoma has been detected in the vertical growth phase and on regional lymph nodes, which correlated with poor prognosis and increased mortality (16, 17). Previous in vivo experiments have provided evidence supporting a crucial role for CXCR4 in the metastasis of melanoma cells (18).Rho GTPases control the dynamics of the actin cytoskeleton during cell migration (19, 20). The activity of Rho GTPases is tightly regulated by guanine-nucleotide exchange factors (GEFs),⁴ which stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating proteins (GAPs), which promote GTP hydrolysis (21, 22), whereas guanine nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of spontaneous activation (23). Therefore, cell migration is finely regulated by the balance between GEF, GAP, and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is well documented (reviewed in Ref. 24), providing control of both cell migration and growth. RhoA and RhoC are highly expressed in colon, breast, and lung carcinoma (25, 26), whereas overexpression of RhoC in melanoma leads to enhancement of cell metastasis (27). CXCL12 activates both RhoA and Rac1 in melanoma cells, and both GTPases play key roles during invasion toward this chemokine (28, 29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and metastasis, in this study we have addressed the question of whether changes in E-cadherin expression on melanoma cells might affect cell invasiveness. We show here that overexpression of E-cadherin leads to impaired melanoma cell invasion to CXCL12, and we provide mechanistic characterization accounting for the decrease in invasion.     

Glycolytic Flux Signals to mTOR through Glyceraldehyde-3-Phosphate Dehydrogenase-Mediated Regulation of Rheb

The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR''s access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.The mTOR complex 1 (mTORC1) signal transduction pathway acts as a central controller of cell growth in mammals (20, 23, 29). mTORC1 integrates a wide range of intracellular and extracellular signals, including insulin, availability of nutrients (glucose and amino acids), cellular energy status, and hypoxia, to regulate protein synthesis and cell growth (11, 12, 17, 36, 46). Many of these environmental cues are integrated into tuberous sclerosis complex (TSC1-TSC2), the major upstream regulator of mTORC1. In response to the absence of insulin and to the low-energy status of cells, the TSC1-TSC2 complex stimulates the GTPase function of Rheb, a small GTPase that acts as a proximal key activator of mTORC1, which leads to the inhibition of Rheb-mediated mTORC1 activation. In contrast, inactivation of the TSC1-TSC2 complex results in the accumulation of GTP-bound Rheb and thus activation of mTORC1 (3, 13, 21, 27, 32, 39). For this reason, both the loss of TSC proteins and the overexpression of Rheb cause hyperactivation of mTORC1 signaling, which is frequently observed in many common human cancers (2, 5, 19, 25, 33). Therefore, a tight regulation of Rheb activity is critical for the proper operation of the mTORC1 pathway in response to environmental cues.Rheb is an atypical member of the Ras superfamily of GTPases (1, 10, 47). As with other small GTPases, the activity of Rheb is regulated by its guanine nucleotide binding status. However, the negative control of GTP-bound Rheb by the TSC1-TSC2 complex has only recently been investigated, and the regulation of the nucleotide binding status of Rheb is not fully understood. A recent study proposed that translationally controlled tumor protein may function as a guanine nucleotide exchange factor for Rheb that causes the accumulation of GTP-bound Rheb (18). GTP-bound Rheb is essential for activating mTOR kinase (21, 28, 38). However, the interaction between Rheb and mTOR does not depend on the GTP binding status of Rheb (30), raising questions regarding the mechanism by which Rheb activates mTORC1. Recently, FKBP38 (immunophilin FK506-binding protein, 38 kDa) was found to be a direct binding partner of Rheb and an inhibitor of mTORC1 (4). GTP-bound Rheb binds FKBP38 and releases FKBP38 from mTORC1, resulting in activation of the mTORC1 pathway. However, there have been conflicting results regarding the effects of nutrient availability on Rheb activity (31, 37, 42, 50) and the effect of these newly identified regulators of Rheb function (44, 45). Thus, the precise molecular mechanisms underlying Rheb regulation and Rheb-mediated mTORC1 activation have remained unclear.In this study, we identified glyceraldehyde-3-phosphate (Gly-3-P) dehydrogenase (GAPDH) as a novel Rheb binding protein and a negative regulator of Rheb. We found that the interaction between GAPDH and Rheb is induced when the glycolytic flux is suppressed under low-glucose conditions to inhibit mTORC1. Here, we provide a molecular mechanism underlying the cross talk between the glycolytic flux and the mTORC1 signaling.     

Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly studied as regulators of the immune response and potential therapeutic agents for autoimmune disorders (1). To date, 15 galectins have been identified and classified according with the structural organization of their distinctive monomeric or dimeric carbohydrate recognition domain for β-galactosides (2, 3). Galectins are secreted by unconventional mechanisms and once outside the cells bind to and cross-link multiple glycoconjugates both at the cell surface and at the extracellular matrix, modulating processes as diverse as cell adhesion, migration, proliferation, differentiation, and apoptosis (4–10). Several galectins have been involved in T cell homeostasis because of their capability to kill thymocytes, activated T cells, and T cell lines (11–16). Pro-apoptotic galectins might contribute to shape the T cell repertoire in the thymus by negative selection, restrict the immune response by eliminating activated T cells at the periphery (1), and help cancer cells to escape the immune system by eliminating cancer-infiltrating T cells (17). They have also a promising therapeutic potential to eliminate abnormally activated T cells and inflammatory cells (1). Studies on the mostly explored galectins, Gal-1, -3, and -9 (14, 15, 18–20), as well as in Gal-2 (13), suggest immunosuppressive complementary roles inducing different pathways to apoptosis. Galectin-8 (Gal-8)⁴ is one of the most widely expressed galectins in human tissues (21, 22) and cancerous cells (23, 24). Depending on the cell context and mode of presentation, either as soluble stimulus or extracellular matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis (6, 7, 9, 10, 22, 25). Its role has been mostly studied in relation to tumor malignancy (23, 24). However, there is some evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or inflammatory disorders. For instance, the intrathymic expression and pro-apoptotic effect of Gal-8 upon CD4^highCD8^high thymocytes suggest a role for Gal-8 in shaping the T cell repertoire (16). Gal-8 could also modulate the inflammatory function of neutrophils (26), Moreover Gal-8-blocking agents have been detected in chronic autoimmune disorders (10, 27, 28). In rheumatoid arthritis, Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid cells, but can be counteracted by a specific rheumatoid version of CD44 (CD44vRA) (27). In systemic lupus erythematosus (SLE), a prototypic autoimmune disease, we recently described function-blocking autoantibodies against Gal-8 (10, 28). Thus it is important to define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with specific integrins, such as α1β1, α3β1, and α5β1 but not α4β1, and as a matrix protein promotes cell adhesion and asymmetric spreading through activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (10). These early effects occur within 5–30 min. However, ERK1/2 signaling supports long term processes such as T cell survival or death, depending on the moment of the immune response. During T cell activation, ERK1/2 contributes to enhance the expression of interleukin-2 (IL-2) required for T cell clonal expansion (29). It also supports T cell survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by other previously activated T cells (30, 31). Later on, ERK1/2 is required for activation-induced cell death, which controls the extension of the immune response by eliminating recently activated and restimulated T cells (32, 33). In activation-induced cell death, ERK1/2 signaling contributes to enhance the expression of FasL and its receptor Fas/CD95 (32, 33), which constitute a preponderant pro-apoptotic system in T cells (34). Here, we ask whether Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling (35) deserves special attention. cAMP/PKA signaling plays an immunosuppressive role in T cells (36) and is altered in SLE (37). Phosphodiesterases (PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA during T cell activation (38, 39). PKA has been described to control the activity of ERK1/2 either positively or negatively in different cells and processes (35). A little explored integration among ERK1/2 and PKA occurs via phosphatidic acid (PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA plays roles in signaling interacting with a variety of targeting proteins that bear PA-binding domains (40). In this way PA recruits Raf-1 to the plasma membrane (41). It is also converted by phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG), which among other functions, recruits and activates the GTPase Ras (42). Both Ras and Raf-1 are upstream elements of the ERK1/2 activation pathway (43). In addition, PA binds to and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP levels and PKA down-regulation (44). The regulation and role of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell homeostasis, because it is also unknown whether galectins stimulate the PLD/PA pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our results for the first time show that a galectin increases the PA levels, down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE activity, and induces an ERK1/2-dependent expression of the pro-apoptotic factor FasL. The enhanced PDE activity induced by Gal-8 is required for the activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces apoptosis in human peripheral blood mononuclear cells (PBMC), especially after activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other galectins the property of killing activated T cells contributing to the T cell homeostasis. The pathway involves a particularly integrated signaling context, engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its susceptibility to inhibition by anti-Gal-8 autoantibodies.     

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis (CME)⁴ is a major mechanism by which cells take up nutrients, control the surface levels of multiple proteins, including ion channels and transporters, and regulate the coupling of signaling receptors to downstream signaling cascades (1-5). In neurons, CME takes on additional specialized roles; it is an important process regulating synaptic vesicle (SV) availability through endocytosis and recycling of SV membranes (6, 7), it shapes synaptic plasticity (8-10), and it is crucial in maintaining synaptic membranes and membrane structure (11).Numerous endocytic accessory proteins participate in CME, interacting with each other and with core components of the endocytic machinery such as clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific modules and peptide motifs (12). One such module is the Eps15 homology domain that binds to proteins bearing NPF motifs (13, 14). Another is the Src homology 3 (SH3) domain, which binds to proline-rich domains in protein partners (15). Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17-19), a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25 (17, 20, 21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16, 22-25). The rich binding capability of intersectin has linked it to various functions from CME (17, 26, 27) and signaling (22, 28, 29) to mitogenesis (30, 31) and regulation of the actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21, 32-34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26, 28, 35, 36). This isoform has all the binding modules of its short (intersectin-s) counterpart but also has additional domains: a DH and a PH domain that provide guanine nucleotide exchange factor (GEF) activity specific for Cdc42 (23, 37) and a C2 domain at the C terminus. Through its GEF activity and binding to actin regulatory proteins, including N-WASP, intersectin-l has been implicated in actin regulation and the development of dendritic spines (19, 23, 24). In addition, because the rest of the binding modules are shared between intersectin-s and -l, it is generally thought that the two intersectin isoforms have the same endocytic functions. In particular, given the well defined role for the invertebrate orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l performs this role in mammalian neurons, which lack intersectin-s. Defining the complement of intersectin functional activities in mammalian neurons is particularly relevant given that the protein is involved in the pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is localized on chromosome 21q22.2 and is overexpressed in DS brains (38). Interestingly, alterations in endosomal pathways are a hallmark of DS neurons and neurons from the partial trisomy 16 mouse, Ts65Dn, a model for DS (39, 40). Thus, an endocytic trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured hippocampal neurons. We find that intersectin-l is localized to the somatodendritic regions of neurons, where it co-localizes with CHC and AP-2 and regulates the uptake of transferrin. Intersectin-l also co-localizes with actin at dendritic spines and disrupting intersectin-l function alters dendritic spine development. In contrast, intersectin-l is absent from presynaptic terminals and has little or no role in SV recycling.