Lunapark ubiquitinates atlastin-2 for the tubular network formation of the endoplasmic reticulum.

Endoplasmic reticulum (ER) tubules are interconnected by three-way junctions, resulting in the formation of a tubular ER network. Lunapark (Lnp) localizes to and stabilizes the three-way junctions. The N-terminal cytoplasmic domain in Lnp has a ubiquitin ligase activity. However, the molecular mechanism of how the ubiquitin ligase activity of Lnp is involved in the formation of the tubular ER network remains unknown. In this study, we examined whether the ER membrane proteins responsible for the formation of the tubular ER network are ubiquitinated by Lnp. We found that atlastin-2 (ATL2), an isoform of the ATL family mediating the generation of the three-way junctions by connecting the ER tubules, is a novel substrate for ubiquitination by Lnp. The localization of Lnp at the three-way junctions is important for ubiquitination of ATL2. Lysine 56, 57, 282 and 302 are the potential ubiquitination sites by Lnp. Silencing ATL2 decreased the number of the three-way junctions, and the expression of the ATL2 mutant in which the lysine residues are substituted with arginine failed to rescue the decrease of the three-way junctions in the ATL2 knocked-down cells. These results suggest that Lnp ubiquitinates ATL2 at the three-way junctions for the proper tubular ER network formation.

CAND1 regulates lunapark for the proper tubular network of the endoplasmic reticulum.

Endoplasmic reticulum (ER) tubules connect each other by three-way junctions, resulting in a tubular ER network. Oligomerization of three-way junction protein lunapark (Lnp) is important for its localization and the three-way junction stability. On the other hand, Lnp has an N-terminal ubiquitin ligase activity domain, which is also important for the three-way junction localization. To understand the mode of action of Lnp, we isolated Cullin-associated and neddylation-dissociated 1 (CAND1), a regulator of Skp1-Cul1-F-box (SCF) ubiquitin ligase, as a Lnp-binding protein by affinity chromatography. CAND1 and Lnp form a higher-molecular-weight complex in vitro, while they do not co-localize at the three-way junctions. CAND1 reduces the auto-ubiquitination activity of Lnp. CAND1 knockdown enhances proteasomal degradation of Lnp and reduces the tubular ER network in mammalian cells. These results suggest that CAND1 has the potency to promote the formation of the higher-molecular-weight complex with Lnp and reduce the auto-ubiquitination activity of Lnp, thereby regulating the three-way junction stability of the tubular ER network.

Harnessing membrane trafficking to promote cancer spreading and invasion: The case of RAB2A.

How cancer disseminates and metastasizes remains an outstanding open question. Emerging evidence indicates that membrane trafficking is frequently harnessed by tumors of epithelial origin to acquire a mesenchymal program of invasiveness. However, the critical molecular hubs used by cancer cells this context have only began to be elucidated. Here, we discussed the results of a recent phenotypic screening that led to the identification of the small GTPase RAB2A, not previously involved in cancer dissemination, as pivotal for the acquisition of pericellular proteolysis, cell dissemination and distant metastatic spreading of human breast cancer. At the cellular levels, RAB2A controls both canonical polarized Golgi-to-Plasma membrane trafficking of the junctional protein E-cadherin, and post-endocytic trafficking of the membrane-bound metalloprotease, MT1-MMP. This finding reveals an unexpected plasticity in the control of diverse trafficking routes exerted by RAB2A through canonical (Golgi stacking) and non-canonical (late endosome recycling) functional interactions, contributing to break established membrane trafficking dogma on the rigorous molecular distinction between polarized Golgi and post endocytic routes. Finally, they suggest that epithelial cancers may specifically select for those molecules that enable them to control multiple trafficking routes, in turn essential for the regulation of activities necessary for acquisition of mesenchymal traits.

RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs.

The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.

The CDC42-interacting protein 4 controls epithelial cell cohesion and tumor dissemination.

The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-ß1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.

The GTPase-activating protein RN-tre controls focal adhesion turnover and cell migration.

BACKGROUND: Integrin-mediated adhesion of cells to the extracellular matrix (ECM) relies on the dynamic formation of focal adhesions (FAs), which are biochemical and mechanosensitive platforms composed of a large variety of cytosolic and transmembrane proteins. During migration, there is a constant turnover of ECM contacts that initially form as nascent adhesions at the leading edge, mature into FAs as actomyosin tension builds up, and are then disassembled at the cell rear, thus allowing for cell detachment. Although the mechanisms of FA assembly have largely been defined, the molecular circuitry that regulates their disassembly still remains elusive. RESULTS: Here, we show that RN-tre, a GTPase-activating protein (GAP) for Rabs including Rab5 and Rab43, is a novel regulator of FA dynamics and cell migration. RN-tre localizes to FAs and to a pool of Rab5-positive vesicles mainly associated with FAs undergoing rapid remodeling. We found that RN-tre inhibits endocytosis of ß1, but not ß3, integrins and delays the turnover of FAs, ultimately impairing ß1-dependent, but not ß3-dependent, chemotactic cell migration. All of these effects are mediated by its GAP activity and rely on Rab5. CONCLUSIONS: Our findings identify RN-tre as the Rab5-GAP that spatiotemporally controls FA remodeling during chemotactic cell migration.

C. elegans AMPKs promote survival and arrest germline development during nutrient stress.

Mechanisms controlling development, growth, and metabolism are coordinated in response to changes in environmental conditions, enhancing the likelihood of survival to reproductive maturity. Much remains to be learned about the molecular basis underlying environmental influences on these processes. C. elegans larvae enter a developmentally dormant state called L1 diapause when hatched into nutrient-poor conditions. The nematode pten homologue daf-18 is essential for maintenance of survival and germline stem cell quiescence during this period (Fukuyama et al., 2006; Sigmond et al., 2008), but the details of the signaling network(s) in which it functions remain to be elucidated. Here, we report that animals lacking both aak-1 and aak-2, which encode the two catalytic α subunits of AMP-activated protein kinase (AMPK), show reduced viability and failure to maintain mitotic quiescence in germline stem cells during L1 diapause. Furthermore, failure to arrest germline proliferation has a long term consequence; aak double mutants that have experienced L1 diapause develop into sterile adults when returned to food, whereas their continuously fed siblings are fertile. Both aak and daf-18 appear to maintain germline quiescence by inhibiting activity of the common downstream target, TORC1 (TOR Complex 1). In contrast, rescue of the lethality phenotype indicates that aak-2 acts not only in the intestine, as does daf-18, but also in neurons, likely promoting survival by preventing energy deprivation during L1 diapause. These results not only provide evidence that AMPK contributes to survival during L1 diapause in a manner distinct from that by which it controls dauer diapause, but they also suggest that AMPK suppresses TORC1 activity to maintain stem cell quiescence.

A novel acetylation cycle of transcription co-activator Yes-associated protein that is downstream of Hippo pathway is triggered in response to SN2 alkylating agents.

Yes-associated protein (YAP) is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes. Although cytoplasmic retention of YAP is known to be mediated by Hippo pathway-dependent phosphorylation, post-translational modifications that regulate YAP in the nucleus remain unclear. Here we report the discovery of a novel cycle of acetylation/deacetylation of nuclear YAP induced in response to S(N)2 alkylating agents. We show that after treatment of cells with the S(N)2 alkylating agent methyl methanesulfonate, YAP phosphorylation mediated by the Hippo pathway is markedly reduced, leading to nuclear translocation of YAP and its acetylation. This YAP acetylation occurs on specific and highly conserved C-terminal lysine residues and is mediated by the nuclear acetyltransferases CBP (CREB binding protein) and p300. Conversely, the nuclear deacetylase SIRT1 is responsible for YAP deacetylation. Intriguingly, we found that YAP acetylation is induced specifically by S(N)2 alkylating agents and not by other DNA-damaging stimuli. These results identify a novel YAP acetylation cycle that occurs in the nucleus downstream of the Hippo pathway. Intriguingly, our findings also indicate that YAP acetylation is involved in responses to a specific type of DNA damage.

RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM) domain-containing (Anks) protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31.

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.

RhoA activation participates in rearrangement of processing bodies and release of nucleated AU-rich mRNAs.

Cytoplasmic ribonucleoprotein granules, known as processing bodies (P-bodies), contain a common set of conserved RNA-processing enzymes, and mRNAs with AU-rich elements (AREs) are delivered to P-bodies for translational silencing. Although the dynamics of P-bodies is physically linked to cytoskeletal network, it is unclear how small GTPases are involved in the P-body regulation and the ARE-mRNA metabolism. We found here that glucose depletion activates RhoA GTPase and alters the P-body dynamics in HeLa cells. These glucose-depleted effects are reproduced by the overexpression of the RhoA-subfamily GTPases and conversely abolished by the inhibition of RhoA activation. Interestingly, both RhoA activation and glucose depletion inhibit the mRNA accumulation and degradation. These findings indicate that RhoA participates in the stress-induced rearrangement of P-bodies and the release of nucleated ARE-mRNAs for their stabilization.

p38 Mitogen-activated protein kinase controls a switch between cardiomyocyte and neuronal commitment of murine embryonic stem cells by activating myocyte enhancer factor 2C-dependent bone morphogenetic protein 2 transcription.

Many studies have shown that it is possible to use culture conditions to direct the differentiation of murine embryonic stem (ES) cells into a variety of cell types, including cardiomyocytes and neurons. However, the molecular mechanisms that control lineage commitment decisions by ES cells remain poorly understood. In this study, we investigated the role of the 3 major mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) in ES cell lineage commitment and showed that the p38 MAPK-specific inhibitor SB203580 blocks the spontaneous differentiation of ES cells into cardiomyocytes and instead induces the differentiation of these ES cells into neurons. Robust p38 MAPK activity between embryoid body culture days 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of p38 MAPK activity at this time results in ES cell differentiation into neurons rather than cardiomyocytes. At the molecular level, inhibition of p38 MAPK activity suppresses the expression of bmp-2 mRNA, whereas treatment of ES cells with bone morphogenetic protein 2 (BMP-2) inhibits the neurogenesis induced by SB203580. Further, luciferase reporter assays and chromatin immunoprecipitation experiments showed that BMP-2 expression in ES cells is regulated directly by the transcription factor myocyte enhancer factor 2C, a well-known substrate of p38 MAPK. Our findings reveal the molecular mechanism by which p38 MAPK activity in ES cells drives their commitment to differentiate preferentially into cardiomyocytes, and the conditions under which these same cells might develop into neurons.

Filamin associates with stress signalling kinases MKK7 and MKK4 and regulates JNK activation.

SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) belongs to the MAPK (mitogen-activated protein kinase) family and is important in many biological contexts. JNK activation is regulated by phosphorylation of specific tyrosine and threonine residues sequentially catalysed by MKK4 and MKK7, which are both dual-specificity MAPKKs (MAPK kinases). Previously, we reported that tyrosine-phosphorylation of JNK by MKK4 precedes threonine-phosphorylation by MKK7, and that both are required for synergistic JNK activation. In the present study, we identify the actin-binding protein-280 (Filamin A) as a presumed 'binder' protein that can bind to MKK7, as well as to MKK4, connecting them in close proximity. We show that Filamin family members A, B and C interact with MKK4 and MKK7, but not with JNK. Filamin A binds to an N-terminal region (residues 31-60) present in the MKK7gamma and MKK7beta splice isoforms, but cannot bind to MKK7alpha which lacks these amino acids. This same N-terminal region is crucial for the intracellular co-localization of MKK7gamma with actin stress fibres and Filamin A. Experiments using Filamin-A-deletion mutants revealed that the MKK7-binding region of Filamin A differs from its MKK4-binding region, and that MKK7gamma (but not MKK7alpha) can form a complex with Filamin A and MKK4. Finally, we used Filamin-A-deficient cells to show that Filamin A enhances MKK7 activation and is important for synergistic stress-induced JNK activation in vivo. Thus Filamin A is a novel member of the group of scaffold proteins whose function is to link two MAPKKs together and promote JNK activation.

CrxOS maintains the self-renewal capacity of murine embryonic stem cells.

Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.

Biochemical characterization of missense mutations in the Arf/Arl-family small GTPase Arl6 causing Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a pleiotropically genetic disorder, whose etiology is linked to cilia. Mutations in the Arf/Arl-family GTPase Arl6 have been recently shown to be responsible for BBS type 3. Here we show that BBS mutations alter the guanine nucleotide-binding properties of Arl6. Specifically, substitution of 31st Threonine to Arginine selectively abrogates the GTP-binding ability of Arl6 without affecting GDP-binding/dissociating properties. Furthermore, all the BBS mutations in Arl6 result in low expression of the mutant proteins, which can be restored by the inhibition of the proteasome. These findings implicate that Arl6 mutants are destabilized and eliminated by the proteasome in cells, probably due to the altered nucleotide-binding properties.

Blockage by SP600125 of Fcepsilon receptor-induced degranulation and cytokine gene expression in mast cells is mediated through inhibition of phosphatidylinositol 3-kinase signalling pathway.

SP600125 is used as a specific inhibitor of c-Jun N-terminal kinase (JNK). We initially aimed to examine physiological roles of JNK in mast cells that play a central role in inflammatory and immediate allergic responses. We found that Fc receptor for IgE (FcepsilonRI)-induced degranulation (serotonin release) and cytokine gene expression [interleukin (IL)-6, tumour necrosis factor-alpha and IL-13] in bone marrow-derived mast cells, were almost completely inhibited by SP600125. However, the time course of FcepsilonRI-induced JNK activation did not correlate with that of serotonin release. Furthermore, FcepsilonRI-induced degranulation and cytokine gene expression were not impaired in a JNK activator, MKK7-deficient mast cells, in which JNK activation was lost. These results indicate that the inhibitory effects by SP600125 are not due to impaired JNK activation. Instead, we found that SP600125 markedly inhibited the FcepsilonRI-induced activation of phosphatidylinositol 3-kinase (PI3K) and Akt, the same as a PI3K inhibitor, wortmannin. Finally, we found that SP600125 specifically inhibits delta form of p110 catalytic subunit (p110delta) of PI3K. Thus, SP600125 exerts its influence on mast cell functions by inhibiting the kinase activity of PI3K, but not JNK.

Tyr-phosphorylation signals translocate RIN3, the small GTPase Rab5-GEF, to early endocytic vesicles.

The small GTPase Rab5 plays a key role in early endocytic pathway, and its activation requires guanine-nucleotide exchange factors (GEFs). Rab5-GEFs share a conserved VPS9 domain for the GEF action, and RIN3 containing additional domains, such as Src-homology 2, RIN-family homology (RH), and Ras-association (RA), was identified as a new Rab5-GEF. However, precise functions of the additional domains and the activation mechanism of RIN3 remain unknown. Here, we found tyrosine-phosphorylation signals are involved in the Rab5-GEF activation. Treatment of HeLa cells with pervanadate translocates RIN3 from cytoplasm to the Rab5-positive vesicles. This RIN3 translocation was applied to various mutants lacking each domain of RIN3. Our present results suggest that a Ras GTPase(s) activated by tyrosine-phosphorylation signals interacts with the inhibitory RA domain, resulting in an active conformation of RIN3 as a Rab5-GEF and that RIN-unique RH domain constitutes a Rab5-binding region for the progress of GEF action.

Characterization of Rab45/RASEF containing EF-hand domain and a coiled-coil motif as a self-associating GTPase.

Rab-family GTPases function as key regulators for membrane traffic. Among them, Rab45/RASEF is an atypical GTPase in that it contains a coiled-coil motif at the mid region and a distinct N-terminal EF-hand domain with C-terminal Rab-homology domain. Here, we provide the initial biochemical characterization and intracellular localization of human Rab45. Rab45 bound guanine nucleotide tri- and di-phosphates through the C-terminal Rab domain. Rab45 was capable of self-interacting, and the self-interaction required the mid region containing the coiled-coil motif. Rab45 expressed in HeLa cells was localized in a small patch in the perinuclear area of the cell, and the localization was regulated by the guanine nucleotide-bound states of Rab45. Interestingly, the mid region, together with Rab domain, appeared to be essential for the characteristic perinuclear localization of Rab45, indicating that the self-interaction may be involved in the intracellular localization of Rab45.

Release of RASSF1C from the nucleus by Daxx degradation links DNA damage and SAPK/JNK activation.

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras-association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia-nuclear bodies (PML-NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML-NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx-RASSF1C complex in PML-NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.

Purification and analysis of RIN family-novel Rab5 GEFs.

The small GTPase Rab5 plays important roles in membrane budding and trafficking in the early endocytic pathways, and the activation of this GTPase is mediated by several guanine nucleotide exchange factors (GEFs) at each of the transport steps. The RIN family has been identified as GEFs for Rab5 and shown to possess unique biochemical properties. The RIN family preferentially interacts with an activated form of Rab5, although it enhances guanine nucleotide exchange reaction. Moreover, biochemical analysis indicates that the RIN family functions as a tetramer. In this chapter, we describe the isolation of the recombinant RIN family via expression in Spodoptera frugiperda (Sf9) insect cells and in mammalian cells. In addition, functional analysis is also provided to assess the physiological properties of the RIN family.