AMPK promotes Arf6 activation in a kinase-independent manner upon glucose starvation.

AMP-activated protein kinase (AMPK) is a crucial cellular nutrient and energy sensor that maintains energy homeostasis. AMPK also governs cancer cell invasion and migration by regulating gene expression and activating multiple cellular signaling pathways. ADP-ribosylation factor 6 (Arf6) can be activated via nucleotide exchange by guanine-nucleotide-exchange factors (GEFs), and its activation also regulates tumor invasion and migration. By studying GEF-mediated Arf6 activation, we have elucidated that AMPK functions as a noncanonical GEF for Arf6 in a kinase-independent manner. Moreover, by examining the physiological role of the AMPK-Arf6 axis, we have determined that AMPK activates Arf6 upon glucose starvation and 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) treatment. We have further identified the binding motif in the C-terminal regulatory domain of AMPK that is responsible for promoting Arf6 activation and, thus, inducing cell migration and invasion. These findings reveal a noncanonical role of AMPK in which its C-terminal regulatory domain serves as a GEF for Arf6 during glucose deprivation.

GAPDH inhibits intracellular pathways during starvation for cellular energy homeostasis.

Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized1, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.

ArfGAP1 acts as a GTPase-activating protein for human ADP-ribosylation factor-like 1 protein.

ADP-ribosylation factors (Arfs) and Arf-like (Arl) GTPases are key regulators of intracellular vesicle trafficking and Golgi structure. Both Arf and Arl proteins cycle between active GTP-bound and inactive GDP-bound forms, where guanine nucleotide exchange factors (GEFs) regulate the exchange of GDP for GTP, whereas GTPase-activating proteins (GAPs) promote the hydrolysis of bound GTP. Human Arl1 is located at the trans-Golgi network (TGN) and regulates the function and structure of the Golgi complex. However, neither GEFs nor GAPs for human Arl1 have been identified. Here, we report that ArfGAP1, an Arf1 GAP, can promote GTP hydrolysis of Arl1. We show that ArfGAP1 directly interacts with GTP-bound Arl1 and exhibits GAP activity toward Arl1 in vitro. Exogenous expression of ArfGAP1, but not ArfGAP2 and ArfGAP3, causes dissociation of endogenous Arl1 from the TGN. In addition, GAP activity-deficient ArfGAP1 fails to regulate the Golgi localization of Arl1. Using an activity pull-down assay, we demonstrated that ArfGAP1 regulates the levels of Arl1-GTP in cells expressing ArfGAP1-myc or with ArfGAP1 knockdown. Finally, we observed that, similar to expression of putative active Arl1 (Arl1QL), ArfGAP1 knockdown impairs endosome-to-TGN retrograde transport of the Shiga toxin B-subunit. Thus, our findings support the idea that ArfGAP1 acts as an Arl1 GAP to regulate the function of Arl1 in vesicle trafficking at the TGN.

The Arl3 and Arl1 GTPases co-operate with Cog8 to regulate selective autophagy via Atg9 trafficking.

The Arl3-Arl1 GTPase cascade plays important roles in vesicle trafficking at the late Golgi and endosomes. Subunits of the conserved oligomeric Golgi (COG) complex, a tethering factor, are important for endosome-to-Golgi transport and contribute to the efficient functioning of the cytoplasm-to-vacuole targeting (Cvt) pathway, a well-known selective autophagy pathway. According to our findings, the Arl3-Arl1 GTPase cascade co-operates with Cog8 to regulate the Cvt pathway via Atg9 trafficking. arl3cog8Δ and arl1cog8Δ exhibit profound defects in aminopeptidase I maturation in rich medium. In addition, the Arl3-Arl1 cascade acts on the Cvt pathway via dynamic nucleotide binding. Furthermore, Atg9 accumulates at the late Golgi in arl3cog8Δ and arl1cog8Δ cells under normal growth conditions but not under starvation conditions. Thus, our results offer insight into the requirement for multiple components in the Golgi-endosome system to determine Atg9 trafficking at the Golgi, thereby regulating selective autophagy.

Unfolded protein response regulates yeast small GTPase Arl1p activation at late Golgi via phosphorylation of Arf GEF Syt1p.

ADP ribosylation factor (Arf) GTPases are key regulators of membrane traffic at the Golgi complex. In yeast, Arf guanine nucleotide-exchange factor (GEF) Syt1p activates Arf-like protein Arl1p, which was accompanied by accumulation of golgin Imh1p at late Golgi, but whether and how this function of Syt1p is regulated remains unclear. Here, we report that the inositol-requiring kinase 1 (Ire1p)-mediated unfolded protein response (UPR) modulated Arl1p activation at late Golgi. Arl1p activation was dependent on both kinase and endo-RNase activities of Ire1p. Moreover, constitutively active transcription factor Hac1p restored the Golgi localization of Arl1p and Imh1p inIRE1-deleted cells. Elucidating the mechanism of Ire1p-Hac1p axis actions, we found that it regulated phosphorylation of Syt1p, which enhances Arl1p activation, recruitment of Imh1p to the Golgi, and Syt1p interaction with Arl1p. Consistent with these findings, the induction of UPR by tunicamycin treatment increases phosphorylation of Syt1p, resulting in Arl1p activation. Thus, these findings clarify how the UPR influences the roles of Syt1p, Arl1p, and Imh1p in Golgi transport.

Mechanism of action of the flippase Drs2p in modulating GTP hydrolysis of Arl1p.

Small GTPase ADP-ribosylation factors (ARFs) are key regulators of membrane trafficking and their activities are determined by guanine-nucleotide-binding status. In Saccharomyces cerevisiae, Arl1p, an ARF-like protein, is responsible for multiple trafficking pathways at the Golgi. The GTP-hydrolysis activity of Arl1p is stimulated by its GTPase-activating protein Gcs1p, and binding with its effector Imh1p protects Arl1p from premature inactivation. However, the mechanism involved in the timing of Arl1p inactivation is unclear. Here, we demonstrate that another Arl1p effector, the lipid flippase Drs2p, is required for Gcs1p-stimulated inactivation of Arl1p. Drs2p is known to be activated by Arl1p and is involved in vesicle formation through its ability to create membrane asymmetry. We found that the flippase activity of Drs2p is required for proper membrane targeting of Gcs1p in vivo. Through modification of the membrane environment, Drs2p promotes the affinity of Gcs1p for the Golgi, where it binds to active Arl1p. Together, Imh1p and Drs2p modulate the activity of Gcs1p by timing its interaction with Arl1p, hence providing feedback regulation of Arl1p activity.

Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p.

ADP ribosylation factors (Arfs) are the central regulators of vesicle trafficking from the Golgi complex. Activated Arfs facilitate vesicle formation through stimulating coat assembly, activating lipid-modifying enzymes and recruiting tethers and other effectors. Lipid translocases (flippases) have been implicated in vesicle formation through the generation of membrane curvature. Although there is no evidence that Arfs directly regulate flippase activity, an Arf-guanine-nucleotide-exchange factor (GEF) Gea2p has been shown to bind to and stimulate the activity of the flippase Drs2p. Here, we provide evidence for the interaction and activation of Drs2p by Arf-like protein Arl1p in yeast. We observed that Arl1p, Drs2p and Gea2p form a complex through direct interaction with each other, and each interaction is necessary for the stability of the complex and is indispensable for flippase activity. Furthermore, we show that this Arl1p-Drs2p-Gea2p complex is specifically required for recruiting golgin Imh1p to the Golgi. Our results demonstrate that activated Arl1p can promote the spatial modulation of membrane organization at the trans-Golgi network through interacting with the effectors Gea2p and Drs2p.

Phosphoglycerate kinase 1 acts as a cargo adaptor to promote EGFR transport to the lysosome.

The epidermal growth factor receptor (EGFR) plays important roles in multiple cellular events, including growth, differentiation, and motility. A major mechanism of downregulating EGFR function involves its endocytic transport to the lysosome. Sorting of proteins into intracellular pathways involves cargo adaptors recognizing sorting signals on cargo proteins. A dileucine-based sorting signal has been identified previously for the sorting of endosomal EGFR to the lysosome, but a cargo adaptor that recognizes this signal remains unknown. Here, we find that phosphoglycerate kinase 1 (PGK1) is recruited to endosomal membrane upon its phosphorylation, where it binds to the dileucine sorting signal in EGFR to promote the lysosomal transport of this receptor. We also elucidate two mechanisms that act in concert to promote PGK1 recruitment to endosomal membrane, a lipid-based mechanism that involves phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and a protein-based mechanism that involves hepatocyte growth factor receptor substrate (Hrs). These findings reveal an unexpected function for a metabolic enzyme and advance the mechanistic understanding of how EGFR is transported to the lysosome.

Syt1p promotes activation of Arl1p at the late Golgi to recruit Imh1p.

In yeast, Arl3p recruits Arl1p GTPase to regulate Golgi function and structure. However, the molecular mechanism involved in regulating activation of Arl1p at the Golgi is unknown. Here, we show that Syt1p promoted activation of Arl1p and recruitment of a golgin protein, Imh1p, to the Golgi. Deletion of SYT1 resulted in the majority of Arl1p being distributed diffusely throughout the cytosol. Overexpression of Syt1p increased Arl1p-GTP production in vivo and the Syt1-Sec7 domain promoted nucleotide exchange on Arl1p in vitro. Syt1p function required the N-terminal region, Sec7 and PH domains. Arl1p, but not Arl3p, interacted with Syt1p. Localization of Syt1p to the Golgi did not require Arl3p. Unlike arl1Δ or arl3Δ mutants, syt1Δ did not show defects in Gas1p transport, cell wall integrity or vacuolar structure. These findings reveal that activation of Arl1p is regulated in part by Syt1p, and imply that Arl1p activation, by using more than one GEF, exerts distinct biological activities at the Golgi compartment.

Ganoderma lucidum Polysaccharides Induce Macrophage-Like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation.

Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT) reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.

The protein kinase Akt acts as a coat adaptor in endocytic recycling.

Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.

ALDH7A1 inhibits the intracellular transport pathways during hypoxia and starvation to promote cellular energy homeostasis.

The aldehyde dehydrogenase (ALDH) family of metabolic enzymes converts aldehydes to carboxylates. Here, we find that the reductive consequence of ALDH7A1 activity, which generates NADH (nicotinamide adenine dinucleotide, reduced form) from NAD, underlies how ALDH7A1 coordinates a broad inhibition of the intracellular transport pathways. Studying vesicle formation by the Coat Protein I (COPI) complex, we elucidate that NADH generated by ALDH7A1 targets Brefeldin-A ADP-Ribosylated Substrate (BARS) to inhibit COPI vesicle fission. Moreover, defining a physiologic role for the broad transport inhibition exerted by ALDH7A1, we find that it acts to reduce energy consumption during hypoxia and starvation to promote cellular energy homeostasis. These findings advance the understanding of intracellular transport by revealing how the coordination of multiple pathways can be achieved, and also defining circumstances when such coordination is needed, as well as uncovering an unexpected way that NADH acts in cellular energetics.

Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction.

BACKGROUND: Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells. RESULTS: Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach. CONCLUSION: Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

Snf1/AMP-activated protein kinase activates Arf3p to promote invasive yeast growth via a non-canonical GEF domain.

Active GTP-bound Arf GTPases promote eukaryotic cell membrane trafficking and cytoskeletal remodelling. Arf activation is accelerated by guanine nucleotide-exchange factors (GEFs) using the critical catalytic glutamate in all known Sec7 domain sequences. Yeast Arf3p, a homologue of mammalian Arf6, is required for yeast invasive responses to glucose depletion. Here we identify Snf1p as a GEF that activates Arf3p when energy is limited. SNF1 is the yeast homologue of AMP-activated protein kinase (AMPK), which is a key regulator of cellular energy homeostasis. As activation of Arf3p does not depend on the Snf1p kinase domain, assay of regulatory domain fragments yield evidence that the C-terminal hydrophobic α-helix core of Snf1p is a non-canonical GEF for Arf3p activation. Thus, our study reveals a novel mechanism for regulating cellular responses to energy deprivation, in particular invasive cell growth, through direct Arf activation by Snf1/AMPK.

Arf3p GTPase is a key regulator of Bud2p activation for invasive growth in Saccharomyces cerevisiae.

The regulation and signaling pathways involved in the invasive growth of yeast have been studied extensively because of their general applicability to fungal pathogenesis. Bud2p, which functions as a GTPase-activating protein (GAP) for Bud1p/Rsr1p, is required for appropriate budding patterns and filamentous growth. The regulatory mechanisms leading to Bud2p activation, however, are poorly understood. In this study, we report that ADP-ribosylation factor 3p (Arf3p) acts as a regulator of Bud2p activation during invasive growth. Arf3p binds directly to the N-terminal region of Bud2p and promotes its GAP activity both in vitro and in vivo. Genetic analysis shows that deletion of BUD1 suppresses the defect of invasive growth in arf3Δ or bud2Δ cells. Lack of Arf3p, like that of Bud2p, causes the intracellular accumulation of Bud1p-GTP. The Arf3p-Bud2p interaction is important for invasive growth and facilitates the Bud2p-Bud1p association in vivo. Finally, we show that under glucose depletion-induced invasion conditions in yeast, more Arf3p is activated to the GTP-bound state, and the activation is independent of Arf3p guanine nucleotide-exchange factor Yel1p. Thus we demonstrate that a novel spatial activation of Arf3p plays a role in regulating Bud2p activation during glucose depletion-induced invasive growth.

The N-terminus of Vps74p is essential for the retention of glycosyltransferases in the Golgi but not for the modulation of apical polarized growth in Saccharomyces cerevisiae.

Vps74p is a member of the PtdIns(4)P-binding protein family. Vps74p interacts with Golgi-resident glycosyltransferases and the coat protein COPI complex to modulate Golgi retention of glycosyltransferases and with the PtdIns(4)P phosphatase Sac1p to modulate PtdIns(4)P homeostasis at the Golgi. Genetic analysis has shown that Vps74p is required for the formation of abnormal elongated buds in cdc34-2 cells. The C-terminal region of Vps74p is required for Vps74p multimerization, Golgi localization, and glycosyltransferase interactions; however, the functional significance of the N-terminal region and three putative phosphorylation sites of Vps74p have not been well characterized. In this study, we demonstrate that Vps74p executes multiple cellular functions using different domains. We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing. Deletion of the N-terminal 90 amino acids, but not the 66 amino acids, of Vps74p impaired its ability to restore the elongated bud phenotype in cdc34-2/vps74Δ cells. Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells. Furthermore, we found that three N-terminal phosphorylation sites contribute to rapamycin hypersensitivity, although these phosphorylation residues are not involved in Vps74p localization, ability to modulate glycosyltransferase retention, or elongated bud formation in cdc34-2 cells. Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions.

Zirconium complexes incorporated with asymmetrical tridentate pincer type mono- and di-anionic pyrrolyl ligands: mechanism and reactivity as catalytic precursors.

The reactions of Zr(NR(2))(4) (1, R = Me; 2, R = Et) with an asymmetrical tridentate pincer type pyrrole ligand precursor [C(4)H(2)NH(2-CH(2)NH(t)Bu)(5-CH(2)NMe(2))] and treatment of the derivatives with either PhNCS or PhNCO have been carried out and characterized. Reacting Zr(NR(2))(4) (1, R = Me; 2, R = Et) with [C(4)H(2)NH(2-CH(2)NH(t)Bu)(5-CH(2)NMe(2))] generates Zr[C(4)H(2)N(2-CH(2)N(t)Bu)(5-CH(2)NMe(2))](NR(2))(2) (3, R = Me; 4, R = Et) in high yield along with the elimination of 2 equiv of dimethylamine or diethylamine, respectively. Interestingly, while changing the solvent from Et(2)O to CH(2)Cl(2), the complex Zr[C(4)H(2)N(2-CH(2)N(t)Bu)(5-CH(2)NMe(2))][C(4)H(2)N(2-CH(2)NH(t)Bu)(5-CH(2)NMe(2))]Cl (5) is produced by undergoing C-Cl bond cleavage. Furthermore, reaction of either 3 or 4 with 1 or 2 equiv of PhNCS or PhNCO yields Zr[C(4)H(2)N(2-CH(2)N(t)Bu)(5-CH(2)NMe(2))](NMe(2))[PhNC(NMe(2))S] (6), Zr[C(4)H(2)N(2-CH(2)N(t)Bu)(5-CH(2)NMe(2))](NEt(2))[PhNC(NEt(2))O] (7) and Zr[C(4)H(2)N(2-CH(2)NH(t)Bu)(5-CH(2)NMe(2))][PhNC(NEt(2))O](3) (8), respectively. All the aforementioned complexes were characterized by (1)H and (13)C NMR spectrometry and the molecular structures of 5, 6, and 8 have been determined by single-crystal X-ray diffractometry. Complexes 4, 5, and 7 initiated the ethylene polymerization in the presence of MAO as the co-catalyst.

CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1).

CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.