Bem1p is a positive regulator of the homotypic fusion of yeast vacuoles

Docked vacuoles are believed to undergo rapid lipid mixing during hemifusion and then a slow, rate-limiting completion of fusion and mixing of lumenal contents. Previous genomic analysis has suggested that Bem1p, a scaffold protein critical for cell polarity, may support vacuole fusion. We now report that bem1Delta strains have fragmented vacuoles (vps class B and C). During in vitro fusion reactions, vacuoles from bem1Delta strains showed a strong reduction in the rate of lipid mixing when compared with vacuoles from the BEM1 parent. The reduction in the overall rate of fusion with bem1Delta vacuoles was modest, consistent with lipid mixing as a non-rate-limiting step in the pathway. Although the fusion of either BEM1 (wild-type) or bem1Delta vacuoles is stimulated by recombinant Bem1p, the lipid mixing of docked bem1Delta vacuoles is highly dependent on rBem1p under certain reaction conditions. Bem1p-stimulated lipid mixing is blocked by well characterized fusion inhibitors including lipid ligands and antibodies to Ypt7p, Vps33p, and Vam3p. Although full-length Bem1p is required for maximal stimulation, a truncation mutant comprising the SH3 domains and the Phox homology (PX) domain retains modest stimulatory activity. In contrast to an earlier report (Han, B. K., Bogomolnaya, L. M., Totten, J. M., Blank, H. M., Dangott, L. J., and Polymenis, M. (2005) Genes Dev. 19, 2606-2618), we did not find phosphorylation of Bem1p at Ser-72 to be required for Bem1p-stimulated fusion. Taken together, Bem1p is a positive regulator of lipid mixing during vacuole hemifusion and fusion.     

Neuraminidase 1 is a negative regulator of lysosomal exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.     

SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.     

Mon2 is a negative regulator of the monomeric G protein, Arl1

Using site-directed mutants of ARL1 predicted to alter nucleotide binding, we examined phenotypes associated with the loss of ARL1 , including effects on membrane traffic and K (+) homeostasis. The GTP-restricted allele, ARL[Q72L] , complemented the membrane traffic phenotype (CPY secretion), but not the K (+) homeostasis phenotypes (sensitivity to hygromycin B, steady-state levels of K (+) , and accumulation of (86) Rb (+) ), while the XTP-restricted mutant, ARL1[D130N] , complemented the ion phenotypes, but not the membrane traffic phenotype. A GDP-restricted allele, ARL1[T32N] , did not effectively complement either phenotype. These results are consistent with a model in which Arl1 has three different conformations in vivo. We also explored the relationship between ARL1 and MON2 using the synthetic lethal phenotype exhibited by these two genes and demonstrated that MON2 is a negative regulator of the GTP-restricted allele of ARL1 , ARL1[Q72L] . Finally, we constructed several new alleles predicted to alter binding of Arl1 to the sole GRIP domain containing protein in yeast, Imh1, and found that ARL1[F52G] and ARL1[Y82G] were unable to complement the loss of ARL1 with respect to either the membrane traffic or K (+) homeostasis phenotypes. Our study expands understanding of the roles of Arl1 in vivo.     

Specificity of Arl2/Arl3 signaling is mediated by a ternary Arl3-effector-GAP complex

Arl2 and Arl3, members of the Arf subfamily of small G proteins, are believed to be involved in ciliary and microtubule-dependent processes. Recently, we could identify RP2, responsible for a variant of X-linked retinitis pigmentosa, as the Arl3-specific GAP. Here, we have characterized Arl2/3 interactions. We show the formation of a ternary complex between Arl3, its cognate GAP RP2 and its retinal effector HRG4. This complex seems to be important for photoreceptor function.     

Drosophila Ndfip is a novel regulator of Notch signaling

In the Drosophila wing, the Nedd4 ubiquitin ligases (E3s), dNedd4 and Su(dx), are important negative regulators of Notch signaling; they ubiquitinate Notch, promoting its endocytosis and turnover. Here, we show that Drosophila Nedd4 family interacting protein (dNdfip) interacts with the Drosophila Nedd4-like E3s. dNdfip expression dramatically enhances dNedd4 and Su(dx)-mediated wing phenotypes and further disrupts Notch signaling. dNdfip colocalizes with Notch in wing imaginal discs and with the late endosomal marker Rab7 in cultured cells. In addition, dNdfip expression in the wing leads to ectopic Notch signaling. Supporting this, expression of dNdfip suppressed Notch(+/-) wing phenotype and knockdown of dNdfip enhanced the Notch(+/-) wing phenotype. The increase in Notch activity by dNdfip is ligand independent as dNdfip expression also suppressed deltex RNAi and Serrate(+/-) wing phenotypes. The opposing effects of dNdfip expression on Notch signaling and its late endosomal localization support a model whereby dNdfip promotes localization of Notch to the limiting membrane of late endosomes allowing for activation, similar to the model previously shown with ectopic Deltex expression. When dNedd4 or Su(dx) are also present, dNdfip promotes their activity in Notch ubiquitination and internalization to the lysosomal lumen for degradation.     

RANKL-induced schlafen2 is a positive regulator of osteoclastogenesis

Osteoclasts are hematopoietic lineage derived-multinucleated cells that resorb bone. Their activity in balance with that of osteoblast is essential for bone homeostasis. Receptor activator of NF-κB ligand (RANKL) is known as an essential cytokine for the osteoclastogenesis, and c-Jun signaling in cooperation with NFAT family is crucial for RANKL-regulated osteoclastogenesis. We show here that schlafen2 (Slfn2), a member of a new family of growth regulatory genes involved in thymocyte development, is critical for osteoclastogenesis. RANKL selectively induces Slfn2 expression in osteoclast precursors via Rac1 signaling pathway. Targeted inhibition of Slfn2 by small interfering RNAs (siRNAs) markedly inhibits the formation of osteoclasts by diminishing the activation of c-Jun and the expression of c-Jun and NFATc1. In contrast, the overexpression of Slfn2 markedly increased phosphorylation and transactivation of c-Jun by RANKL. Together, these results indicate that Slfn2 has an essential role in osteoclastogenesis, functioning upstream of c-Jun and NFATc1.     

HOOKLESS1 is a positive regulator in Arabidopsis thermomorphogenesis

<正>Dear Editor,With the inevitable trend of global warming, it is urgent to understand how plants sense and respond to temperature increases for designing new crop varieties that can tolerate high ambient temperature. In Arabidopsis thaliana, high ambient temperature promotes hypocotyl elongation in seedlings and stimulates petiole elongation and hyponasty in rosette leaves. These changes in architecture are collectively     

Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance

The RPW8 locus from Arabidopsis thaliana Ms-0 includes two functional paralogous genes ( RPW8.1 and RPW8.2 ) and confers broad-spectrum resistance via the salicylic acid-dependent signaling pathway to the biotrophic fungal pathogens Golovinomyces spp. that cause powdery mildew diseases on multiple plant species. To identify proteins involved in regulation of the RPW8 protein function, a yeast two-hybrid screen was performed using RPW8.2 as bait. The 14-3-3 isoform lambda (designated GF14λ) was identified as a potential RPW8.2 interactor. The RPW8.2–GF14λ interaction was specific and engaged the C-terminal domain of RPW8.2, which was confirmed by pulldown assays. The physiological impact of the interaction was revealed by knocking down GF14λ by T-DNA insertion, which compromised basal and RPW8-mediated resistance to powdery mildew. In addition, over-expression of GF14λ resulted in hypersensitive response-like cell death and enhanced resistance to powdery mildew via the salicylic acid-dependent signaling pathway. The results from this study suggest that GF14λ may positively regulate the RPW8.2 resistance function and play a role in enhancing basal resistance in Arabidopsis.     

Hrs is a positive regulator of VEGF and insulin signaling

Both VEGF and insulin are implicated in the pathogenesis of diabetic retinopathy. While it has been established for many years that the number of cell surface receptors impacts upon VEGF and insulin action, little is known about the precise machinery and proteins driving VEGF-R2 and IR degradation. Here, we investigate the role of Hepatocyte growth factor-Regulated tyrosine kinase Substrate (Hrs), a regulator of RTK trafficking, in VEGF and insulin signaling. We report that ectopic expression of Hrs increases VEGF-R2 and IR number and tyrosine phosphorylation, leading to amplification of their downstream signaling. The UIM (Ubiquitin Interacting Motif) domain of Hrs is required for Hrs-induced increases in VEGF-R2, but not in IR. Furthermore, Hrs is tyrosine-phosphorylated in response to VEGF and insulin. We show that the UIM domain is required for Hrs phosphorylation in response to VEGF, but not to insulin. Importantly, Hrs co-localizes with both VEGF-R2 and IR and co-immunoprecipitates with both in a manner independent of the Hrs-UIM domain. Finally, we demonstrate that Hrs inhibits Nedd4-mediated VEGF-R2 degradation and acts additively with Grb10. We conclude that Hrs is a positive regulator of VEGF-R2 and IR signaling and that ectopic expression of Hrs protects both VEGF-R2 and IR from degradation.     

Cthrc1 is a positive regulator of osteoblastic bone formation

Background

Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.

Methodology/Principal Findings

We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.

Conclusions/Significance

Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.     

TIMAP is a positive regulator of pulmonary endothelial barrier function

TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.     

Drosophila Src42A is a negative regulator of RTK signaling

The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. Here we show that an extragenic suppressor of Raf, Su(Raf)1, encodes a Drosophila Src family gene Src42A. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Our study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed.     

Menin is a regulator of the stress response in Drosophila melanogaster

Menin, the product of the multiple endocrine neoplasia type I gene, has been implicated in several biological processes, including the control of gene expression and apoptosis, the modulation of mitogen-activated protein kinase pathways, and DNA damage sensing or repair. In this study, we have investigated the function of menin in the model organism Drosophila melanogaster. We show that Drosophila lines overexpressing menin or an RNA interference for this gene develop normally but are impaired in their response to several stresses, including heat shock, hypoxia, hyperosmolarity and oxidative stress. In the embryo subjected to heat shock, this impairment was characterized by a high degree of developmental arrest and lethality. The overexpression of menin enhanced the expression of HSP70 in embryos and interfered with its down-regulation during recovery at the normal temperature. In contrast, the inhibition of menin with RNA interference reduced the induction of HSP70 and blocked the activation of HSP23 upon heat shock, Menin was recruited to the Hsp70 promoter upon heat shock and menin overexpression stimulated the activity of this promoter in embryos. A 70-kDa inducible form of menin was expressed in response to heat shock, indicating that menin is also regulated in conditions of stress. The induction of HSP70 and HSP23 was markedly reduced or absent in mutant embryos harboring a deletion of the menin gene. These embryos, which did not express the heat shock-inducible form of menin, were also hypersensitive to various conditions of stress. These results suggest a novel role for menin in the control of the stress response and in processes associated with the maintenance of protein integrity.     

The deubiquitinase JOSD2 is a positive regulator of glucose metabolism

Cancer cells undergo complex metabolic alterations. The mechanisms underlying the tuning of cancer metabolism are under active investigation. Here, we identify the uncharacterized deubiquitinase JOSD2 as a positive regulator of cancer cell proliferation by displaying comprehensive effects on glucose catabolism. We found that JOSD2 directly controls a metabolic enzyme complex that includes Aldolase A, Phosphofructokinase-1 and Phosphoglycerate dehydrogenase, in vitro and in vivo. Further, JOSD2 expression, but not a catalytically inactive mutant, deubiquitinates and stabilizes the enzyme complex, thereby enhancing their activities and the glycolytic rate. This represents a selective JOSD2 feature that is not shared among other Machado–Joseph disease DUBs or observed in nontransformed cells. JOSD2 deficiency displays cytostatic effects and reduces glycolysis in a broad spectrum of tumor cells of distinct origin and its expression correlates with poor prognosis in non-small cell lung cancer. Overall, our study provides evidence for a previously unknown biological mechanism in which JOSD2 integrates glucose and serine metabolism with potential therapeutic implications.Subject terms: Cancer metabolism, Lung cancer