Molecular Biology of the Cell

EGF Transregulates Opioid Receptors through EGFR-mediated GRK2 Phosphorylation and Activation.

Publication Date: 2008 May 7 PMID: 18463167
Authors: Chen, Y. - Long, H. - Wu, Z. - Jiang, X. - Ma, L.
Journal: Mol Biol Cell

Monitoring Editor: Richard Assoian G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. Following EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild type GRK2 in an EGFR tyrosine kinase activity-dependent manner, while EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of micro-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.

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A Multi-Component Assembly Pathway Contributes to the Formation of Acentrosomal Microtubule Arrays in Interphase Drosophila cells.

Publication Date: 2008 May 7 PMID: 18463166
Authors: Rogers, G. C. - Rusan, N. M. - Peifer, M. - Rogers, S. L.
Journal: Mol Biol Cell

Monitoring Editor: Tim Stearns In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both gamma-tubulins. However, gamma-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted gamma-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila.

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High Resolution Crystal Structure and in vivo Function of a Kinesin-2 Homolog in Giardia intestinalis.

Publication Date: 2008 May 7 PMID: 18463165
Authors: Hoeng, J. C. - Dawson, S. C. - House, S. A. - Sagolla, M. S. - Pham, J. K. - Mancuso, J. J. - Lowe, J. - Cande, W. Z.
Journal: Mol Biol Cell

Monitoring Editor: Tim Stearns A critical component of flagellar assembly, the kinesin-2 heterotrimeric complex powers the anterograde movement of proteinaceous rafts along the outer doublet of axonemes in intraflagellar transport (IFT). We present the first high-resolution structures of a kinesin-2 motor domain and an ATP hydrolysis deficient motor domain mutant from the parasitic protist Giardia intestinalis. The high-resolution crystal structures of Giardia intestinalis wild-type kinesin-2 (GiKIN2a) motor domain with its docked neck linker and the hydrolysis deficient mutant GiKIN2aT104N were solved in a complex with ADP and Mg(2+) at 1.6 A and 1.8 A resolutions, respectively. These high-resolution structures provide unique insight into the nucleotide coordination within the active site. Giardia intestinalis has eight flagella, and we demonstrate that both kinesin-2 homologues and IFT proteins localize to both cytoplasmic and membrane-bound regions of axonemes with foci at cell body exit points and the distal flagellar tips. We demonstrate that the T104N mutation causes GiKIN2a to act as a rigor mutant in vitro. Overexpression of GiKIN2aT104N results in significant inhibition of flagellar assembly in the caudal, ventral, and posterolateral flagellar pairs. Thus we confirm the conserved evolutionary structure and functional role of kinesin-2 as the anterograde IFT motor in Giardia intestinalis.

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Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11.

Publication Date: 2008 May 7 PMID: 18463164
Authors: Murzik, U. - Hemmerich, P. - Weidtkamp-Peters, S. - Ulbricht, T. - Bussen, W. - Hentschel, J. - von Eggeling, F. - Melle, C.
Journal: Mol Biol Cell

Monitoring Editor: M. Bishr Omary S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11 we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, inluding DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatialy associated with sites of DNA double-strand break repair. Furthermore, while the expression of p21(WAF1/CIP1) was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knock-down. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA double-strand break repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle.

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Regulation of Microtubule Assembly and Organization in Mitosis by the AAA+ ATPase Pontin.

Publication Date: 2008 May 7 PMID: 18463163
Authors: Ducat, D. - Kawaguchi, S. I. - Liu, H. - Yates, J. R. 3rd - Zheng, Y.
Journal: Mol Biol Cell

Monitoring Editor: Stephen Doxsey To identify novel proteins important for microtubule assembly in mitosis, we have used a centrosome-based complementation assay to enrich for proteins with mitotic functions. An RNAi-based screen of these proteins allowed us to uncover 13 novel mitotic regulators. We carried out in-depth analyses of one of these proteins, Pontin, which is known to have a number of functions in interphase, including chromatin remodeling, DNA repair, and transcription. We show that reduction of Pontin by RNAi resulted in defects in spindle assembly in Drosophila S2 cells and in a number of mammalian tissue culture cell lines. Further characterization of Pontin in Xenopus egg extracts demonstrates that Pontin interacts with the gamma-tubulin ring complex (gammaTuRC). Because depletion of Pontin leads to defects in the assembly and organization of microtubule arrays in egg extracts, our studies suggest that Pontin has a mitosis-specific function in regulating microtubule assembly.

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Signal-dependent Regulation of Transcription by Histone Deacetylase 7 (HDAC7) Involves Recruitment to PML Nuclear Bodies.

Publication Date: 2008 May 7 PMID: 18463162
Authors: Gao, C. - Cheng, X. - Lam, M. - Liu, Y. - Liu, Q. - Chang, K. S. - Kao, H. Y.
Journal: Mol Biol Cell

Monitoring Editor: A. Gregory Matera Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic subnuclear compartments that play roles in several cellular processes including apoptosis, transcriptional regulation, and DNA repair. Histone deacetylase 7 (HDAC7) is a potent corepressor that inhibits transcription by MEF2 transcription factors. We show here that endogenous HDAC7 and PML interact and partially colocalize in PML NBs. Tumor necrosis factor alpha (TNF-alpha) treatment recruits HDAC7 to PML NBs and enhances association of HDAC7 with PML in human umbilical vein endothelial cells (HUVECs). Consequently, TNF-alpha promotes dissociation of HDAC7 from MEF2 transcription factors and the promoters of MEF2 target genes such as matrix metalloproteinase-10 (MMP-10), leading to accumulation of MMP-10 mRNA. Conversely, knockdown of PML enhances the association between HDAC7 and MEF2 and decreases MMP-10 mRNA accumulation. Accordingly, ectopic expression of PML recruits HDAC7 to PML NBs and leads to activation of MEF2 reporter activity. Notably, siRNA knockdown of PML decreases basal and TNF-alpha-induced MMP-10 mRNA accumulation. Our results reveal a novel mechanism by which PML sequesters HDAC7 to relieve repression and up-regulate gene expression.

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The Involvement of the Tyrosine Kinase c-Src in the Regulation of Reactive Oxygen Species (ROS) Generation Mediated by NADPH oxidase-1 (Nox1).

Publication Date: 2008 May 7 PMID: 18463161
Authors: Gianni, D. - Bohl, B. - Courtneidge, S. A. - Bokoch, G. M.
Journal: Mol Biol Cell

Monitoring Editor: Jonathan Chernoff NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been shown to function as second messenger molecules. To date 7 members of this family have been reported, including Nox1-5 and Duox1 and 2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and requires two cytosolic regulators, NoxO1 and NoxA1, as well as the binding of Rac1 GTPase, for its activity. In this study, we investigate the role of the tyrosine kinase c-Src in the regulation of ROS formation by Nox1. We show that c-Src induces Nox1-mediated ROS generation in the HT29 human colon carcinoma cell line through a Rac-dependent mechanism. Treatment of HT29 cells with the Src inhibitor PP2, expression of a kinase-inactive form of c-Src, and c-Src depletion by siRNA reduce both ROS generation and the levels of active Rac1. This is associated with decreased Src-mediated phosphorylation and activation of the Rac1-GEF Vav2. Consistent with this, Vav2 siRNA that specifically reduces endogenous Vav2 protein is able to dramatically decrease Nox1-dependent ROS generation and completely abolish c-Src-induced Nox1 activity. Taken together, these results establish c-Src as an important regulator of Nox1 activity, and may provide insight into the mechanisms of tumor formation in colon cancers.

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Interactions among Ytm1, Erb1, and Nop7 Required for Assembly of the Nop7-Subcomplex in Yeast Preribosomes.

Publication Date: 2008 Apr 30 PMID: 18448671
Authors: Tang, L. - Sahasranaman, A. - Jakovljevic, J. - Schleifman, E. - Woolford, J. L. Jr
Journal: Mol Biol Cell

Monitoring Editor: Thomas Fox In S. cerevisiae, more than 180 assembly factors associate with preribosomes to enable folding of pre-rRNA, recruitment of ribosomal proteins, and processing of pre-rRNAs to produce mature ribosomes. To examine the molecular architecture of preribosomes and to connect this structure to functions of each assembly factor, assembly subcomplexes have been purified from preribosomal particles. The Nop7-subcomplex contains three assembly factors: Nop7, Erb1, and Ytm1, each of which is necessary for conversion of 27SA3 pre-rRNA to 27SBS pre-rRNA. However, interactions among these three proteins and mechanisms of their recruitment and function in pre-rRNPs are poorly understood. Here we show that Ytm1, Erb1, and Nop7 assemble into preribosomes in an interdependent manner. We identified which domains within Ytm1, Erb1, and Nop7 are necessary for their interaction with each other and are sufficient for recruitment of each protein into preribosomes. Dominant negative effects on growth and ribosome biogenesis caused by overexpressing truncated Ytm1, Erb1, or Nop7 constructs, and recessive phenotypes of the truncated proteins revealed not only interaction domains but also other domains potentially important for each protein to function in ribosome biogenesis. Our data suggest a model for the architecture of the Nop7-subcomplex and provide potential functions of domains of each protein.

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Nucleophosmin Is a Binding Partner of Nucleostemin in Human Osteosarcoma Cells.

Publication Date: 2008 Apr 30 PMID: 18448670
Authors: Ma, H. - Pederson, T.
Journal: Mol Biol Cell

Monitoring Editor: Wendy Bickmore Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells as well as in many tumors and tumor-derived cell lines. In coimmunoprecipitation experiments nucleostemin is recovered with the tumor suppressor p53 and more recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway. Here we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high degree of colocalization in the granular component of the nucleolus during interphase and both proteins associated with pre-nucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that NS and NPM coreside in complexes and yeast two-hybrid experiments confirmed that they are interactive proteins, revealing the NPM-interactive region to be the 46 amino acid N-terminal domain of NS. In bimolecular fluorescence complementation (BiFC) studies bright nucleolar signals were observed, indicating that these two proteins directly interact in the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production.

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Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T Cells: Puncta and Distal Caps.

Publication Date: 2008 Apr 30 PMID: 18448669
Authors: Barr, V. A. - Bernot, K. M. - Srikanth, S. - Gwack, Y. - Balagopalan, L. - Regan, C. K. - Helman, D. J. - Sommers, C. L. - Oh-Hora, M. - Rao, A. - Samelson, L. E.
Journal: Mol Biol Cell

Monitoring Editor: Carl-Henrik Heldin The proteins STIM1 and Orai1are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells following engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum (ER). Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca(+2) influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca(+2) channel components to existing and newly forming immunological synapses.

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Interaction between Epsin/Yap180 Adaptors and the Scaffolds Ede1/Pan1 Is Required for Endocytosis.

Publication Date: 2008 Apr 30 PMID: 18448668
Authors: Maldonado-Baez, L. - Dores, M. R. - Perkins, E. M. - Drivas, T. G. - Hicke, L. - Wendland, B.
Journal: Mol Biol Cell

Monitoring Editor: Howard Riezman The spatial and temporal regulation of the interactions among the approximately 60 proteins required for endocytosis are under active investigation in many labs. We have identified the interaction between monomeric clathrin adaptors and endocytic scaffold proteins as a critical prerequisite for the recruitment and/or spatiotemporal dynamics of endocytic proteins at early and late stages of internalization. Quadruple deletion yeast cells (DeltaDeltaDeltaDelta) lacking four putative adaptors, Ent1/2 and Yap1801/2 (homologues of epsin and AP180/CALM proteins), with a plasmid encoding Ent1 or Yap1802 mutants, have defects in endocytosis and growth at 37 degrees C. Live-cell imaging revealed that the dynamics of the early- and late-acting scaffold proteins Ede1 and Pan1, respectively, depend upon adaptor interactions mediated by adaptor NPF motifs binding to scaffold EH domains. These results suggest that adaptor/scaffold interactions regulate transitions from early to late events, and that clathrin adaptor/scaffold protein interaction is essential for clathrin-mediated endocytosis.

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Access to Ribosomal Protein Rpl25p by the Signal Recognition Particle Is Required for Efficient Cotranslational Translocation.

Publication Date: 2008 Apr 30 PMID: 18448667
Authors: Dalley, J. A. - Selkirk, A. - Pool, M. R.
Journal: Mol Biol Cell

Monitoring Editor: Reid Gilmore Targeting of proteins to the endoplasmic reticulum (ER) occurs cotranslationally necessitating the interaction of the signal recognition particle (SRP) and the translocon with the ribosome. Biochemical and structural studies implicate ribosomal protein Rpl25p as a major ribosome interaction site for both these factors. Here we characterize an RPL25GFP fusion, which behaves as a dominant mutant leading to defects in cobut not post-translational translocation in vivo. In these cells, ribosomes still interact with ER membrane and the translocon, but are defective in binding SRP. Overexpression of SRP can restore ribosome binding of SRP, but only partially rescues growth and translocation defects. Our results indicate that Rpl25p plays a critical role in the recruitment of SRP to the ribosome.

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A Raft-derived, Pak1-regulated Entry Participates in {alpha}2{beta}1 Integrin-dependent Sorting to Caveosomes.

Publication Date: 2008 Apr 30 PMID: 18448666
Authors: Karjalainen, M. - Kakkonen, E. - Upla, P. - Paloranta, H. - Kankaanpaa, P. - Liberali, P. - Renkema, G. H. - Hyypia, T. - Heino, J. - Marjomaki, V.
Journal: Mol Biol Cell

Monitoring Editor: Jean Gruenberg We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulo-vesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized SV40 (SV40). Furthermore, the internalized endosomes do not contain GPI-anchored proteins or flotillin 1 suggesting that clustered alpha2beta1 integrin does not enter the GEEC (GPI-anchored protein enriched endosomal compartment) or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by Pak1, Rac1, PI3 kinase, PLC, and actin but not by dynamin 2 in SAOS-alpha2beta1 cells. An amiloride analog, EIPA, blocks infection, causes integrin accumulation in early tubulovesicular structures and prevents their structural maturation into multivesicular structures. Our results altogether suggest that alpha2beta1 integrin clustering defines its own entry pathway which is Pak1 dependent but clathrin and caveolin independent and which is able to sort cargo to caveosomes.

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Golgi-resident Small GTPase Rab33B Interacts with Atg16L and Modulates Autophagosome Formation.

Publication Date: 2008 Apr 30 PMID: 18448665
Authors: Itoh, T. - Fujita, N. - Kanno, E. - Yamamoto, A. - Yoshimori, T. - Fukuda, M.
Journal: Mol Biol Cell

Monitoring Editor: Akihiko Nakano Macroautophagy is a mechanism of degradation of cytoplasmic components in all eukaryotic cells. In the process of macroautophagy cytoplasmic components are wrapped by double-membrane structures called autophagosomes, whose formation involves unique membrane dynamics, i.e., de novo formation of a double-membrane sac called the isolation membrane and its elongation. However, the precise regulatory mechanism of isolation membrane formation and elongation remains unknown. In this study we showed that Golgi-resident small GTPase Rab33B (and Rab33A) specifically interacts with Atg16L, an essential factor in isolation membrane formation, in a GTP-dependent manner. Expression of a GTPase-deficient mutant Rab33B (Rab33B-Q92L) induced the lipidation of LC3, which is an essential process in autophagosome formation, even under nutrient-rich conditions, and attenuated macroautophagy, as judged by the degradation of p62/SQSTM1 (sequestosome 1). In addition, overexpression of the Rab33B-binding domain of Atg16L suppressed autophagosome formation. Our findings suggest that Rab33 modulates autophagosome formation through interaction with Atg16L.

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InCytes from MBC, May 2008.

Publication Date: 2008 May PMID: 18443187
Authors:
Journal: Mol Biol Cell

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Different Ubiquitin Signals Act at the Golgi and Plasma Membrane to Direct GAP1 Trafficking.

Publication Date: 2008 Apr 23 PMID: 18434603
Authors: Risinger, A. L. - Kaiser, C. A.
Journal: Mol Biol Cell

Monitoring Editor: David Drubin The high capacity general amino acid permease, Gap1p, in S. cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.

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A Novel Role for Cdk1/cyclin B in Regulating B-Raf Activation at Mitosis.

Publication Date: 2008 Apr 23 PMID: 18434602
Authors: Borysov, S. I. - Guadagno, T. M.
Journal: Mol Biol Cell

Monitoring Editor: Yixian Zheng MAPK activity is important during mitosis for spindle assembly and maintenance of the spindle checkpoint arrest. We previously identified B-Raf as a critical activator of the MAPK cascade during mitosis in Xenopus egg extracts and showed that B-Raf activation is regulated in an M-phase dependent manner. The mechanism that mediates B-Raf activation at mitosis has not been elucidated. Interestingly, activation of 95 kDa B-Raf at mitosis does not require phosphorylation of Thr-599 and Ser-602 residues (Thr-633 and Ser-636 in Xenopus B-Raf), previously shown to be essential for B-Raf activation by Ras. Instead, we provide evidence for Cdk1/cyclin B in mediating mitotic activation of B-Raf. In particular, Cdk1/cyclin B complexes associate with B-Raf at mitosis in Xenopus egg extracts and contribute to its phosphorylation. Mutagenesis and in vitro kinase assays demonstrated that Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144, which is part of a conserved Cdk1 preferential consensus site (S(144)PQK). Importantly, phosphorylation of Ser-144 is absolutely required for mitotic activation of B-Raf and subsequent activation of the MAPK cascade. However, substitution of a phospho-mimicking amino acid at Ser-144 failed to produce a constitutive active B-Raf indicating that, in addition of Ser-144 phosphorylation, other regulatory events may be needed to activate B-Raf at mitosis. Taken together, our data reveals a novel cell cycle mechanism for activating the B-Raf/MEK/MAPK cascade.

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Genetic Complementation Screen Identifies a Map Kinase Phosphatase, MKP3, as a Regulator of Dopamine Transporter Trafficking.

Publication Date: 2008 Apr 23 PMID: 18434601
Authors: Mortensen, O. V. - Larsen, M. B. - Prasad, B. M. - Amara, S. G.
Journal: Mol Biol Cell

Monitoring Editor: Jean Gruenberg The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization we used a genetic complementation screen in Xenopus oocytes to identify a MAP kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits Protein kinase C-induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38 and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin dependent endocytosis.

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Essential Role of GAK (auxilin-2) in Developing and Mature Mice.

Publication Date: 2008 Apr 23 PMID: 18434600
Authors: Lee, D. W. - Zhao, X. - Yim, Y. I. - Eisenberg, E. - Greene, L. E.
Journal: Mol Biol Cell

Monitoring Editor: Robert Parton Hsc70 with its cochaperone, either auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, since synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters all of which turn on during embryonic development. Deletion of GAK from brain, liver or skin dramatically altered the histology of these tissues causing the mice to die shortly after birth. Furthermore, by using a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis.

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UNC-108/Rab2 Regulates Post-endocytic Trafficking in C. elegans.

Publication Date: 2008 Apr 23 PMID: 18434599
Authors: Chun, D. K. - McEwen, J. M. - Burbea, M. - Kaplan, J. M.
Journal: Mol Biol Cell

Monitoring Editor: Sandra Lemmon Following endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway, and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways are differentially regulated. Here we identify UNC-108/Rab2 as a regulator of post-endocytic trafficking in both neurons and coelomocytes. Mutations in the C. elegans Rab2 gene unc-108, caused the GFP-tagged glutamate receptor, GLR-1 (GLR-1::GFP), to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, post-endocytic trafficking of the marker Texas Red-BSA was delayed. These results demonstrate that UNC-108/Rab2 regulates post-endocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative post-endocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking.

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GRASP55 Regulates Golgi Ribbon Formation.

Publication Date: 2008 Apr 23 PMID: 18434598
Authors: Feinstein, T. N. - Linstedt, A. D.
Journal: Mol Biol Cell

Monitoring Editor: Benjamin Glick Recent work indicates that MEK1 signaling at the G2/M cell cycle transition unlinks the contiguous mammalian Golgi apparatus and that this regulates cell cycle progression. Here we sought to determine the role in this pathway of GRASP55, a Golgi-localized target of MEK/ERK phosphorylation at mitosis. In support of the hypothesis that GRASP55 is inhibited in late G2 phase causing unlinking of the Golgi ribbon, we found that HeLa cells depleted of GRASP55 show a fragmented Golgi similar to control cells arrested in G2 phase. In the absence of GRASP55 Golgi stack length is shortened but Golgi stacking, compartmentalization, and transport appear normal. Absence of GRASP55 was also sufficient to suppress the requirement for MEK1 in the G2/M transition, a requirement which we previously found depends on an intact Golgi ribbon. Further, mimicking mitotic phosphorylation of GRASP55 using aspartic acid substitutions is sufficient to unlink the Golgi apparatus in a gene replacement assay. Our results implicate MEK1/ERK regulation of GRASP55-mediated Golgi linking as a control point in cell cycle progression.

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Coat-Tether Interaction in Golgi Organization.

Publication Date: 2008 Apr 23 PMID: 18434597
Authors: Guo, Y. - Punj, V. - Sengupta, D. - Linstedt, A. D.
Journal: Mol Biol Cell

Monitoring Editor: Patrick Brennwald Biogenesis of the Golgi apparatus is likely mediated by the COPI vesicle coat complex but the mechanism is poorly understood. Modeling of the COPI subunit betaCOP based on the clathrin adaptor AP2 suggested that the betaCOP C-terminus forms an appendage domain with a conserved FW binding pocket motif. On gene replacement after knockdown, versions of betaCOP with a mutated FW motif or flanking basic residues yielded a defect in Golgi organization reminiscent of that occurring in the absence of the vesicle tether p115. Indeed, betaCOP bound p115 and this depended on the betaCOP FW motif. Further, the interaction depended on E19E21 in the p115 head domain and inverse charge substitution blocked Golgi biogenesis in intact cells. Finally, Golgi assembly in permeabilized cells was significantly reduced by inhibitors containing intact, but not mutated, betaCOP FW or p115 EE motifs. Thus, Golgi organization depends on mutually interacting domains in betaCOP and p115 suggesting that vesicle tethering at the Golgi involves p115 binding to the COPI coat.

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An Mep2-dependent Transcriptional Profile Links Permease Function to Gene Expression during Pseudohyphal Growth in Saccharomyces cerevisiae.

Publication Date: 2008 Apr 23 PMID: 18434596
Authors: Rutherford, J. C. - Chua, G. - Hughes, T. - Cardenas, M. E. - Heitman, J.
Journal: Mol Biol Cell

Monitoring Editor: Carole Parent The ammonium permease Mep2 is required for the induction of pseudohyphal growth, a process in S. cerevisiae that occurs in response to nutrient limitation. Mep2 has both a transport and a regulatory function, supporting models in which Mep2 acts as a sensor of ammonium availability. Potentially similar ammonium permease dependent regulatory cascades operate in other fungi, and may also function in animals via the homologous Rh proteins; however, little is known about the molecular mechanisms that mediate ammonium sensing. We show that Mep2 is localized to the cell surface during pseudohyphal growth, and is required for both filamentous and invasive growth. Analysis of site-directed Mep2 mutants in residues lining the ammonia conducting channel reveal separation of function alleles (transport and signaling defective; transport-proficient/signaling-defective), indicating transport is necessary but not sufficient to sense ammonia. Further, Mep2 overexpression enhances differentiation under normally repressive conditions and induces a transcriptional profile that is consistent with activation of the MAP kinase pathway. This finding is supported by epistasis analysis establishing that the known role of the MAP kinase pathway in pseudohyphal growth is linked to Mep2 function. Taken together, these data strengthen the model that Mep2-like proteins are nutrient sensing transceptors that govern cellular differentiation.

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Global Regulation of the Interphase Microtubule System by Abundantly Expressed Op18/Stathmin.

Publication Date: 2008 Apr 23 PMID: 18434595
Authors: Sellin, M. E. - Holmfeldt, P. - Stenmark, S. - Gullberg, M.
Journal: Mol Biol Cell

Monitoring Editor: Yixian Zheng Op18/stathmin (Op18), a conserved microtubule depolymerizing and tubulin heterodimer binding protein, is a major interphase regulator of tubulin monomer-polymer partitioning in diverse cell types in which Op18 is abundant. Here we addressed the question of whether the microtubule regulatory function of Op18 includes regulation of tubulin heterodimer synthesis. We used two human cell model systems, K562 and Jurkat, combined with strategies for regulatable overexpression or depletion of Op18. Although Op18 depletion caused extensive overpolymerization and increased microtubule content in both cell types, we did not detect any alteration in polymer stability. Interestingly, however, we found that Op18 mediates positive regulation of tubulin heterodimer content in Jurkat cells, which was not observed in K562 cells. By analysis of cells treated with microtubule-poisoning drugs, we found that Jurkat cells regulate tubulin mRNA levels by a posttranscriptional mechanism similarly to normal primary cells, while this mechanism is nonfunctional in K562 cells. We present evidence that Op18 mediates posttranscriptional regulation of tubulin mRNA in Jurkat cells through the same basic autoregulatory mechanism as microtubule-poisoning drugs. This, combined with potent regulation of tubulin monomer-polymer partitioning, enables Op18 to exert global regulation of the microtubule system.

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Insulin Stimulates Phosphatidylinositol 3-Phosphate Production via the Activation of Rab5.

Publication Date: 2008 Apr 30 PMID: 18434594
Authors: Lodhi, I. J. - Bridges, D. - Chiang, S. H. - Zhang, Y. - Cheng, A. - Geletka, L. M. - Weisman, L. S. - Saltiel, A. R.
Journal: Mol Biol Cell

Monitoring Editor: Francis Barr Phosphatidylinositol 3-phosphate (PI(3)P) plays an important role in insulin-stimulated glucose uptake. Insulin promotes the production of PI(3)P at the plasma membrane by a process dependent on TC10 activation. Here, we report that insulin-stimulated PI(3)P production requires the activation of Rab5, a small GTPase that plays a critical role in phosphoinositide synthesis and turnover. This activation occurs at the plasma membrane, and is downstream of TC10. TC10 stimulates Rab5 activity via the recruitment of GAPEX-5, a VPS9 domain-containing guanyl nucleotide exchange factor that forms a complex with TC10. While overexpression of plasma membrane-localized GAPEX-5 or constitutively active Rab5 promotes PI(3)P formation, knockdown of GAPEX-5 or overexpression of a dominant negative Rab5 mutant blocks the effects of insulin or TC10 on this process. Concomitant with its effect on PI(3)P levels, the knockdown of GAPEX-5 blocks insulin-stimulated Glut4 translocation and glucose uptake. Together, these studies suggest that the TC10/GAPEX-5/Rab5 axis mediates insulin-stimulated production of PI(3)P, which regulates trafficking of Glut4 vesicles.

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The RING Domain of cIAP1 Mediates the Degradation of RING-bearing IAPs by Distinct Pathways.

Publication Date: 2008 Apr 23 PMID: 18434593
Authors: Cheung, H. H. - Plenchette, S. - Kern, C. J. - Mahoney, D. J. - Korneluk, R. G.
Journal: Mol Biol Cell

Monitoring Editor: Kunxin Luo The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of downregulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP and Livin, while sparing NAIP and Survivin which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 upregulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and ubiquitin-independent pathways.

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Regulation of the Candida albicans Cell Wall Damage Response by Transcription Factor Sko1 and PAS Kinase Psk1.

Publication Date: 2008 Apr 23 PMID: 18434592
Authors: Rauceo, J. M. - Blankenship, J. R. - Fanning, S. - Hamaker, J. J. - Deneault, J. S. - Smith, F. J. - Nantel, A. - Mitchell, A. P.
Journal: Mol Biol Cell

Monitoring Editor: Kerry Bloom The environmental niche of each fungus places distinct functional demands on the cell wall. Hence cell wall regulatory pathways may be highly divergent. We have pursued this hypothesis through analysis of C. albicans transcription factor mutants that are hypersensitive to caspofungin, an inhibitor of beta-1,3-glucan synthase. We report here that mutations in SKO1 cause this phenotype. C. albicans Sko1 undergoes Hog1-dependent phosphorylation after osmotic stress, like its S. cerevisiae ortholog, thus arguing that this Hog1-Sko1 relationship is conserved. However, Sko1 has a distinct role in the response to cell wall inhibition because: (1) sko1 mutants are much more sensitive to caspofungin than hog1 mutants; (2) Sko1 does not undergo detectable phosphorylation in response to caspofungin; (3) SKO1 transcript levels are induced by caspofungin in both wild-type and hog1 mutant strains; (4) sko1 mutants are defective in expression of caspofungin-inducible genes that are not induced by osmotic stress. Upstream Sko1 regulators were identified from a panel of caspofungin-hypersensitive protein kinase-defective mutants. Our results show that protein kinase Psk1 is required for expression of SKO1 and of Sko1-dependent genes in response to caspofungin. Thus Psk1 and Sko1 lie in a newly described signal transduction pathway.

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Aurora a Phosphorylates MCAK to Control Ran-dependent Spindle Bipolarity.

Publication Date: 2008 Apr 23 PMID: 18434591
Authors: Zhang, X. - Ems-McClung, S. C. - Walczak, C. E.
Journal: Mol Biol Cell

Monitoring Editor: Stephen Doxsey During mitosis MCAK localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic and pole localized MCAK are regulated is currently not clear. In this study we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity.

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The Endocytic Coat Protein ARH Associates with Motor and Centrosomal Proteins and Is Involved in Centrosome Assembly and Cytokinesis.

Publication Date: 2008 Apr 16 PMID: 18417616
Authors: Lehtonen, S. - Shah, M. - Nielsen, R. - Iino, N. - Ryan, J. J. - Zhou, H. - Farquhar, M. G.
Journal: Mol Biol Cell

Monitoring Editor: Yixian Zheng Numerous proteins involved in endocytosis at the plasma membrane have been shown to be present at novel intracellular locations and to have previously unrecognized functions. ARH (Autosomal Recessive Hypercholesterolemia) is an endocytic clathrin-associated adaptor protein that sorts members of the LDL receptor superfamily (LDLR, megalin, LRP). We report here that ARH also associates with centrosomes in several cell types. ARH interacts with centrosomal (gamma-tubulin and GPC2 and GPC3) and motor proteins (dynein heavy and intermediate chains). ARH cofractionates with gamma-tubulin on isolated centrosomes, and gamma-tubulin and ARH interact on isolated membrane vesicles. During mitosis, ARH sequentially localizes to the nuclear membrane, kinetochores, spindle poles and the midbody. ARH(-/-) embryonic fibroblasts (MEFs) show smaller or absent centrosomes suggesting ARH plays a role in centrosome assembly. Rat-1 fibroblasts depleted of ARH by siRNA and ARH(-/-) MEFs exhibit a slower rate of growth and prolonged cytokinesis. Taken together the data suggest that the defects in centrosome assembly in ARH depleted cells may give rise to cell cycle and mitotic/cytokinesis defects. We propose that ARH participates in centrosomal and mitotic dynamics by interacting with centrosomal proteins. Whether the centrosomal and mitotic functions of ARH are related to its endocytic role remains to be established.

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The Subcellular Distribution of Calnexin is Mediated by PACS-2.

Publication Date: 2008 Apr 16 PMID: 18417615
Authors: Myhill, N. - Lynes, E. M. - Nanji, J. A. - Blagoveshchenskaya, A. D. - Fei, H. - Simmen, K. C. - Cooper, T. J. - Thomas, G. - Simmen, T.
Journal: Mol Biol Cell

Monitoring Editor: Adam Linstedt Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 224 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.

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Glypican-1 Regulates Anaphase Promoting Complex/Cyclosome (APC/C) Substrates and Cell Cycle Progression in Endothelial Cells.

Publication Date: 2008 Apr 30 PMID: 18417614
Authors: Qiao, D. - Yang, X. - Meyer, K. - Friedl, A.
Journal: Mol Biol Cell

Monitoring Editor: Richard Assoian Glypican-1 (GPC1), a member of the mammalian glypican family of heparan sulfate proteoglycans, is highly expressed in glioma blood vessel endothelial cells (ECs). In this study, we investigated the role of GPC1 in EC replication by manipulating GPC1 expression in cultured mouse brain ECs. Moderate GPC1 overexpression stimulates EC growth, but proliferation is significantly suppressed when GPC1 expression is either knocked down or the molecule is highly overexpressed. Flow cytometric and biochemical analyses show that high or low expression of GPC1 cause cell cycle arrest at the G2 phase of the cell cycle and/or mitosis, accompanied by endoreduplication and consequently polyploidization. We further show that GPC1 inhibits the anaphase promoting complex/cyclosome (APC/C) mediated degradation of mitotic cyclins and securin. High-levels of GPC1 induce metaphase arrest and centrosome overproduction, alterations which are mimicked by overexpression of cyclin B1 and cyclin A, respectively. These observations suggest that GPC1 regulates EC cell cycle progression at least partially by modulating APC/C mediated degradation of mitotic cyclins and securin.

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Redundant Roles of BIG2 and BIG1, Guanine-nucleotide Exchange Factors for ARFs in Membrane Traffic between the TGN and endosomes.

Publication Date: 2008 Apr 16 PMID: 18417613
Authors: Ishizaki, R. - Shin, H. W. - Mitsuhashi, H. - Nakayama, K.
Journal: Mol Biol Cell

Monitoring Editor: Akihiko Nakano BIG2 and BIG1 are closely related guanine-nucleotide exchange factors (GEFs) for ADP-ribosylation factors (ARFs) and are involved in the regulation of membrane traffic through activating ARFs and recruiting coat protein complexes, such as the COPI complex and the AP-1 clathrin adaptor complex. Although both ARF-GEFs are associated mainly with the trans-Golgi network (TGN) and BIG2 is also associated with recycling endosomes, it is unclear whether BIG2 and BIG1 share some roles in membrane traffic. We here show that knockdown of both BIG2 and BIG1 by RNAi causes mislocalization of a subset of proteins associated with the TGN and recycling endosomes and blocks retrograde transport of furin from late endosomes to the TGN. Similar mislocalization and protein transport block, including furin, were observed in cells depleted of AP-1. Taken together with previous reports, these observations indicate that BIG2 and BIG1 play redundant roles in trafficking between the TGN and endosomes that involves the AP-1 complex.

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The Rho GDI Rdi1 Regulates Rho GTPases by Distinct Mechanisms.

Publication Date: 2008 Apr 16 PMID: 18417612
Authors: Tiedje, C. - Sakwa, I. - Just, U. - Hofken, T.
Journal: Mol Biol Cell

Monitoring Editor: Daniel Lew The small GTP-binding proteins of the Rho family are implicated in various cell functions including establishment and maintenance of cell polarity. Activity of Rho GTPases is not only regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast S. cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1 and Rho4, and regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase (PAK) Cla4. Following membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3beta (GSK-3beta) homolog Ygk3, vacuolar proteases and the proteasome. Taken together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.

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Gcn4 Is Required for the Response to Peroxide Stress in the Yeast Saccharomyces cerevisiae.

Publication Date: 2008 Apr 16 PMID: 18417611
Authors: Mascarenhas, C. - Edwards-Ingram, L. C. - Zeef, L. - Shenton, D. - Ashe, M. P. - Grant, C. M.
Journal: Mol Biol Cell

Monitoring Editor: Thomas Fox An oxidative stress occurs when reactive oxygen species overwhelm the cellular antioxidant defenses. We have examined the regulation of protein synthesis in S. cerevisiae in response to oxidative stress induced by exposure to hydroperoxides (hydrogen peroxide, cumene hydroperoxide), a thiol oxidant (diamide) and a heavy metal (cadmium). Examination of translational activity indicates that these oxidants inhibit translation at the initiation and post-initiation phases. Inhibition of translation initiation in response to hydroperoxides is entirely dependent on phosphorylation of the alpha subunit of eIF2 by the Gcn2 kinase. Activation of Gcn2 is mediated by uncharged tRNA since mutation of its HisRS domain abolishes regulation in response to hydroperoxides. Furthermore, Gcn4 is translationally up-regulated in response to H2O2 and is required for hydroperoxide resistance. We used transcriptional profiling to identify a wide-range of genes which mediate this response as part of the Gcn4-dependent H2O2-regulon. In contrast to hydroperoxides, regulation of translation initiation in response to cadmium and diamide depends on both Gcn2 and the eIF4E binding protein Eap1. Thus, the response to oxidative stress is mediated by oxidant-specific regulation of translation initiation and we suggest that this is an important mechanism underlying the ability of cells to adapt to different oxidants.

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Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization During Filamentous Growth.

Publication Date: 2008 Apr 16 PMID: 18417610
Authors: Bharucha, N. - Ma, J. - Dobry, C. J. - Lawson, S. K. - Yang, Z. - Kumar, A.
Journal: Mol Biol Cell

Monitoring Editor: Charles Boone The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving MAPK and PKA signaling modules, wherein yeast cells form interconnected and elongated chains. As standard strains of yeast are nonfilamentous, we constructed a unique set of 125 kinase-YFP chimeras in the filamentous Sigma1278b strain for this study. In total, we identified six cytoplasmic kinases (Bcy1p, Fus3p, Ksp1p, Kss1p, Sks1p, Tpk2p) that localize predominantly to the nucleus during filamentous growth. These kinases form part of an interdependent, localization-based regulatory network: deletion of each individual kinase, or loss of kinase activity, disrupts the nuclear translocation of at least two other kinases. In particular, this study highlights a previously unknown function for the kinase Ksp1p, indicating the essentiality of its nuclear translocation during yeast filamentous growth. Thus, the localization of Ksp1p and the other kinases identified here is tightly controlled during filamentous growth, representing an overlooked regulatory component of this stress response.

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Identification of a Novel Protein MICS1 that is Involved in Maintenance of Mitochondrial Morphology and Apoptotic Release of cytochrome c.

Publication Date: 2008 Apr 16 PMID: 18417609
Authors: Oka, T. - Sayano, T. - Tamai, S. - Yokota, S. - Kato, H. - Fujii, G. - Mihara, K.
Journal: Mol Biol Cell

Monitoring Editor: Janet Shaw Mitochondrial morphology dynamically changes in a balance of membrane fusion and fission in response to the environment, cell cycle, and apoptotic stimuli. Here, we report that a novel mitochondrial protein, MICS1, is involved in mitochondrial morphology in specific cristae structures and the apoptotic release of cytochrome c from the mitochondria. MICS1 is an inner membrane protein with a cleavable presequence and multiple transmembrane segments, and belongs to the Bi-1 super family. MICS1 down-regulation causes mitochondrial fragmentation and cristae disorganization, and stimulates the release of proapoptotic proteins. Expression of the anti-apoptotic protein Bcl-XL does not prevent morphological changes of mitochondria caused by MICS1 down-regulation, indicating that MICS1 plays a role in maintaining mitochondrial morphology separately from the function in apoptotic pathways. MICS1 overproduction induces mitochondrial aggregation, and partially inhibits cytochrome c release during apoptosis, regardless of the occurrence of Bax targeting. MICS1 is cross-linked to cytochrome c without disrupting membrane integrity. Thus, MICS1 facilitates the tight association of cytochrome c with the inner membrane. Furthermore, under low serum condition, the delay in apoptotic release of cytochrome c correlates with MICS1 upregulation without significant changes in mitochondrial morphology, suggesting that MICS1 individually functions in mitochondrial morphology and cytochrome c release.

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HOPS Proofreads the trans-SNARE Complex for Yeast Vacuole Fusion.

Publication Date: 2008 Apr 16 PMID: 18385512
Authors: Starai, V. J. - Hickey, C. M. - Wickner, W.
Journal: Mol Biol Cell

Monitoring Editor: Thomas F.J. Martin The fusion of yeast vacuoles, like other organelles, requires a Rab-family GTPase (Ypt7p), a Rab effector and an SM protein (HOPS complex), and SNAREs. The central 0-layer of the 4 bundled vacuolar SNAREs requires the wild-type 3 glutaminyl (Q) and 1 arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the Km for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at 2 levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, as it suppresses the fusion of trans-SNARE complexes formed without the N-terminal PX domain of Vam7p (Qc). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Qa) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.

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Distinct Angiogenic Mediators Are Required for Basic Fibroblast Growth Factor- and Vascular Endothelial Growth Factor-induced Angiogenesis: The Role of Cytoplasmic Tyrosine Kinase c-Abl in Tumor Angiogenesis.

Publication Date: 2008 May PMID: 18353972
Authors: Yan, W. - Bentley, B. - Shao, R.
Journal: Mol Biol Cell

Signaling pathways engaged by angiogenic factors bFGF and VEGF in tumor angiogenesis are not fully understood. The current study identifies cytoplasmic tyrosine kinase c-Abl as a key factor differentially mediating bFGF- and VEGF-induced angiogenesis in microvascular endothelial cells. STI571, a c-Abl kinase inhibitor, only inhibited bFGF- but not VEGF-induced angiogenesis. bFGF induced membrane receptor cooperation between integrin beta(3) and FGF receptor, and triggered a downstream cascade including FAK, c-Abl, and MAPK. This signaling pathway is different from one utilized by VEGF that includes integrin beta(5), VEGF receptor-2, Src, FAK, and MAPK. Ectopic expression of wild-type c-Abl sensitized angiogenic response to bFGF, but kinase dead mutant c-Abl abolished this activity. Furthermore, the wild-type c-Abl enhanced angiogenesis in both Matrigel implantation and tumor xenograft models. These data provide novel insights into c-Abl's differential functions in mediating bFGF- and VEGF-induced angiogenesis.

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Src-mediated Cortactin Phosphorylation Regulates Actin Localization and Injurious Blebbing in Acinar Cells.

Publication Date: 2008 May PMID: 18353971
Authors: Singh, V. P. - McNiven, M. A.
Journal: Mol Biol Cell

Suprastimulation of pancreatic acini is a well-known model for pancreatitis, and it is characterized by actin reorganization and cell blebbing. Currently, however, the mechanisms underlying regulation of these aberrant cytoskeletal and membrane dynamics and how they contribute to cell injury are unclear. We observed that suprastimulation results in a rapid activation of Src and relocalization of the actin-binding protein cortactin from the apical to the basolateral domain at the necks of membrane blebs. Furthermore, Src-mediated cortactin tyrosine phosphorylation was markedly increased after suprastimulation. Pretreatment of acini with Src inhibitors or expression of a cortactin tyrosine phospho-inhibitory mutant reduced actin redistribution and bleb formation induced by suprastimulation in vitro. Importantly, inhibition of Src activity in rat models of suprastimulation-induced pancreatitis substantially reduced disease severity, as indicated by a reduction in serum amylase and pancreatic edema and a striking improvement in tissue histology. These findings indicate a novel, disease-relevant role for Src-mediated cortactin phosphorylation in aberrant reorganization of the actin cytoskeleton, a mechanism that is likely to have implications in other types of cell injury. In addition, they suggest a potential use for Src inhibitors as an approach to reduce cell injury.

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Functional Redundancy of the B9 Proteins and Nephrocystins in Caenorhabditis elegans Ciliogenesis.

Publication Date: 2008 May PMID: 18337471
Authors: Williams, C. L. - Winkelbauer, M. E. - Schafer, J. C. - Michaud, E. J. - Yoder, B. K.
Journal: Mol Biol Cell

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS.

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Alg13p, the Catalytic Subunit of the Endoplasmic Reticulum UDP-GlcNAc Glycosyltransferase, Is a Target for Proteasomal Degradation.

Publication Date: 2008 May PMID: 18337470
Authors: Averbeck, N. - Gao, X. D. - Nishimura, S. - Dean, N.
Journal: Mol Biol Cell

The second step of dolichol-linked oligosaccharide synthesis in the N-linked glycosylation pathway at the endoplasmic reticulum (ER) membrane is catalyzed by an unusual hetero-oligomeric UDP-N-acetylglucosamine transferase that in most eukaryotes is comprised of at least two subunits, Alg13p and Alg14p. Alg13p is the cytosolic and catalytic subunit that is recruited to the ER by the membrane protein Alg14p. We show that in Saccharomyces cerevisiae, cytosolic Alg13p is very short-lived, whereas membrane-associated Alg13 is relatively stable. Cytosolic Alg13p is a target for proteasomal degradation, and the failure to degrade excess Alg13p leads to glycosylation defects. Alg13p degradation does not require ubiquitin but instead, requires a C-terminal domain whose deletion results in Alg13p stability. Conversely, appending this sequence onto normally long-lived beta-galactosidase causes it to undergo rapid degradation, demonstrating that this C-terminal domain represents a novel and autonomous degradation motif. These data lead to the model that proteasomal degradation of excess unassembled Alg13p is an important quality control mechanism that ensures proper protein complex assembly and correct N-linked glycosylation.

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Prolyl Hydroxylase PHD3 Activates Oxygen-dependent Protein Aggregation.

Publication Date: 2008 May PMID: 18337469
Authors: Rantanen, K. - Pursiheimo, J. - Hogel, H. - Himanen, V. - Metzen, E. - Jaakkola, P. M.
Journal: Mol Biol Cell

The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability.

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Constitutive Activation of Chaperone-mediated Autophagy in Cells with Impaired Macroautophagy.

Publication Date: 2008 May PMID: 18337468
Authors: Kaushik, S. - Massey, A. C. - Mizushima, N. - Cuervo, A. M.
Journal: Mol Biol Cell

Three different types of autophagy-macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA)-contribute to degradation of intracellular components in lysosomes in mammalian cells. Although some level of basal macroautophagy and CMA activities has been described in different cell types and tissues, these two pathways are maximally activated under stress conditions. Activation of these two pathways is often sequential, suggesting the existence of some level of cross-talk between both stress-related autophagic pathways. In this work, we analyze the consequences of blockage of macroautophagy on CMA activity. Using mouse embryonic fibroblasts deficient in Atg5, an autophagy-related protein required for autophagosome formation, we have found that blockage of macroautophagy leads to up-regulation of CMA, even under basal conditions. Interestingly, different mechanisms contribute to the observed changes in CMA-related proteins and the consequent activation of CMA during basal and stress conditions in these macroautophagy-deficient cells. This work supports a direct cross-talk between these two forms of autophagy, and it identifies changes in the lysosomal compartment that underlie the basis for the communication between both autophagic pathways.

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Soft Substrate Up-regulates the Interaction of STIM1 with Store-operated Ca2+ Channels That Lead to Normal Epithelial Cell Apoptosis.

Publication Date: 2008 May PMID: 18337467
Authors: Chiu, W. T. - Tang, M. J. - Jao, H. C. - Shen, M. R.
Journal: Mol Biol Cell

We have demonstrated that soft substrate induced apoptosis in polarized cells, but not in transformed cells by disturbance of Ca(2+) homeostasis. This study aims to further investigate the regulatory mechanisms underlying the disruption of Ca(2+)-signaling integrity in soft substrate-induced epithelial apoptosis. Soft substrate up-regulated the store-operated Ca(2+) (SOC) entry across the plasma membrane of normal cervical epithelial cells, which resulted in increased cytosolic Ca(2+) levels. Concomitantly, soft substrate induced the aggregation and translocation of stromal interacting molecule 1 (STIM1) toward the cell periphery to colocalize with Orai1, an essential pore subunit of SOC channel, detected by fluorescence resonance energy transfer approach and confocal image analyses. The disturbed Ca(2+) homeostasis resulted in the activation of mu-calpain, which cleaved alpha-spectrin, induced actin disorganization, and caused apoptosis. In contrast, soft substrate did not disturb Ca(2+) homeostasis or induce apoptosis in cervical cancer cells. Chelating extracellular Ca(2+) by EGTA and down-regulated SOC entry by small interfering RNA targeting STIM1 or inhibitors targeting Ca(2+)-binding site of calpain significantly inhibited soft substrate-induced activation of mu-calpain and epithelial cell apoptosis. Thus, soft substrate up-regulates the interaction of STIM1 with SOC channels, which results in the activation of mu-calpain and subsequently induces normal epithelial cell apoptosis.

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Apical accumulation of rho in the neural plate is important for neural plate cell shape change and neural tube formation.

Publication Date: 2008 May PMID: 18337466
Authors: Kinoshita, N. - Sasai, N. - Misaki, K. - Yonemura, S.
Journal: Mol Biol Cell

Although Rho-GTPases are well-known regulators of cytoskeletal reorganization, their in vivo distribution and physiological functions have remained elusive. In this study, we found marked apical accumulation of Rho in developing chick embryos undergoing folding of the neural plate during neural tube formation, with similar accumulation of activated myosin II. The timing of accumulation and biochemical activation of both Rho and myosin II was coincident with the dynamics of neural tube formation. Inhibition of Rho disrupted its apical accumulation and led to defects in neural tube formation, with abnormal morphology of the neural plate. Continuous activation of Rho also altered neural tube formation. These results indicate that correct spatiotemporal regulation of Rho is essential for neural tube morphogenesis. Furthermore, we found that a key morphogenetic signaling pathway, the Wnt/PCP pathway, was implicated in the apical accumulation of Rho and regulation of cell shape in the neural plate, suggesting that this signal may be the spatiotemporal regulator of Rho in neural tube formation.

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Interplay between S-Cyclin-dependent Kinase and Dbf4-dependent Kinase in Controlling DNA Replication through Phosphorylation of Yeast Mcm4 N-Terminal Domain.

Publication Date: 2008 May PMID: 18321994
Authors: Devault, A. - Gueydon, E. - Schwob, E.
Journal: Mol Biol Cell

Cyclin-dependent (CDK) and Dbf4-dependent (DDK) kinases trigger DNA replication in all eukaryotes, but how these kinases cooperate to regulate DNA synthesis is largely unknown. Here, we show that budding yeast Mcm4 is phosphorylated in vivo during S phase in a manner dependent on the presence of five CDK phosphoacceptor residues within the N-terminal domain of Mcm4. Mutation to alanine of these five sites (mcm4-5A) abolishes phosphorylation and decreases replication origin firing efficiency at 22 degrees C. Surprisingly, the loss of function mcm4-5A mutation confers cold and hydroxyurea sensitivity to DDK gain of function conditions (mcm5/bob1 mutation or DDK overexpression), implying that phosphorylation of Mcm4 by CDK somehow counteracts negative effects produced by ectopic DDK activation. Deletion of the S phase cyclins Clb5,6 is synthetic lethal with mcm4-5A and mimics its effects on DDK up mutants. Furthermore, we find that Clb5 expressed late in the cell cycle can still suppress the lethality of clb5,6Delta bob1 cells, whereas mitotic cyclins Clb2, 3, or 4 expressed early cannot. We propose that the N-terminal extension of eukaryotic Mcm4 integrates regulatory inputs from S-CDK and DDK, which may play an important role for the proper assembly or stabilization of replisome-progression complexes.

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Collagen Fibril Flow and Tissue Translocation Coupled to Fibroblast Migration in 3D Collagen Matrices.

Publication Date: 2008 May PMID: 18321993
Authors: Miron-Mendoza, M. - Seemann, J. - Grinnell, F.
Journal: Mol Biol Cell

In nested collagen matrices, human fibroblasts migrate from cell-containing dermal equivalents into surrounding cell-free outer matrices. Time-lapse microscopy showed that in addition to cell migration, collagen fibril flow occurred in the outer matrix toward the interface with the dermal equivalent. Features of this flow suggested that it depends on the same cell motile machinery that normally results in cell migration. Collagen fibril flow was capable of producing large-scale tissue translocation as shown by closure of a approximately 1-mm gap between paired dermal equivalents in floating, nested collagen matrices. Our findings demonstrate that when fibroblasts interact with collagen matrices, tractional force exerted by the cells can couple to matrix translocation as well as to cell migration.

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The Yak1 Kinase Is Involved in the Initiation and Maintenance of Hyphal Growth in Candida albicans.

Publication Date: 2008 May PMID: 18321992
Authors: Goyard, S. - Knechtle, P. - Chauvel, M. - Mallet, A. - Prevost, M. C. - Proux, C. - Coppee, J. Y. - Schwartz, P. - Dromer, F. - Park, H. - Filler, S. G. - Janbon, G. - d'Enfert, C.
Journal: Mol Biol Cell

Members of the dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) family perform a variety of functions in eukaryotes. We used gene disruption, targeted pharmacologic inhibition, and genome-wide transcriptional profiling to dissect the function of the Yak1 DYRK in the human fungal pathogen Candida albicans. C. albicans strains with mutant yak1 alleles showed defects in the yeast-to-hypha transition and in maintaining hyphal growth. They also could not form biofilms. Despite their in vitro filamentation defect, C. albicans yak1Delta/yak1Delta mutants remained virulent in animal models of systemic and oropharyngeal candidiasis. Transcriptional profiling showed that Yak1 was necessary for the up-regulation of only a subset of hypha-induced genes. Although downstream targets of the Tec1 and Bcr1 transcription factors were down-regulated in the yak1Delta/yak1Delta mutant, TEC1 and BCR1 were not. Furthermore, 63% of Yak1-dependent, hypha-specific genes have been reported to be negatively regulated by the transcriptional repressor Tup1 and inactivation of TUP1 in the yak1Delta/yak1Delta mutant restored filamentation, suggesting that Yak1 may function upstream of Tup1 in governing hyphal emergence and maintenance.

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The Integrin-coupled Signaling Adaptor p130Cas Suppresses Smad3 Function in Transforming Growth Factor-{beta} Signaling.

Publication Date: 2008 May PMID: 18321991
Authors: Kim, W. - Seok Kang, Y. - Soo Kim, J. - Shin, N. Y. - Hanks, S. K. - Song, W. K.
Journal: Mol Biol Cell

Reciprocal cooperative signaling by integrins and growth factor receptors at G1 phase during cell cycle progression is well documented. By contrast, little is known about the cross-talk between integrin and transforming growth factor (TGF)-beta signaling. Here, we show that integrin signaling counteracts the inhibitory effects of TGF-beta on cell growth and that this effect is mediated by p130Cas (Crk-associated substrate, 130 kDa). Adhesion to fibronectin or laminin reduces TGF-beta-induced Smad3 phosphorylation and thus inhibits TGF-beta-mediated growth arrest; loss of p130Cas abrogates these effects. Loss and gain of function studies demonstrated that, once tyrosine-phosphorylated via integrin signaling, p130Cas binds to Smad3 and reduces phosphorylation of Smad3. That in turn leads to inhibition of p15 and p21 expression and facilitation of cell cycle progression. Thus, p130Cas-mediated control of TGF-beta/Smad signaling may provide an additional clue to the mechanism underlying resistance to TGF-beta-induced growth inhibition.

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Aurora A Regulates the Activity of HURP by Controlling the Accessibility of Its Microtubule-binding Domain.

Publication Date: 2008 May PMID: 18321990
Authors: Wong, J. - Lerrigo, R. - Jang, C. Y. - Fang, G.
Journal: Mol Biol Cell

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

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Cleavage of Mcd1 by Caspase-like Protease Esp1 Promotes Apoptosis in Budding Yeast.

Publication Date: 2008 May PMID: 18321989
Authors: Yang, H. - Ren, Q. - Zhang, Z.
Journal: Mol Biol Cell

Over the last decade, yeast has been used successfully as a model system for studying the molecular mechanism of apoptotic cell death. Here, we report that Mcd1, the yeast homology of human cohesin Rad21, plays an important role in hydrogen peroxide-induced apoptosis in yeast. On induction of cell death, Mcd1 is cleaved and the C-terminal fragment is translocated from nucleus into mitochondria, causing the decrease of mitochondrial membrane potential and the amplification of cell death in a cytochrome c-dependent manner. We further demonstrate that the caspase-like protease Esp1 has dual functions and that it is responsible for the cleavage of Mcd1 during the hydrogen peroxide-induced apoptosis. When apoptosis is induced, Esp1 is released from the anaphase inhibitor Pds1. The activated Esp1 acts as caspase-like protease for the cleavage of Mcd1, which enhances the cell death via its translocation from nucleus to mitochondria.

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The Atg16L Complex Specifies the Site of LC3 Lipidation for Membrane Biogenesis in Autophagy.

Publication Date: 2008 May PMID: 18321988
Authors: Fujita, N. - Itoh, T. - Omori, H. - Fukuda, M. - Noda, T. - Yoshimori, T.
Journal: Mol Biol Cell

Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an approximately 800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L inhibits autophagosome formation. Mechanistically, the site of LC3 lipidation is determined by the membrane localization of the Atg16L complex as well as the interaction of Atg12 with Atg3, the E2 enzyme for the LC3 lipidation process. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. We propose that the Atg16L complex is a new type of E3-like enzyme that functions as a scaffold for LC3 lipidation by dynamically localizing to the putative source membranes for autophagosome formation.

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Regulation and Targeting of the Fission Yeast Formin cdc12p in Cytokinesis.

Publication Date: 2008 May PMID: 18305104
Authors: Yonetani, A. - Lustig, R. J. - Moseley, J. B. - Takeda, T. - Goode, B. L. - Chang, F.
Journal: Mol Biol Cell

Formins are conserved actin nucleators which promote the assembly of actin filaments for the formation of diverse actin structures. In fission yeast Schizosaccharomyces pombe, the formin cdc12p is required specifically in assembly of the actin-based contractile ring during cytokinesis. Here, using a mutational analysis of cdc12p, we identify regions of cdc12p responsible for ring assembly and localization. Profilin-binding residues of the FH1 domain regulate actin assembly and processive barbed-end capping by the FH2 domain. Studies using photobleaching (FRAP) and sensitivity to latrunculin A treatment show that profilin binding modulates the rapid dynamics of actin and cdc12p within the ring in vivo. Visualized by functional GFP-fusion constructs expressed from the endogenous promoter, cdc12p appears in a small number of cytoplasmic motile spot structures that deliver the formin to the ring assembly site, without detectable formation of an intermediate band of "nodes." The FH3/DID region directs interphase spot localization, while an N-terminal region and the FH1-FH2 domains of cdc12p can target its localization to the ring. Mutations in putative DID and DAD regions do not alter regulation, suggesting that cdc12p is not regulated by a canonical autoinhibition mechanism. Our findings provide insights into the regulation of formin activity and the mechanisms of contractile ring dynamics and assembly.

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Stathmin Activity Influences Sarcoma Cell Shape, Motility, and Metastatic Potential.

Publication Date: 2008 May PMID: 18305103
Authors: Belletti, B. - Nicoloso, M. S. - Schiappacassi, M. - Berton, S. - Lovat, F. - Wolf, K. - Canzonieri, V. - D'Andrea, S. - Zucchetto, A. - Friedl, P. - Colombatti, A. - Baldassarre, G.
Journal: Mol Biol Cell

The balanced activity of microtubule-stabilizing and -destabilizing proteins determines the extent of microtubule dynamics, which is implicated in many cellular processes, including adhesion, migration, and morphology. Among the destabilizing proteins, stathmin is overexpressed in different human malignancies and has been recently linked to the regulation of cell motility. The observation that stathmin was overexpressed in human recurrent and metastatic sarcomas prompted us to investigate stathmin contribution to tumor local invasiveness and distant dissemination. We found that stathmin stimulated cell motility in and through the extracellular matrix (ECM) in vitro and increased the metastatic potential of sarcoma cells in vivo. On contact with the ECM, stathmin was negatively regulated by phosphorylation. Accordingly, a less phosphorylable stathmin point mutant impaired ECM-induced microtubule stabilization and conferred a higher invasive potential, inducing a rounded cell shape coupled with amoeboid-like motility in three-dimensional matrices. Our results indicate that stathmin plays a significant role in tumor metastasis formation, a finding that could lead to exploitation of stathmin as a target of new antimetastatic drugs.

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Multiplexed Dendritic Targeting of {alpha} Calcium Calmodulin-dependent Protein Kinase II, Neurogranin, and Activity-regulated Cytoskeleton-associated Protein RNAs by the A2 Pathway.

Publication Date: 2008 May PMID: 18305102
Authors: Gao, Y. - Tatavarty, V. - Korza, G. - Levin, M. K. - Carson, J. H.
Journal: Mol Biol Cell

In neurons, many different RNAs are targeted to dendrites where local expression of the encoded proteins mediates synaptic plasticity during learning and memory. It is not known whether each RNA follows a separate trafficking pathway or whether multiple RNAs are targeted to dendrites by the same pathway. Here, we show that RNAs encoding alpha calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein are coassembled into the same RNA granules and targeted to dendrites by the same cis/trans-determinants (heterogeneous nuclear ribonucleoprotein [hnRNP] A2 response element and hnRNP A2) that mediate dendritic targeting of myelin basic protein RNA by the A2 pathway in oligodendrocytes. Multiplexed dendritic targeting of different RNAs by the same pathway represents a new organizing principle for coordinating gene expression at the synapse.

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Genome-wide Expression Profiling, In Vivo DNA Binding Analysis, and Probabilistic Motif Prediction Reveal Novel Abf1 Target Genes during Fermentation, Respiration, and Sporulation in Yeast.

Publication Date: 2008 May PMID: 18305101
Authors: Schlecht, U. - Erb, I. - Demougin, P. - Robine, N. - Borde, V. - Nimwegen, E. V. - Nicolas, A. - Primig, M.
Journal: Mol Biol Cell

The autonomously replicating sequence binding factor 1 (Abf1) was initially identified as an essential DNA replication factor and later shown to be a component of the regulatory network controlling mitotic and meiotic cell cycle progression in budding yeast. The protein is thought to exert its functions via specific interaction with its target site as part of distinct protein complexes, but its roles during mitotic growth and meiotic development are only partially understood. Here, we report a comprehensive approach aiming at the identification of direct Abf1-target genes expressed during fermentation, respiration, and sporulation. Computational prediction of the protein's target sites was integrated with a genome-wide DNA binding assay in growing and sporulating cells. The resulting data were combined with the output of expression profiling studies using wild-type versus temperature-sensitive alleles. This work identified 434 protein-coding loci as being transcriptionally dependent on Abf1. More than 60% of their putative promoter regions contained a computationally predicted Abf1 binding site and/or were bound by Abf1 in vivo, identifying them as direct targets. The present study revealed numerous loci previously unknown to be under Abf1 control, and it yielded evidence for the protein's variable DNA binding pattern during mitotic growth and meiotic development.

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The Nup358-RanGAP Complex Is Required for Efficient Importin {alpha}/{beta}-dependent Nuclear Import.

Publication Date: 2008 May PMID: 18305100
Authors: Hutten, S. - Flotho, A. - Melchior, F. - Kehlenbach, R. H.
Journal: Mol Biol Cell

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin alpha/beta-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin beta, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin beta strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin beta recycling and reformation of novel import complexes.

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The Clathrin Adaptor Gga2p Is a Phosphatidylinositol 4-phosphate Effector at the Golgi Exit.

Publication Date: 2008 May PMID: 18287542
Authors: Demmel, L. - Gravert, M. - Ercan, E. - Habermann, B. - Muller-Reichert, T. - Kukhtina, V. - Haucke, V. - Baust, T. - Sohrmann, M. - Kalaidzidis, Y. - Klose, C. - Beck, M. - Peter, M. - Walch-Solimena, C.
Journal: Mol Biol Cell

Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.

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Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase.

Publication Date: 2008 May PMID: 18287541
Authors: Burakov, A. V. - Zhapparova, O. N. - Kovalenko, O. V. - Zinovkina, L. A. - Potekhina, E. S. - Shanina, N. A. - Weiss, D. G. - Kuznetsov, S. A. - Nadezhdina, E. S.
Journal: Mol Biol Cell

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-DeltaT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-DeltaT have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

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GRIM-19 Is Essential for Maintenance of Mitochondrial Membrane Potential.

Publication Date: 2008 May PMID: 18287540
Authors: Lu, H. - Cao, X.
Journal: Mol Biol Cell

GRIM-19 was found to copurify with complex I of mitochondrial respiratory chain and subsequently was demonstrated to be involved in complex I assembly and activity. To further understand its function in complex I, we dissected its functional domains by generating a number of deletion, truncation, and point mutants. The mitochondrial localization sequences were located at the N-terminus. Strikingly, deletion of residues 70-80, 90-100, or the whole C-terminal region (70-144) led to a loss of mitochondrial transmembrane potential (DeltaPsim). However, similar deletions of another two complex I subunits, NDUFA9 and NDUFS3, did not show such effect. We also found that deletion of the last 10 residues affected GRIM-19's ability to be assembled to complex I. We constructed a dominant-negative mutant containing the N-terminal 60 and the last C-terminal 10 residues, which could be assembled into complex I, but failed to maintain normal DeltaPsim. Cells overexpressing this mutant did not spontaneously undergo cell death, but were sensitized to apoptosis induced by cell death agents. Our results demonstrate that GRIM-19 is required for electron transfer activity of complex I, and disruption of DeltaPsim by GRIM-19 mutants enhances the cells' sensitivity to apoptotic stimuli.

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Yeast ARV1 Is Required for Efficient Delivery of an Early GPI Intermediate to the First Mannosyltransferase during GPI Assembly and Controls Lipid Flow from the Endoplasmic Reticulum.

Publication Date: 2008 May PMID: 18287539
Authors: Kajiwara, K. - Watanabe, R. - Pichler, H. - Ihara, K. - Murakami, S. - Riezman, H. - Funato, K.
Journal: Mol Biol Cell

Glycosylphosphatidylinositol (GPI), covalently attached to many eukaryotic proteins, not only acts as a membrane anchor but is also thought to be a sorting signal for GPI-anchored proteins that are associated with sphingolipid and sterol-enriched domains. GPI anchors contain a core structure conserved among all species. The core structure is synthesized in two topologically distinct stages on the leaflets of the endoplasmic reticulum (ER). Early GPI intermediates are assembled on the cytoplasmic side of the ER and then are flipped into the ER lumen where a complete GPI precursor is synthesized and transferred to protein. The flipping process is predicted to be mediated by a protein referred as flippase; however, its existence has not been proven. Here we show that yeast Arv1p is an important protein required for the delivery of an early GPI intermediate, GlcN-acylPI, to the first mannosyltransferase of GPI synthesis in the ER lumen. We also provide evidence that ARV1 deletion and mutations in other proteins involved in GPI anchor synthesis affect inositol phosphorylceramide synthesis as well as the intracellular distribution and amounts of sterols, suggesting a role of GPI anchor synthesis in lipid flow from the ER.

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Bap31 Is an Itinerant Protein That Moves between the Peripheral Endoplasmic Reticulum (ER) and a Juxtanuclear Compartment Related to ER-associated Degradation.

Publication Date: 2008 May PMID: 18287538
Authors: Wakana, Y. - Takai, S. - Nakajima, K. I. - Tani, K. - Yamamoto, A. - Watson, P. - Stephens, D. J. - Hauri, H. P. - Tagaya, M.
Journal: Mol Biol Cell

Certain endoplasmic reticulum (ER)-associated degradation (ERAD) substrates with transmembrane domains are segregated from other ER proteins and sorted into a juxtanuclear subcompartment, known as the ER quality control compartment. Bap31 is an ER protein with three transmembrane domains, and it is assumed to be a cargo receptor for ER export of some transmembrane proteins, especially those prone to ERAD. Here, we show that Bap31 is a component of the ER quality control compartment and that it moves between the peripheral ER and a juxtanuclear ER or ER-related compartment distinct from the conventional ER-Golgi intermediate compartment. The third and second transmembrane domains of Bap31 are principally responsible for the movement to and recycling from the juxtanuclear region, respectively. This cycling was blocked by depolymerization of microtubules and disruption of dynein-dynactin function. Overexpression of Sar1p and Arf1 mutants affected Bap31 cycling, suggesting that this cycling pathway is related to the conventional vesicular transport pathways.

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Paralemmin-1, a Modulator of Filopodia Induction Is Required for Spine Maturation.

Publication Date: 2008 May PMID: 18287537
Authors: Arstikaitis, P. - Gauthier-Campbell, C. - Carolina Gutierrez Herrera, R. - Huang, K. - Levinson, J. N. - Murphy, T. H. - Kilimann, M. W. - Sala, C. - Colicos, M. A. - El-Husseini, A.
Journal: Mol Biol Cell

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.

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The rim101 pathway is involved in rsb1 expression induced by altered lipid asymmetry.

Publication Date: 2008 May PMID: 18287536
Authors: Ikeda, M. - Kihara, A. - Denpoh, A. - Igarashi, Y.
Journal: Mol Biol Cell

Biological membranes consist of lipid bilayers. The lipid compositions between the two leaflets of the plasma membrane differ, generating lipid asymmetry. Maintenance of proper lipid asymmetry is physiologically quite important, and its collapse induces several cellular responses including apoptosis and platelet coagulation. Thus, a change in lipid asymmetry must be restored to maintain "lipid asymmetry homeostasis." However, to date no lipid asymmetry-sensing proteins or any related downstream signaling pathways have been identified. We recently demonstrated that expression of the putative yeast sphingoid long-chain base transporter/translocase Rsb1 is induced when glycerophospholipid asymmetry is altered. Using mutant screening, we determined that the pH-responsive Rim101 pathway, the protein kinase Mck1, and the transcription factor Mot3 all act in lipid asymmetry signaling, and that the Rim101 pathway was activated in response to a change in lipid asymmetry. The activated transcription factor Rim101 induces Rsb1 expression via repression of another transcription repressor, Nrg1. Changes in lipid asymmetry are accompanied by cell surface exposure of negatively charged phospholipids; we speculate that the Rim101 pathway recognizes the surface charges.

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