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1.  The 90-kDa Heat Shock Protein Stabilizes the Polysomal Ribonuclease 1 mRNA Endonuclease to Degradation by the 26S Proteasome 
Molecular Biology of the Cell  2008;19(2):546-552.
The polysomal ribonuclease 1 (PMR1) mRNA endonuclease forms a selective complex with its translating substrate mRNAs where it is activated to initiate mRNA decay. Previous work showed tyrosine phosphorylation is required for PMR1 targeting to this polysome-bound complex, and it identified c-Src as the responsible kinase. c-Src phosphorylation occurs in a distinct complex, and the current study shows that 90-kDa heat shock protein (Hsp90) is also recovered with PMR1 and c-Src. Hsp90 binding to PMR1 is inhibited by geldanamycin, and geldanamycin stabilizes substrate mRNA to PMR1-mediated decay. PMR1 is inherently unstable and geldanamycin causes PMR1 to rapidly disappear in a process that is catalyzed by the 26S proteasome. We present a model where Hsp90 interacts transiently to stabilize PMR1 in a manner similar to its interaction with c-Src, thus facilitating the tyrosine phosphorylation and targeting of PMR1 to polysomes.
doi:10.1091/mbc.E07-08-0774
PMCID: PMC2230583  PMID: 18045990
2.  The 90-kDa Heat Shock Protein Stabilizes the Polysomal Ribonuclease 1 mRNA Endonuclease to Degradation by the 26S Proteasome 
Molecular biology of the cell  2007;19(2):546-552.
The polysomal ribonuclease 1 (PMR1) mRNA endonuclease forms a selective complex with its translating substrate mRNAs where it is activated to initiate mRNA decay. Previous work showed tyrosine phosphorylation is required for PMR1 targeting to this polysome-bound complex, and it identified c-Src as the responsible kinase. c-Src phosphorylation occurs in a distinct complex, and the current study shows that 90-kDa heat shock protein (Hsp90) is also recovered with PMR1 and c-Src. Hsp90 binding to PMR1 is inhibited by geldanamycin, and geldanamycin stabilizes substrate mRNA to PMR1-mediated decay. PMR1 is inherently unstable and geldanamycin causes PMR1 to rapidly disappear in a process that is catalyzed by the 26S proteasome. We present a model where Hsp90 interacts transiently to stabilize PMR1 in a manner similar to its interaction with c-Src, thus facilitating the tyrosine phosphorylation and targeting of PMR1 to polysomes.
doi:10.1091/mbc.E07-08-0774
PMCID: PMC2230583  PMID: 18045990
3.  Nudel Modulates Kinetochore Association and Function of Cytoplasmic Dynein in M Phase 
Molecular Biology of the Cell  2007;18(7):2656-2666.
The microtubule-based motor cytoplasmic dynein/dynactin is a force generator at the kinetochore. It also transports proteins away from kinetochores to spindle poles. Regulation of such diverse functions, however, is poorly understood. We have previously shown that Nudel is critical for dynein-mediated protein transport, whereas mitosin, a kinetochore protein that binds Nudel, is involved in retention of kinetochore dynein/dynactin against microtubule-dependent stripping. Here we demonstrate that Nudel is required for robust localization of dynein/dynactin at the kinetochore. It localizes to kinetochores after nuclear envelope breakdown, depending mostly (∼78%) on mitosin and slightly on dynein/dynactin. Depletion of Nudel by RNA interference (RNAi) or overexpression of its mutant incapable of binding either Lis1 or dynein heavy chain abolishes the kinetochore protein transport and mitotic progression. Similar to mitosin RNAi, Nudel RNAi also leads to increased stripping of kinetochore dynein/dynactin in the presence of microtubules. Taking together, our results suggest a dual role of kinetochore Nudel: it activates dynein-mediated protein transport and, when interacting with both mitosin and dynein, stabilizes kinetochore dynein/dynactin against microtubule-dependent stripping to facilitate the force generation function of the motor.
doi:10.1091/mbc.E06-04-0345
PMCID: PMC1924840  PMID: 17494871
4.  Ihh/Gli2 Signaling Promotes Osteoblast Differentiation by Regulating Runx2 Expression and Function 
Molecular Biology of the Cell  2007;18(7):2411-2418.
Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.
doi:10.1091/mbc.E06-08-0743
PMCID: PMC1924839  PMID: 17442891
5.  The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting 
Molecular Biology of the Cell  2007;18(7):2728-2734.
The RNA component of the signal recognition particle (SRP) is universally required for cotranslational protein targeting. Biochemical studies have shown that SRP RNA participates in the central step of protein targeting by catalyzing the interaction of the SRP with the SRP receptor (SR). SRP RNA also accelerates GTP hydrolysis in the SRP·SR complex once formed. Using a reverse-genetic and biochemical analysis, we identified mutations in the E. coli SRP protein, Ffh, that abrogate the activity of the SRP RNA and cause corresponding targeting defects in vivo. The mutations in Ffh that disrupt SRP RNA activity map to regions that undergo dramatic conformational changes during the targeting reaction, suggesting that the activity of the SRP RNA is linked to the major conformational changes in the signal sequence-binding subunit of the SRP. In this way, the SRP RNA may coordinate the interaction of the SRP and the SR with ribosome recruitment and transfer to the translocon, explaining why the SRP RNA is an indispensable component of the protein targeting machinery.
doi:10.1091/mbc.E07-02-0117
PMCID: PMC1924838  PMID: 17507650
7.  Anchoring of Protein Kinase A-Regulatory Subunit IIα to Subapically Positioned Centrosomes Mediates Apical Bile Canalicular Lumen Development in Response to Oncostatin M but Not cAMP 
Molecular Biology of the Cell  2007;18(7):2745-2754.
Oncostatin M and cAMP signaling stimulate apical surface-directed membrane trafficking and apical lumen development in hepatocytes, both in a protein kinase A (PKA)-dependent manner. Here, we show that oncostatin M, but not cAMP, promotes the A-kinase anchoring protein (AKAP)-dependent anchoring of the PKA regulatory subunit (R)IIα to subapical centrosomes and that this requires extracellular signal-regulated kinase 2 activation. Stable expression of the RII-displacing peptide AKAP-IS, but not a scrambled peptide, inhibits the association of RIIα with centrosomal AKAPs and results in the repositioning of the centrosome from a subapical to a perinuclear location. Concomitantly, common endosomes, but not apical recycling endosomes, are repositioned from a subapical to a perinuclear location, without significant effects on constitutive or oncostatin M-stimulated basolateral-to-apical transcytosis. Importantly, however, the expression of the AKAP-IS peptide completely blocks oncostatin M-, but not cAMP-stimulated apical lumen development. Together, the data suggest that centrosomal anchoring of RIIα and the interrelated subapical positioning of these centrosomes is required for oncostatin M-, but not cAMP-mediated, bile canalicular lumen development in a manner that is uncoupled from oncostatin M-stimulated apical lumen-directed membrane trafficking. The results also imply that multiple PKA-mediated signaling pathways control apical lumen development and that subapical centrosome positioning is important in some of these pathways.
doi:10.1091/mbc.E06-08-0732
PMCID: PMC1924835  PMID: 17494870
8.  Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA 
Molecular Biology of the Cell  2007;18(7):2636-2645.
Cotranslational protein targeting to membranes is regulated by two GTPases in the signal recognition particle (SRP) and the SRP receptor; association between the two GTPases is slow and is accelerated 400-fold by the SRP RNA. Intriguingly, the otherwise universally conserved SRP RNA is missing in a novel chloroplast SRP pathway. We found that even in the absence of an SRP RNA, the chloroplast SRP and receptor GTPases can interact efficiently with one another; the kinetics of interaction between the chloroplast GTPases is 400-fold faster than their bacterial homologues, and matches the rate at which the bacterial SRP and receptor interact with the help of SRP RNA. Biochemical analyses further suggest that the chloroplast SRP receptor is pre-organized in a conformation that allows optimal interaction with its binding partner, so that conformational changes during complex formation are minimized. Our results highlight intriguing differences between the classical and chloroplast SRP and SRP receptor GTPases, and help explain how the chloroplast SRP pathway can mediate efficient targeting of proteins to the thylakoid membrane in the absence of the SRP RNA, which plays an indispensable role in all the other SRP pathways.
doi:10.1091/mbc.E07-01-0037
PMCID: PMC1924832  PMID: 17475780
9.  The Conserved Spc7 Protein Is Required for Spindle Integrity and Links Kinetochore Complexes in Fission Yeast  
Molecular Biology of the Cell  2007;18(7):2441-2454.
Spc7, a member of the conserved Spc105/KNL-1 family of kinetochore proteins, was identified as an interaction partner of the EB1 homologue Mal3. Spc7 associates with the central centromere region of the chromosome but does not affect transcriptional silencing. Here, we show that Spc7 is required for the integrity of the spindle as well as for targeting of MIND but not of Ndc80 complex components to the kinetochore. Spindle defects in spc7 mutants were severe ranging from the inability to form a bipolar spindle in early mitosis to broken spindles in midanaphase B. spc7 mutant phenotypes were partially rescued by extra α-tubulin or extra Mal2. Thus, Spc7 interacts genetically with the Mal2-containing Sim4 complex.
doi:10.1091/mbc.E06-08-0738
PMCID: PMC1924829  PMID: 17442892
10.  A Cytosolic Splice Variant of Cab45 Interacts with Munc18b and Impacts on Amylase Secretion by Pancreatic Acini 
Molecular Biology of the Cell  2007;18(7):2473-2480.
We identified in a yeast two-hybrid screen the EF-hand Ca2+-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, we characterize a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind 45Ca2+, and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca2+. In this assay, Cab45b also binds the Munc18a isoform in a Ca2+-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca2+-triggered zymogen secretion. Furthermore, we show that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca2+-induced amylase release from streptolysin-O–permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini.
doi:10.1091/mbc.E06-10-0950
PMCID: PMC1924827  PMID: 17442889
11.  A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis 
Molecular Biology of the Cell  2007;18(7):2455-2462.
Type I interferons (IFN-α/β) induce apoptosis in certain tumor cell lines but not others. Here we describe a mutation in STAT2 that confers an apoptotic effect in tumor cells in response to type I IFNs. This mutation was introduced in a conserved motif, PYTK, located in the STAT SH2 domain, which is shared by STAT1, STAT2, and STAT3. To test whether the tyrosine in this motif might be phosphorylated and affect signaling, Y631 of STAT2 was mutated to phenylalanine (Y631F). Although it was determined that Y631 was not phosphorylated, the Y631F mutation conferred sustained signaling and induction of IFN-stimulated genes. This prolonged IFN response was associated with sustained tyrosine phosphorylation of STAT1 and STAT2 and their mutual association as heterodimers, which resulted from resistance to dephosphorylation by the nuclear tyrosine phosphatase TcPTP. Finally, cells bearing the Y631F mutation in STAT2 underwent apoptosis after IFN-α stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-α could account for one difference between tumor cell lines that undergo IFN-α–induced apoptosis compared with those that display an antiproliferative response but do not die.
doi:10.1091/mbc.E06-09-0843
PMCID: PMC1924825  PMID: 17442890
12.  Cholesterol Controls Lipid Endocytosis through Rab11 
Molecular Biology of the Cell  2007;18(7):2667-2677.
Cellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We examined the endocytosis of several lipid probes in subconfluent and confluent CHO cells. In subconfluent cells, fluorescent lipid probes including poly(ethylene glycol)derivatized cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol, and fluorescent sphingomyelin analogs were internalized to pericentriolar recycling endosomes. This accumulation was not observed in confluent cells. Internalization of fluorescent lactosylceramide was not affected by cell confluency, suggesting that the endocytosis of specific membrane components is affected by cell confluency. The crucial role of cellular cholesterol in cell confluency–dependent endocytosis was suggested by the observation that the fluorescent sphingomyelin was transported to recycling endosomes when cellular cholesterol was depleted in confluent cells. To understand the molecular mechanism(s) of cell confluency– and cholesterol-dependent endocytosis, we examined intracellular distribution of rab small GTPases. Our results indicate that rab11 but not rab4, altered intracellular localization in a cell confluency–associated manner, and this alteration was dependent on cell cholesterol. In addition, the expression of a constitutive active mutant of rab11 changed the endocytic route of lipid probes from early to recycling endosomes. These results thus suggest that cholesterol controls endocytic routes of a subset of membrane lipids through rab11.
doi:10.1091/mbc.E06-10-0924
PMCID: PMC1924824  PMID: 17475773
13.  Coordination of Hpr1 and Ubiquitin Binding by the UBA Domain of the mRNA Export Factor Mex67 
Molecular Biology of the Cell  2007;18(7):2561-2568.
The ubiquitin-associated (UBA) domain of the mRNA nuclear export receptor Mex67 helps in coordinating transcription elongation and nuclear export by interacting both with ubiquitin conjugates and specific targets, such as Hpr1, a component of the THO complex. Here, we analyzed substrate specificity and ubiquitin selectivity of the Mex67 UBA domain. UBA-Mex67 is formed by three helices arranged in a classical UBA fold plus a fourth helix, H4. Deletion or mutation of helix H4 strengthens the interaction between UBA-Mex67 and ubiquitin, but it decreases its affinity for Hpr1. Interaction with Hpr1 is required for Mex67 UBA domain to bind polyubiquitin, possibly by inducing an H4-dependent conformational change. In vivo, deletion of helix H4 reduces cotranscriptional recruitment of Mex67 on activated genes, and it also shows an mRNA export defect. Based on these results, we propose that H4 functions as a molecular switch that coordinates the interaction of Mex67 with ubiquitin bound to specific substrates, defines the selectivity of the Mex67 UBA domain for polyubiquitin, and prevents its binding to nonspecific substrates.
doi:10.1091/mbc.E07-02-0153
PMCID: PMC1924821  PMID: 17475778
14.  A Novel AAK1 Splice Variant Functions at Multiple Steps of the Endocytic Pathway 
Molecular Biology of the Cell  2007;18(7):2698-2706.
Phosphorylation is a critical step in regulating receptor transport through the endocytic pathway. AAK1 is a serine/threonine kinase that is thought to coordinate the recruitment of AP-2 to receptors containing tyrosine-based internalization motifs by phosphorylating the μ2 subunit. Here we have identified a long form of AAK1 (AAK1L) that contains an extended C-terminus that encodes an additional clathrin-binding domain (CBD2) consisting of multiple low-affinity interaction motifs. Protein interaction studies demonstrate that AAK1L CBD2 directly binds clathrin. However, in vitro kinase assays reveal little difference between AAK1 isoforms in their basal or clathrin-stimulated kinase activity toward the AP-2 μ2 subunit. However, overexpression of AAK1L CBD2 impairs transferrin endocytosis, confirming an endocytic role for AAK1. Surprisingly, CBD2 overexpression or AAK1 depletion by RNA interference significantly impairs transferrin recycling from the early/sorting endosome. These observations suggest that AAK1 functions at multiple steps of the endosomal pathway by regulating transferrin internalization and its rapid recycling back to the plasma membrane from early/sorting endosome.
doi:10.1091/mbc.E06-09-0831
PMCID: PMC1924820  PMID: 17494869
15.  A Knockout Mouse Approach Reveals that TCTP Functions as an Essential Factor for Cell Proliferation and Survival in a Tissue- or Cell Type–specific Manner 
Molecular Biology of the Cell  2007;18(7):2525-2532.
Translationally controlled Tumor Protein (TCTP) is an evolutionally highly conserved protein which has been implicated in many cellular functions that are related to cell growth, death, and even the allergic response of the host. To address the physiological roles of TCTP, we generated TCTP knockout mice by targeted gene disruption. Heterozygous mutants appeared to be developmentally normal. However, homozygous mutants (TCTP−/−) were embryonic lethal. TCTP−/− embryos were smaller in size than the control littermates at all postimplantation stages examined. Although TCTP is widely expressed in both extraembryonic and embryonic tissues, the most prominent defect of the TCTP−/− embryo at embryonic stage day 5.5 (E5.5) was in its epiblast, which had a reduced number of cells compared with wild-type controls. The knockout embryos also suffered a higher incidence of apoptosis in epiblast starting about E6.5 and subsequently died around E9.5–10.5 with a severely disorganized structure. Last, we demonstrated that TCTP−/− and control mouse embryonic fibroblasts manifested similar proliferation activities and apoptotic sensitivities to various death stimuli. Taken together, our results suggest that despite that TCTP is widely expressed in many tissues or cell types, it appears to regulate cell proliferation and survival in a tissue- or cell type–specific manner.
doi:10.1091/mbc.E07-02-0188
PMCID: PMC1924818  PMID: 17475776
16.  Binding of CAP70 to Inducible Nitric Oxide Synthase and Implications for the Vectorial Release of Nitric Oxide in Polarized Cells 
Molecular Biology of the Cell  2007;18(7):2768-2777.
In this article we analyze the mechanisms by which the C-terminal four amino acids of inducible nitric oxide synthase (NOS2) interact with proteins that contain PDZ (PSD-95/DLG/ZO-1) domains resulting in the translocation of NOS2 to the cellular apical domain. It has been reported that human hepatic NOS2 associates to EBP50, a protein with two PDZ domains present in epithelial cells. We describe herein that NOS2 binds through its four carboxy-terminal residues to CAP70, a protein that contains four PDZ modules that is targeted to apical membranes. Interestingly, this interaction augments both the cytochrome c reductase and ·NO-synthase activities of NOS2. Binding of CAP70 to NOS2 also results in an increase in the population of active NOS2 dimers. In addition, CAP70 participates in the correct subcellular targeting of NOS2 in a process that is also dependent on the acylation state of the N-terminal end of NOS2. Hence, nonpalmitoylated NOS2 is unable to progress toward the apical side of the cell despite its interaction with either EBP50 or CAP70. Likewise, if we abrogate the interaction of NOS2 with either EBP50 or CAP70 by fusing the GFP reporter to the carboxy-terminal end of NOS2 palmitoylation is not sufficient to confer an apical targeting.
doi:10.1091/mbc.E06-12-1102
PMCID: PMC1924814  PMID: 17507652
17.  Cytotoxic Necrotizing Factor 1 Prevents Apoptosis via the Akt/IκB Kinase Pathway: Role of Nuclear Factor-κB and Bcl-2 
Molecular Biology of the Cell  2007;18(7):2735-2744.
Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by some pathogenic strains of Escherichia coli that specifically activates Rho, Rac, and Cdc42 GTPases. We previously reported that this toxin prevents the ultraviolet-B–induced apoptosis in epithelial cells, with a mechanism that remained to be defined. In this work, we show that the proteasomal degradation of the Rho GTPase is necessary to achieve cell death protection, because inhibition of Rho degradation abolishes the prosurvival activity of CNF1. We hypothesize that Rho inactivation allows the activity of Rac to become dominant. This in turn leads to stimulation of the phosphoinositide 3-kinase/Akt/IκB kinase/nuclear factor-κB prosurvival pathway and to a remarkable modification in the architecture of the mitochondrial network, mainly consisting in the appearance of elongated and interconnected mitochondria. Importantly, we found that Bcl-2 silencing reduces the ability of CNF1 to protect cells against apoptosis and that it also prevents the CNF1-induced mitochondrial changes. It is worth noting that the ability of a bacterial toxin to induce such a remodeling of the mitochondrial network is herein reported for the first time. The possible pathophysiological relevance of this finding is discussed.
doi:10.1091/mbc.E06-10-0910
PMCID: PMC1924812  PMID: 17507655
18.  Rab1b Interacts with GBF1 and Modulates both ARF1 Dynamics and COPI Association 
Molecular Biology of the Cell  2007;18(7):2400-2410.
Assembly of the cytosolic coat protein I (COPI) complex at the ER–Golgi interface is directed by the ADP ribosylation factor1 (Arf1) and its guanine nucleotide exchange factor (GBF1). Rab1b GTPase modulates COPI recruitment, but the molecular mechanism underlying this action remains unclear. Our data reveal that in vivo expression of the GTP-restricted Rab1b mutant (Rab1Q67L) increased the association of GBF1 and COPI to peripheral structures localized at the ER exit sites (ERES) interface. Active Rab1b also stabilized Arf1 on Golgi membranes. Furthermore, we characterized GBF1 as a new Rab1b effector, and showed that its N-terminal domain was involved in this interaction. Rab1b small interfering RNA oligonucleotide assays suggested that Rab1b was required for GBF1 membrane association. To further understand how Rab1b functions in ER-to-Golgi transport, we analyzed GFP-Rab1b dynamics in HeLa cells. Time-lapse microscopy indicated that the majority of the Rab1b-labeled punctuated structures are relatively short-lived with limited-range movements. FRAP of Golgi GFP-Rab1bwt showed rapid recovery (t1/2 120 s) with minimal dependence on microtubules. Our data support a model where Rab1b-GTP induces GBF1 recruitment at the ERES interface and at the Golgi complex where it is required for COPII/COPI exchange or COPI vesicle formation, respectively.
doi:10.1091/mbc.E06-11-1005
PMCID: PMC1924811  PMID: 17429068
19.  Depletion of the Nucleolar Protein Nucleostemin Causes G1 Cell Cycle Arrest via the p53 Pathway 
Molecular Biology of the Cell  2007;18(7):2630-2635.
Nucleostemin (NS) is a nucleolar protein expressed in adult and embryo-derived stem cells, transformed cell lines, and tumors. NS decreases when proliferating cells exit the cell cycle, but it is unknown how NS is controlled, and how it participates in cell growth regulation. Here, we show that NS is down-regulated by the tumor suppressor p14ARF and that NS knockdown elevates the level of tumor suppressor p53. NS knockdown led to G1 cell cycle arrest in p53-positive cells but not in cells in which p53 was genetically deficient or depleted by small interfering RNA knockdown. These results demonstrate that, in the cells investigated, the level of NS is regulated by p14ARF and the control of the G1/S transition by NS operates in a p53-dependent manner.
doi:10.1091/mbc.E07-03-0244
PMCID: PMC1924810  PMID: 17494866
20.  Multiple Interactions Drive Adaptor-Mediated Recruitment of the Ubiquitin Ligase Rsp5 to Membrane Proteins In Vivo and In Vitro 
Molecular Biology of the Cell  2007;18(7):2429-2440.
Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain “PY” motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY–WW interactions is required for the ubiquitination of Smf1.
doi:10.1091/mbc.E07-01-0011
PMCID: PMC1924809  PMID: 17429078
21.  MTOC Reorientation Occurs during FcγR-mediated Phagocytosis in Macrophages 
Molecular Biology of the Cell  2007;18(7):2389-2399.
Cell polarization is essential for targeting signaling elements and organelles to active plasma membrane regions. In a few specialized cell types, cell polarity is enhanced by reorientation of the MTOC and associated organelles toward dynamic membrane sites. Phagocytosis is a highly polarized process whereby particles >0.5 μm are internalized at stimulated regions on the cell surface of macrophages. Here we provide detailed evidence that the MTOC reorients toward the site of particle internalization during phagocytosis. We visualized MTOC proximity to IgG-sRBCs in fixed RAW264.7 cells, during live cell imaging using fluorescent chimeras to label the MTOC and using frustrated phagocytosis assays. MTOC reorientation in macrophages is initiated by FcγR ligation and is complete within 1 h. Polarization of the MTOC toward the phagosome requires the MT cytoskeleton and dynein motor activity. cdc42, PI3K, and mPAR-6 are all important signaling molecules for MTOC reorientation during phagocytosis. MTOC reorientation was not essential for particle internalization or phagolysosome formation. However Golgi reorientation in concert with MTOC reorientation during phagocytosis implicates MTOC reorientation in antigen processing events in macrophages.
doi:10.1091/mbc.E06-12-1128
PMCID: PMC1924806  PMID: 17442887
22.  Inhibition of Integrin-mediated Crosstalk with Epidermal Growth Factor Receptor/Erk or Src Signaling Pathways in Autophagic Prostate Epithelial Cells Induces Caspase-independent Death 
Molecular Biology of the Cell  2007;18(7):2481-2490.
In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via α3β1 and α6β4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via α3β1, but not α6β4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through α3β1 and α6β4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.
doi:10.1091/mbc.E06-04-0261
PMCID: PMC1924805  PMID: 17475774
23.  PI4P Promotes the Recruitment of the GGA Adaptor Proteins to the Trans-Golgi Network and Regulates Their Recognition of the Ubiquitin Sorting Signal 
Molecular Biology of the Cell  2007;18(7):2646-2655.
Phosphatidylinositol 4 phosphate (PI4P) is highly enriched in the trans-Golgi network (TGN). Here we establish that PI4P is a key regulator of the recruitment of the GGA clathrin adaptor proteins to the TGN and that PI4P has a novel role in promoting their recognition of the ubiquitin (Ub) sorting signal. Knockdown of PI4KIIα by RNA interference (RNAi), which depletes the TGN′s PI4P, impaired the recruitment of the GGAs to the TGN. GGAs bind PI4P primarily through their GAT domain, in a region called C-GAT, which also binds Ub but not Arf1. We identified two basic residues in the GAT domain that are essential for PI4P binding in vitro and for the recruitment of GGAs to the TGN in vivo. Unlike wild-type GGA, GGA with mutated GATs failed to rescue the abnormal TGN phenotype of the GGA RNAi-depleted cells. These residues partially overlap with those that bind Ub, and PI4P increased the affinity of the GAT domain for Ub. Because the recruitment of clathrin adaptors and their cargoes to the TGN is mediated through a web of low-affinity interactions, our results show that the dual roles of PI4P can promote specific GGA targeting and cargo recognition at the TGN.
doi:10.1091/mbc.E06-10-0897
PMCID: PMC1924815  PMID: 17494868
24.  Rapid and Reversible Nuclear Accumulation of Cytoplasmic tRNA in Response to Nutrient Availability 
Molecular Biology of the Cell  2007;18(7):2678-2686.
Cytoplasmic tRNAs have recently been found to accumulate in the nucleus during amino acid starvation in yeast. The mechanism and regulation by which tRNAs return to the nucleus are unclear. Here, we show accumulation of cytoplasmic tRNA in the nucleus also occurs during glucose starvation. Nuclear accumulation of tRNA in response to acute glucose or amino acid starvation is rapid, reversible, requires no new transcription, and is independent of the aminoacylation status of tRNA. Gradual depletion of nutrients also results in the accrual of tRNA in the nucleus. Distinct signal transduction pathways seem to be involved in the accumulation of cytoplasmic tRNA in the nucleus in response to amino acid versus glucose starvation. These findings suggest tRNA nucleocytoplasmic distribution may play a role in gene expression in response to nutritional stress.
doi:10.1091/mbc.E07-01-0006
PMCID: PMC1924813  PMID: 17475781
25.  Urokinase Receptor Cleavage: A Crucial Step in Fibroblast-to-Myofibroblast Differentiation 
Molecular Biology of the Cell  2007;18(7):2716-2727.
Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation.
doi:10.1091/mbc.E06-10-0912
PMCID: PMC1924808  PMID: 17507651

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