bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitogen-activated protein kinase kinase activity is required for the G
2
/M transition of the cell cycle in mammalian fibroblasts
Authors: Wright Jocelyn H. , Munar Erlynda , Jameson Damon R. , Andreassen Paul R. , Margolis Robert L. , Seger Rony , Krebs Edwin G.
Published: July 2002

The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G
0
, the level of MAPKK activity was observed to affect the kinetics of progression through both the G
1
and G
2
phases of the cell cycle in NIH 3T3 cells. Ectopic expression of dominant-negative forms of MAPKK1, which was previously shown to inhibit G
0
/G
1
progression, was found to also delay progression of cells through G
2
. In addition, treatment of cells with the specific MAPKK inhibitor PD 98059 during a synchronous S phase arrested the cells in the following G
2
phase. These data demonstrate a novel role for the MAPK cascade in progression from G
2
into mitosis in NIH 3T3 cells.
RNase E is required for the maturation of
ssrA
RNA and normal
ssrA
RNA peptide-tagging activity
Authors: Lin-Chao Sue , Wei Chia-Li , Lin Yi-Tzu
Published: July 2002

During recent studies of ribonucleolytic “degradosome” complexes of
Escherichia coli
, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is
ssrA
(for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt
ssrA
RNA is generated by RNase E cleavage at the CCA-3′ terminus of a 457-nt
ssrA
RNA precursor and that interference with this cleavage
in vivo
leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature
ssrA
RNA and for normal
ssrA
RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in
E. coli
, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3′ end of
ssrA
RNA.
Hypoglossal canal size and hominid speech
Authors: DeGusta David , Gilbert W. Henry , Turner Scott P.
Published: July 2002

The mammalian hypoglossal canal transmits the nerve that supplies
the motor innervation to the tongue. Hypoglossal canal size has
previously been used to date the origin of human-like speech
capabilities to at least 400,000 years ago and to assign modern human
vocal abilities to Neandertals. These conclusions are based on the
hypothesis that the size of the hypoglossal canal is indicative of
speech capabilities. This hypothesis is falsified here by the finding
of numerous nonhuman primate taxa that have hypoglossal canals in the
modern human size range, both absolutely and relative to oral cavity
volume. Specimens of
Australopithecus afarensis, Australopithecus
africanus,
and
Australopithecus boisei
also have
hypoglossal canals that, both absolutely and relative to oral cavity
volume, are equal in size to those of modern humans. The basis for the
hypothesis that hypoglossal canal size is indicative of speech was the
assumption that hypoglossal canal size is correlated with hypoglossal
nerve size, which in turn is related to tongue function. This
assumption is probably incorrect, as we found no apparent correlation
between the size of the hypoglossal nerve, or the number of axons it
contains, and the size of the hypoglossal canal in a sample of
cadavers. Our data demonstrate that the size of the hypoglossal canal
does not reflect vocal capabilities or language usage. Thus the date of
origin for human language and the speech capabilities of Neandertals
remain open questions.
Selenium redox biochemistry of zinc–sulfur coordination sites in proteins and enzymes
Authors: Jacob Claus , Maret Wolfgang , Vallee Bert L.
Published: July 2002

Selenium has been increasingly recognized as an essential element in biology and medicine. Its biochemistry resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidation states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc–sulfur centers as possible targets of selenium redox biochemistry. Selenium compounds release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Aromatic selenium compounds are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidation state +2), benzeneselenenyl chloride (oxidation state 0) and selenocystamine (oxidation state −1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, respectively. Selenium compounds also catalyze the release of zinc from MT in peroxidation and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of
t
-butyl hydroperoxide, suggesting that this type of redox chemistry may be employed in biology for the control of metal metabolism. Moreover, selenium compounds are likely targets for zinc/thiolate coordination centers
in vivo
, because the reactions are only partially suppressed by excess glutathione. This specificity and the potential to undergo catalytic reactions at low concentrations suggests that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compounds in antiinflammatory and anticarcinogenic agents.
The vacuolating toxin from
Helicobacter pylori
forms hexameric pores in lipid bilayers at low pH
Authors: Czajkowsky Daniel M. , Iwamoto Hideki , Cover Timothy L. , Shao Zhifeng
Published: July 2002

Pathogenic strains of
Helicobacter pylori
secrete a cytotoxin, VacA, that in the presence of weak bases, causes osmotic swelling of acidic intracellular compartments enriched in markers for late endosomes and lysosomes. The molecular mechanisms by which VacA causes this vacuolation remain largely unknown. At neutral pH, VacA is predominantly a water-soluble dodecamer formed by two apposing hexamers. In this report, we show by using atomic force microscopy that below pH ≈5, VacA associates with anionic lipid bilayers to form hexameric membrane-associated complexes. We propose that water-soluble dodecameric VacA proteins disassemble at low pH and reassemble into membrane-spanning hexamers. The surface contour of the membrane-bound hexamer is strikingly similar to the outer surface of the soluble dodecamer, suggesting that the VacA surface in contact with the membrane is buried within the dodecamer before protonation. In addition, electrophysiological measurements indicate that, under the conditions determined by atomic force microscopy for membrane association, VacA forms pores across planar lipid bilayers. This low pH-triggered pore formation is likely a critical step in VacA activity.
Position-dependent inhibition of class-switch recombination by PGK-neorcassettes inserted into the immunoglobulin heavy chain constant region locus
Authors: Seidl Katherine J. , Manis John P. , Bottaro Andrea , Zhang Jue , Davidson Laurie , Kisselgof Alison , Oettgen Hans , Alt Frederick W.
Published: July 2002

The Ig heavy chain (IgH) constant region (CH) genes are organized from 5′ to 3′ in the order Cμ, Cδ, Cγ3, Cγ1, Cγ2b, Cγ2a, Cɛ, and Cα. Expression of CHgenes downstream of Cδ involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding CHgene. Previously, we demonstrated that insertion of a PGK-neorcassette at two sites downstream of Cα inhibits, in cultured B cells,GT of and CSR to a subset of CHgenes (including Cγ3, Cγ2a, Cγ2b, and Cɛ) that lie as far as 120 kb upstream. Here we show that insertion of the PGK-neorcassette in place of sequences in the Iγ2b locus inhibits GT of and CSR to the upstream Cγ3 gene, but has no major effect on the downstream Cγ2a and Cɛ genes. Moreover, replacement of the Cɛ exons with a PGK-neorcassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream Cγ3, Cγ2b, and Cγ2a genes. As with the PGK-neorinsertions 3′ of Cα studied previously, the Cγ1 and Cα genes were less affected by these mutations both in culture and in mice, whereas the Cγ2b gene appeared less affectedin vivo. Our findings support the existence of a long-range 3′ IgH regulatory region required for GT of and CSR to multiple CHgenes and suggest that PGK-neorcassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent CHgenes upstream of the insertion.
Exploitation of host cells by enteropathogenic
Escherichia
coli
Authors: Vallance B. A. , Finlay B. B.
Published: July 2002

Microbial pathogens have evolved many ingenious ways to infect
their hosts and cause disease, including the subversion and
exploitation of target host cells. One such subversive microbe is
enteropathogenic
Escherichia coli
(EPEC). A major cause
of infantile diarrhea in developing countries, EPEC poses a significant
health threat to children worldwide. Central to EPEC-mediated disease
is its colonization of the intestinal epithelium. After initial
adherence, EPEC causes the localized effacement of microvilli and
intimately attaches to the host cell surface, forming characteristic
attaching and effacing (A/E) lesions. Considered the prototype for a
family of A/E lesion-causing bacteria, recent
in vitro
studies of EPEC have revolutionized our understanding of how these
pathogens infect their hosts and cause disease. Intimate attachment
requires the type III-mediated secretion of bacterial proteins, several
of which are translocated directly into the infected cell, including
the bacteria's own receptor (Tir). Binding to this membrane-bound,
pathogen-derived protein permits EPEC to intimately attach to mammalian
cells. The translocated EPEC proteins also activate signaling pathways
within the underlying cell, causing the reorganization of the host
actin cytoskeleton and the formation of pedestal-like structures
beneath the adherent bacteria. This review explores what is known about
EPEC's subversion of mammalian cell functions and how this knowledge
has provided novel insights into bacterial pathogenesis and
microbe-host interactions. Future studies of A/E pathogens in animal
models should provide further insights into how EPEC exploits not only
epithelial cells but other host cells, including those of the immune
system, to cause diarrheal disease.
Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia inTrichuris muris-infected mice
Authors: Richard Mélisande , Grencis Richard K. , Humphreys Neil E. , Renauld Jean-Christophe , Van Snick Jacques
Published: July 2002

Production of neutralizing anti-IL-9 antibodies was induced in mice by immunization with mouse IL-9 coupled to ovalbumin. In the six mouse strains tested, a strong and long-lasting anti-IL-9 response developed with seric inhibitory titers of 10−3to 10−5, as measured in anin vitroIL-9-dependent cell proliferation assay.In vivo, this immunization completely abrogated the increase in mast-cell protease-1 levels as well as the eosinophilia observed in mice after implantation of an IL-9-secreting tumor. We took advantage of this method to assess the role of IL-9 in infections with nematodeTrichuris muris, where IL-9 production correlates with the resistant phenotype. C57BL/6 mice, which normally expel the parasite, became susceptible after anti-IL-9 immunization, demonstrating that IL-9 plays a critical role in this model. In addition, neutralization of IL-9 also inhibited parasite-induced blood eosinophilia. Taken together, the present data demonstrate the potency of our strategy to antagonize IL-9in vivoand shows that this cytokine plays a major role in resistance againstT. murisinfection.
Nitric oxide negatively regulates c-Jun N-terminal kinase/stress-activated protein kinase by means ofS-nitrosylation
Authors: Park Hee-Sae , Huh Sung-Ho , Kim Mi-Sung , Lee Soo Hwan , Choi Eui-Ju
Published: July 2002

NO, produced froml-arginine in a reaction catalyzed by NO synthase, is an endogenous free radical with multiple functions in mammalian cells. Here, we demonstrate that endogenously produced NO can suppress c-Jun N-terminal kinase (JNK) activation in intact cells. Treatment of BV-2 murine microglial cells with IFN-γ induced endogenous NO production, concomitantly suppressing JNK1 activation. Similarly, IFN-γ induced suppression of JNK1 activation in RAW264.7 murine macrophage cells and rat alveolar macrophages. The IFN-γ-induced suppression of JNK1 activation in BV-2, RAW264.7, or rat alveolar macrophage cells was completely prevented byNG-nitro-l-arginine, a NO synthase inhibitor. Interestingly, the IFN-γ-induced suppression of JNK1 activation was not affected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase. 8-Bromo-cGMP, a membrane-permeant analogue of cGMP, did not change JNK1 activation in intact cells either. In contrast,S-nitro-N-acetyl-dl-penicillamine (SNAP), a NO donor, inhibited JNK1 activityin vitro. Furthermore, a thiol reducing agent, DTT, reversed not only thein vitroinhibition of JNK1 activity by SNAP but also thein vivosuppression of JNK1 activity by IFN-γ. Substitution of serine for cysteine-116 in JNK1 abolished the inhibitory effect of IFN-γ or SNAP on JNK1 activityin vivoorin vitro, respectively. Moreover, IFN-γ enhanced endogenousS-nitrosylation of JNK1 in RAW264.7 cells. Collectively, our data suggest that endogenous NO mediates the IFN-γ-induced suppression of JNK1 activation in macrophage cells by means of a thiol-redox mechanism.
A family of chromatin remodeling factors related to Williams syndrome transcription factor
Authors: Bochar Daniel A. , Savard Julie , Wang Weidong , Lafleur David W. , Moore Paul , Côté Jacques , Shiekhattar Ramin
Published: July 2002

Chromatin remodeling complexes have been implicated in the disruption or reformation of nucleosomal arrays resulting in modulation of transcription, DNA replication, and DNA repair. Here we report the isolation of WCRF, a new chromatin-remodeling complex from HeLa cells. WCRF is composed of two subunits, WCRF135, the human homolog of
Drosophila
ISWI, and WCRF180, a protein related to the Williams syndrome transcription factor. WCRF180 is a member of a family of proteins sharing a putative heterochromatin localization domain, a PHD finger, and a bromodomain, prevalent in factors involved in regulation of chromatin structure.
The Value of Cognitive Grammar in Typological Studies: the Case of
Norwegian and Russian Passive, Middle and Reflexive
Authors: Enger Hans-Olav , Nesset Tore
Published: July 2002

This paper shows that Cognitive Grammar is valuable for typological
studies. The Norwegian and Russian reflexive-middle-passive systems are
analysed comprehensively and compared. Whereas Cognitive Grammar is
compatible with the typological tradition (represented by Kemmer), it needs
amendment in certain important respects. The main theoretical contribution
of the paper is that the Cognitive Grammar notions of “instantiation”,
“prototype”, “entrenchment” and “extension” are useful; without these
notions, interesting generalizations about the Norwegian and Russian systems
are inexpressible. The main empirical contribution of the paper is that a
full-fledged analysis of the Russian system is presented. In addition,
Kemmer's analysis of Norwegian is improved upon, partly because findings
from the generative tradition are incorporated.
Age‐Related Patterns of Factors Associated with Completed Suicide in Men with Alcohol Dependence
Authors: Conner Kenneth R. , Duberstein  , Conwell Yeates
Published: July 2002

Seven factors associated with completed suicide among male alcoholics were investigated to determine if victims of differing ages would show different patterns of factors. Data on 56 male alcoholic suicide victims were collected post‐mortem. Subjects were divided into three age groups: 21–34, 35–49, and ≥ 50 years. Consistent with the hypotheses, unemployment and recent loss due to separation from a domestic partner were more likely with younger age, and serious medical problems and mood disorders were associated with increased age. There were no differences on other presumed risk factors among the age groups. Results suggest that factors associated with suicide may cluster in male alcoholics of differing ages, with implications for risk recognition and intervention.
Growth-inhibitory Activity and Downregulation of the Class II Tumor-suppressor Gene H-rev107 in Tumor Cell Lines and Experimental Tumors
Authors: Sers Christine , Emmenegger Urban , Husmann Knut , Bucher Katharina , Andres Ann-Catherine , Schäfer Reinhold
Published: July 2002

The H-rev107 gene is a new class II tumor suppressor, as defined by its reversible downregulation and growth-inhibiting capacity in HRAS transformed cell lines. Overexpression of the H-rev107 cDNA in HRAS-transformed ANR4 hepatoma cells or in FE-8 fibroblasts resulted in 75% reduction of colony formation. Cell populations of H-rev107 transfectants showed an attenuated tumor formation in nude mice. Cells explanted from tumors or maintained in cell culture for an extended period of time no longer exhibited detectable levels of the H-rev107 protein, suggesting strong selection against H-rev107 expression in vitro and in vivo. Expression of the truncated form of H-rev107 lacking the COOH-terminal membrane associated domain of 25 amino acids, had a weaker inhibitory effect on proliferation in vitro and was unable to attenuate tumor growth in nude mice. The H-rev107 mRNA is expressed in most adult rat tissues, and immunohistochemical analysis showed expression of the protein in differentiated epithelial cells of stomach, of colon and small intestine, in kidney, bladder, esophagus, and in tracheal and bronchial epithelium. H-rev107 gene transcription is downregulated in rat cell lines derived from liver, kidney, and pancreatic tumors and also in experimental mammary tumors expressing a RAS transgene. In colon carcinoma cell lines only minute amounts of protein were detectable. Thus, downregulation of H-rev107 expression may occur at the level of mRNA or protein.
Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells
Authors: Dharmawardhane Suranganie , Sanders Luraynne C. , Martin Stuart S. , Daniels R. Hugh , Bokoch Gary M.
Published: July 2002

The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics.

We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src–transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.
Pex17p of Saccharomyces cerevisiae Is a Novel Peroxin and Component of the Peroxisomal Protein Translocation Machinery
Authors: Huhse Bettina , Rehling Peter , Albertini Markus , Blank Lars , Meller Karl , Kunau Wolf-H.
Published: July 2002

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83–92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants (“ghosts”). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.
Transient Mitochondrial Depolarizations Reflect Focal Sarcoplasmic Reticular Calcium Release in Single Rat Cardiomyocytes
Authors: Duchen Michael R. , Leyssens Anne , Crompton Martin
Published: July 2002

Digital imaging of mitochondrial potential in single rat cardiomyocytes revealed transient depolarizations of mitochondria discretely localized within the cell, a phenomenon that we shall call “flicker.” These events were usually highly localized and could be restricted to single mitochondria, but they could also be more widely distributed within the cell. Contractile waves, either spontaneous or in response to depolarization with 50 mM K+, were associated with propagating waves of mitochondrial depolarization, suggesting that propagating calcium waves are associated with mitochondrial calcium uptake and consequent depolarization. Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria. Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 μM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter. These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.
Release of cAMP Gating by the α6β4 Integrin Stimulates Lamellae Formation and the Chemotactic Migration of Invasive Carcinoma Cells
Authors: O'Connor Kathleen L. , Shaw Leslie M. , Mercurio Arthur M.
Published: July 2002

The α6β4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949–960). We demonstrate here using MDA-MB-435 breast carcinoma cells that α6β4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by α6β4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon α6β4 expression. Both lamellae formation and chemotactic migration are inhibited or “gated” by cAMP and our results reveal that a critical function of α6β4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that α6β4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.
Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK
Authors: Yamakita Yoshihiko , Totsukawa Go , Yamashiro Shigeko , Fry David , Zhang Xiaoe , Hanks Steven K. , Matsumura Fumio
Published: July 2002

At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130CAS (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading.

We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.
Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts
Authors: Angers-Loustau Alexandre , Côté Jean-François , Charest Alain , Dowbenko Donald , Spencer Susan , Lasky Laurence A. , Tremblay Michel L.
Published: July 2002

In this article, we show that, in transfected COS-1 cells, protein tyrosine phosphatase (PTP)-PEST translocates to the membrane periphery following stimulation by the extracellular matrix protein fibronectin. When plated on fibronectin, PTP-PEST (−/−) fibroblasts display a strong defect in motility. 3 h after plating on fibronectin, the number and size of vinculin containing focal adhesions were greatly increased in the homozygous PTP-PEST mutant cells as compared with heterozygous cells. This phenomenon appears to be due in part to a constitutive increase in tyrosine phosphorylation of p130CAS, a known PTP-PEST substrate, paxillin, which associates with PTP-PEST in vitro, and focal adhesion kinase (FAK). Another effect of this constitutive hyperphosphorylation, consistent with the focal adhesion regulation defect, is that (−/−) cells spread faster than the control cell line when plated on fibronectin. In the PTP-PEST (−/−) cells, an increase in affinity for the SH2 domains of Src and Crk towards p130CAS was also observed. In (−/−) cells, we found a significant increase in the level of tyrosine phosphorylation of PSTPIP, a cleavage furrow–associated protein that interacts physically with all PEST family members. An effect of PSTPIP hyperphosphorylation appears to be that some cells remain attached at the site of the cleavage furrow for an extended period of time. In conclusion, our data suggest PTP-PEST plays a dual role in cell cytoskeleton organization, by promoting the turnover of focal adhesions required for cell migration, and by directly or indirectly regulating the proline, serine, threonine phosphatase interacting protein (PSTPIP) tyrosine phosphorylation level which may be involved in regulating cleavage furrow formation or disassembly during normal cell division.
Specific Myosin Heavy Chain Mutations Suppress Troponin I Defects in Drosophila Muscles
Authors: Kronert William A. , Acebes Angel , Ferrús Alberto , Bernstein Sanford I.
Published: July 2002

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.
Vamp-7 Mediates Vesicular Transport from Endosomes to Lysosomes
Authors: Advani Raj J. , Yang Bin , Prekeris Rytis , Lee Kelly C. , Klumperman Judith , Scheller Richard H.
Published: July 2002

A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O–permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome.
Domains in the 1α Dynein Heavy Chain Required for Inner Arm Assembly and Flagellar Motility in Chlamydomonas
Authors: Myster Steven H. , Knott Julie A. , Wysocki Katrina M. , O'Toole Eileen , Porter Mary E.
Published: July 2002

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1α Dhc of the I1 inner dynein arm. The 1α Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH2- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1α Dhcs were recovered. The truncated 1α Dhc fragments lacked the dynein motor domain, but still assembled with the 1β Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1α motor domain in the I1 structure and further revealed the role of the 1α Dhc in flagellar motility and phototactic behavior.
A Role for Centrin 3 in Centrosome Reproduction
Authors: Middendorp Sandrine , Küntziger Thomas , Abraham Yann , Holmes Simon , Bordes Nicole , Paintrand Michel , Paoletti Anne , Bornens Michel
Published: July 2002

Centrosome reproduction by duplication is essential for the bipolarity of cell division, but the molecular basis of this process is still unknown. Mutations in Saccharomyces cerevisiae CDC31 gene prevent the duplication of the spindle pole body (SPB). The product of this gene belongs to the calmodulin super-family and is concentrated at the half bridge of the SPB. We present a functional analysis of HsCEN3, a human centrin gene closely related to the CDC31 gene. Tran- sient overexpression of wild-type or mutant forms of HsCen3p in human cells demonstrates that centriole localization depends on a functional fourth EF-hand, but does not produce mitotic phenotype. However, injection of recombinant HsCen3p or of RNA encoding HsCen3p in one blastomere of two-cell stage Xenopus laevis embryos resulted in undercleavage and inhibition of centrosome duplication. Furthermore, HsCEN3 does not complement mutations or deletion of CDC31 in S. cerevisiae, but specifically blocks SPB duplication, indicating that the human protein acts as a dominant negative mutant of CDC31. Several lines of evidence indicate that HsCen3p acts by titrating Cdc31p-binding protein(s).

Our results demonstrate that, in spite of the large differences in centrosome structure among widely divergent species, the centrosome pathway of reproduction is conserved.
Autophagic Tubes
Authors: Müller Oliver , Sattler Tanja , Flötenmeyer Matthias , Schwarz Heinz , Plattner Helmut , Mayer Andreas
Published: July 2002

Many intracellular compartments of eukaryotic cells do not adopt a spherical shape, which would be expected in the absence of mechanisms organizing their structure. However, little is known about the principles determining the shape of organelles. We have observed very defined structural changes of vacuoles, the lysosome equivalents of yeast. The vacuolar membrane can form a large tubular invagination from which vesicles bud off into the lumen of the organelle. Formation of the tube is regulated via the Apg/Aut pathway. Its lumen is continuous with the cytosol, making this inverse budding reaction equivalent to microautophagocytosis. The tube is highly dynamic, often branched, and defined by a sharp kink of the vacuolar membrane at the site of invagination. The tube is formed by vacuoles in an autonomous fashion. It persists after vacuole isolation and, therefore, is independent of surrounding cytoskeleton. There is a striking lateral heterogeneity along the tube, with a high density of transmembrane particles at the base and a smooth zone devoid of transmembrane particles at the tip where budding occurs. We postulate a lateral sorting mechanism along the tube that mediates a depletion of large transmembrane proteins at the tip and results in the inverse budding of lipid-rich vesicles into the lumen of the organelle.
Identification of an Outer Segment Targeting Signal in the Cooh Terminus of Rhodopsin Using Transgenic Xenopus laevis
Authors: Tam Beatrice M. , Moritz Orson L. , Hurd Lawrence B. , Papermaster David S.
Published: July 2002

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and α adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.
Stromelysin-1 Regulates Adipogenesis during Mammary Gland Involution
Authors: Alexander Caroline M. , Selvarajan Sushma , Mudgett John , Werb Zena
Published: July 2002

The matrix metalloproteinase MMP-3/stromelysin-1 (Str1) is highly expressed during mammary gland involution induced by weaning. During involution, programmed cell death of the secretory epithelium takes place concomitant with the repopulation of the mammary fat pad with adipocytes. In this study, we have used a genetic approach to determine the role of Str1 during mammary involution. Although Str1 has been shown to induce unscheduled apoptosis when expressed ectopically during late pregnancy (Alexander, C.M., E.W. Howard, M.J. Bissell, and Z. Werb. 1996. J. Cell Biol. 135:1669–1677), we found that during post-lactational involution, mammary glands from transgenic mice that overexpress the tissue inhibitor of metalloproteinases, TIMP-1 (TO), or mice carrying a targeted mutation in Str1 showed accelerated differentiation and hypertrophy of adipocytes, while epithelial apoptosis was unaffected. These data suggest that matrix metalloproteinases (MMPs) do not induce unscheduled epithelial cell death after weaning, but instead alter the stromal microenvironment. We used adipogenic 3T3-L1 cells as a cell culture model to test the function of MMPs during adipocyte differentiation. Fibroblastic 3T3-L1 progenitor cells expressed very low levels of MMPs or TIMPs. The transcription of a number of MMP and TIMP mRNAs [Str1, MT1-MMP, (MMP-14) collagenase-3 (MMP-13), gelatinase A (MMP-2), and TIMP-1, -2 and -3] was induced in committed preadipocytes, but only differentiated adipocytes expressed an activated MMP, gelatinase A. The addition of MMP inhibitors (GM 6001 and TIMP-1) dramatically accelerated the accumulation of lipid during differentiation. We conclude that MMPs, especially Str1, determine the rate of adipocyte differentiation during involutive mammary gland remodeling.
Regulation of Op18 during Spindle Assembly in Xenopus Egg Extracts
Authors: Budde Priya Prakash , Kumagai Akiko , Dunphy William G. , Heald Rebecca
Published: July 2002

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.
Cytoplasmic Dynein Regulation by Subunit Heterogeneity and Its Role in Apical Transport
Authors: Tai Andrew W. , Chuang Jen-Zen , Sung Ching-Hwa
Published: July 2002

Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia.
Human Vam6p promotes lysosome clustering and fusion in vivo
Authors: Caplan Steve , Hartnell Lisa M. , Aguilar Rubén C. , Naslavsky Naava , Bonifacino Juan S.
Published: July 2002

Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH2 terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2–3 μm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.
Fyn tyrosine kinase is a downstream mediator of Rho/PRK2 function in keratinocyte cell–cell adhesion
Authors: Calautti Enzo , Grossi Maddalena , Mammucari Cristina , Aoyama Yumi , Pirro Maria , Ono Yoshitaka , Li Jie , Dotto G. Paolo
Published: July 2002

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell–cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of β- and γ-catenin, which is associated with the establishment of keratinocyte cell–cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell–cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of β- and γ-catenin and p120ctn. The positive effects of activated Rho on cell–cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell–cell adhesion and induces tyrosine phosphorylation of β- and γ-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell–cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.
Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells
Authors: Barbero Pierre , Bittova Lenka , Pfeffer Suzanne R.
Published: July 2002

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.