bluuebunny/crossref_metadata_2025 huggingface dataset:
The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes.
Authors: Spinardi L , Ren Y L , Sanders R , Giancotti F G
Published: August 2013

The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.
Suppression of a sec63 mutation identifies a novel component of the yeast endoplasmic reticulum translocation apparatus.
Authors: Kurihara T , Silver P
Published: August 2013

Mutations in the SEC63 gene are associated with defects in protein translocation into the endoplasmic reticulum (ER) as well as in nuclear protein localization in Saccharomyces cerevisiae. To identify proteins that might interact and/or function with SEC63p, we cloned a high copy suppressor (HSS1) of the temperature-sensitive lethal phenotype of the sec63-101 mutant. HSS1 is an allele-specific sec63 suppressor that encodes an integral ER membrane glycoprotein of 206 amino acids with the N-terminus in the ER lumen and C-terminal region in the cytoplasm. Haploid strains disrupted for HSS1 are temperature-sensitive for growth and accumulate precursor forms of Kar2p and invertase. The HSS1 null allele is synthetically lethal in combination with mutations affecting ER translocation. We propose that HSS1p is important for ER translocation and interacts with previously identified components of the yeast translocation apparatus. HSS1 is identical to SEC66, which encodes a glycoprotein complexed with SEC62p and SEC63p.
Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins.
Authors: Gibbons B H , Asai D J , Tang W J , Hays T S , Gibbons I R
Published: August 2013

Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of cytoplasmic dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the dynein heavy chain genes identified in Drosophila, indicating that the radiation of the dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in cytoplasmic dynein have been more constrained than those in the axonemal dyneins.
The essential yeast Tcp1 protein affects actin and microtubules.
Authors: Ursic D , Sedbrook J C , Himmel K L , Culbertson M R
Published: August 2013

Previously, we showed that the yeast Saccharomyces cerevisiae cold-sensitive mutation tcp1-1 confers growth arrest concomitant with cytoskeletal disorganization and disruption of microtubule-mediated processes. We have identified two new recessive mutations, tcp1-2 and tcp1-3, that confer heat- and cold-sensitive growth. Cells carrying tcp1 alleles were analyzed after exposure to the appropriate restrictive temperatures by cell viability tests, differential contrast microscopy, fluorescent, and immunofluorescent microscopy of DNA, tubulin, and actin and by determining the DNA content per cell. All three mutations conferred unique phenotypes indicative of cytoskeletal dysfunction. A causal relationship between loss of Tcp1p function and the development of cytoskeletal abnormalities was established by double mutant analyses. Novel phenotypes indicative of allele-specific genetic interactions were observed when tcp1-1 was combined in the same strain with tub1-1, tub2-402, act1-1, and act1-4, but not with other tubulin or actin mutations or with mutations in other genes affecting the cytoskeleton. Also, overproduction of wild-type Tcp1p partially suppressed growth defects conferred by act1-1 and act1-4. Furthermore, Tcp1p was localized to the cytoplasm and the cell cortex. Based on our results, we propose that Tcp1p is required for normal development and function of actin and microtubules either through direct or indirect interaction with the major cytoskeletal components.
Molecular characterization of Xenopus laevis DP proteins.
Authors: Girling R , Bandara L R , Ormondroyd E , Lam E W , Kotecha S , Mohun T , La Thangue N B
Published: August 2013

It is widely believed that in mammalian cells the cellular transcription factor (DRTF1/E2F integrates cell-cycle events with the transcription apparatus by interacting with important regulators of the cell cycle, such as the retinoblastoma gene product (pRb) and related proteins, cyclins, and cyclin-dependent kinases. Here, we have defined DRTF1/E2F in Xenopus laevis that, like its mammalian counterpart, specifically binds to the E2F site, is regulated during development, and interacts with pRb and related proteins. We have isolated cDNAs that encode the functional homologue of mammalian DP-1, X1 DP-1, together with a close relative, X1 DP-2. X1 DP-1, which is highly conserved with murine DP-1, is a major DNA binding component of X1 DRTF1/E2F. Both DP-1 and DP-2 synergistically interact with members of the E2F family of proteins, E2F-1, E2F-2, and E2F-3, to generate DNA binding complexes that specifically recognize the E2F site and functionally interact with E2F-1 in E2F site-dependent transcriptional activation of cellular genes. DP-1 and DP-2 encode maternally stored transcripts that are expressed during early development. In the adult however, the expression of DP-1 and DP-2 is tissue restricted. This study therefore defines a new family of transcription factors, the DP proteins, members of which can interact combinatorially with E2F proteins to generate an array of DNA binding complexes that integrate cell-cycle progression with the transcription apparatus through the E2F binding site. The tissue-specific expression of DP family members suggests that the combination of DP/E2F heterodimers that constitute DRTF1/E2F is influenced by the phenotype of the cell.
Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle.
Authors: Flucher B E , Andrews S B , Daniels M P
Published: August 2013

The relationship between the molecular composition and organization of the triad junction and the development of excitation-contraction (E-C) coupling was investigated in cultured skeletal muscle. Action potential-induced calcium transients develop concomitantly with the first expression of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR), which are colocalized in clusters from the time of their earliest appearance. These DHPR/RyR clusters correspond to junctional domains of the transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), respectively. Thus, at first contact T-tubules and SR form molecularly and structurally specialized membrane domains that support E-C coupling. The earliest T-tubule/SR junctions show structural characteristics of mature triads but are diverse in conformation and typically are formed before the extensive development of myofibrils. Whereas the initial formation of T-tubule/SR junctions is independent of association with myofibrils, the reorganization into proper triads occurs as junctions become associated with the border between the A band and the I band of the sarcomere. This final step in triad formation manifests itself in an increased density and uniformity of junctions in the cytoplasm, which in turn results in increased calcium release and reuptake rates.
Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae.
Authors: Berkower C , Loayza D , Michaelis S
Published: August 2013

STE6, a member of the ATP binding cassette (ABC) transporter superfamily, is a membrane protein required for the export of the a-factor mating pheromone in Saccharomyces cerevisiae. To initiate a study of the intracellular trafficking of STE6, we have examined its half-life and localization. We report here that STE6 is metabolically unstable in a wild-type strain, and that this instability is blocked in a pep4 mutant, suggesting that degradation of STE6 occurs in the vacuole and is dependent upon vacuolar proteases. In agreement with a model whereby STE6 is routed to the vacuole via endocytosis from the plasma membrane, we show that degradation of STE6 is substantially reduced at nonpermissive temperature in mutants defective in delivery of proteins to the plasma membrane (sec6) or in endocytosis (end3 and end4). Whereas STE6 appears to undergo constitutive internalization from the plasma membrane, as do the pheromone receptors STE2 and STE3, we show that two other proteins, the plasma membrane ATPase (PMA1) and the general amino acid permease (GAP1), are significantly more stable than STE6, indicating that rapid turnover in the vacuole is not a fate common to all plasma membrane proteins in yeast. Investigation of STE6 partial molecules (half- and quarter-molecules) indicates that both halves of STE6 contain sufficient information to mediate internalization. Examination of STE6 localization by indirect immunofluorescence indicates that STE6 is found in a punctate, possibly vesicular, intracellular pattern, distinct from the rim-staining pattern characteristic of PMA1. The punctate pattern is consistent with the view that most of the STE6 molecules present in a cell at any given moment could be en route either to or from the plasma membrane. In a pep4 mutant, STE6 is concentrated in the vacuole, providing further evidence that the vacuole is the site of STE6 degradation, while in an end4 mutant STE6 exhibits rim-staining, indicating that it can accumulate in the plasma membrane when internalization is blocked. Taken together, the results presented here suggest that STE6 first travels to the plasma membrane and subsequently undergoes endocytosis and degradation in the vacuole, with perhaps only a transient residence at the plasma membrane; an alternative model, in which STE6 circumvents the plasma membrane, is also discussed.
Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.
Authors: Li G , Rungger-Brändle E , Just I , Jonas J C , Aktories K , Wollheim C B
Published: August 2013

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane.
The MPM-2 antibody inhibits mitogen-activated protein kinase activity by binding to an epitope containing phosphothreonine-183.
Authors: Taagepera S , Dent P , Her J H , Sturgill T W , Gorbsky G J
Published: August 2013

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition.
Beta-centractin: characterization and distribution of a new member of the centractin family of actin-related proteins.
Authors: Clark S W , Staub O , Clark I B , Holzbaur E L , Paschal B M , Vallee R B , Meyer D I
Published: August 2013

An examination of human-expressed sequence tags indicated the existence of an isoform of centractin, an actin-related protein localized to microtubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. The protein encoded represents the first example of multiple isoforms of an actin-related protein in a single organism. Northern analysis using centractin-specific probes revealed three species of mRNA in HeLa cells that could encode centractin isoforms. One mRNA encodes the previously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag encodes a new member of the centractin family, beta-centractin. A probe specific for alpha-centractin hybridized to the third species of mRNA observed (referred to as gamma-centractin). Comparisons of Northern blots of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, whereas the expression of gamma-centractin appears to be tissue specific. The amino acid sequence of beta-centractin, deduced from the cDNA, indicates a 91% identity with alpha-centractin, increasing to 96% similarity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poorly with beta-centractin, new antibodies were produced and combined with two-dimensional gel electrophoresis to discriminate the two isoforms. Using this system, the subcellular distribution of the alpha- and beta-isoforms were determined. Both isoforms were found predominantly in the cytosolic fraction as a part of a previously identified 20S complex (referred to as the dynactin complex) with no evidence for a free pool of either isoform. The isoforms were found in a constant ratio of approximately 15:1 (alpha:beta) in the dynactin complex.
Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast.
Authors: al-Khodairy F , Fotou E , Sheldrick K S , Griffiths D J , Lehmann A R , Carr A M
Published: August 2013

To investigate the mechanisms that ensure the dependency relationships between cell cycle events and to investigate the checkpoints that prevent progression through the cell cycle after DNA damage, we have isolated mutants defective in the checkpoint and feedback control pathways. We report the isolation and characterization of 11 new loci that define distinct classes of mutants defective in one or more of the checkpoint and feedback control pathways. Two mutants, rad26.T12 and rad27.T15, were selected for molecular analysis. The null allele of the rad26 gene (rad26.d) shares the phenotype reported for the "checkpoint rad" mutants rad1, rad3, rad9, rad17, and hus1, which are defective in the radiation checkpoint and in the feedback controls that ensure the order of cell cycle events. The null allele of the rad27 gene (rad27.d) defines a new class of Schizosaccharomyces pombe mutant. The rad27 complementing gene codes for a putative protein kinase that is required for cell cycle arrest after DNA damage but not for the feedback control that links mitosis to the completion of prior DNA synthesis (the same gene has recently been described by Walworth et al. (1993) as chk1). These properties are similar to those of the rad9 gene of Saccharomyces cerevisiae. A comparative analysis of the radiation responses in rad26.d, rad26.T12, and rad27.d cells has revealed the existence of two separable responses to DNA damage controlled by the "checkpoint rad" genes. The first, G2 arrest, is defective in rad27.d and rad26.d but is unaffected in rad26.T12 cells. The second response is not associated with G2 arrest after DNA damage and is defective in rad26.d and rad26.T12 but not rad27.d cells. A study of the radiation sensitivity of these mutants through the cell cycle suggests that this second response is associated with S phase and that the checkpoint rad mutants, in addition to an inability to arrest mitosis after radiation, are defective in an S phase radiation checkpoint.
Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase.
Authors: Hisanaga S , Matsuoka Y , Nishizawa K , Saito T , Inagaki M , Hirokawa N
Published: August 2013

Phosphorylation of neurofilament-L protein (NF-L) by the catalytic subunit of cAMP-dependent protein kinase (A-kinase) inhibits the reassembly of NF-L and disassembles filamentous NF-L. The effects of phosphorylation by A-kinase on native neurofilaments (NF) composed of three distinct subunits: NF-L, NF-M, and NF-H, however, have not yet been described. In this paper, we examined the effects of phosphorylation of NF proteins by A-kinase on both native and reassembled filaments containing all three NF subunits. In the native NF, A-kinase phosphorylated each NF subunit with stoichiometries of 4 mol/mol for NF-L, 6 mol/mol for NF-M, and 4 mol/mol for NF-H. The extent of NF-L phosphorylation in the native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.
Distinct and overlapping ligand specificities of the alpha 3A beta 1 and alpha 6A beta 1 integrins: recognition of laminin isoforms.
Authors: Delwel G O , de Melker A A , Hogervorst F , Jaspars L H , Fles D L , Kuikman I , Lindblom A , Paulsson M , Timpl R , Sonnenberg A
Published: August 2013

The ligand specificity of the alpha 3A beta 1 integrin was analyzed using K562 cells transfected with full-length alpha 3A cDNA and was compared with that of alpha 6A beta 1 in similarly transfected K562 cells. Clones were obtained that showed comparable surface expression of either alpha 3A beta 1 or alpha 6A beta 1 integrins. Those expressing alpha 3A beta 1 attached to and spread on immunopurified human kalinin and cellular matrices containing human kalinin, which is a particular isoform of laminin. In addition, alpha 3A transfectants adhered to bovine kidney laminins possessing a novel A chain variant. Binding to kalinin was blocked by a monoclonal antibody against the A chain constituent of kalinin and adhesion to both kalinin and kidney laminins by anti-alpha 3 and beta 1 monoclonal antibodies. The alpha 3A transfected cells bound more strongly to kalinin and bovine kidney laminins after treatment with the beta 1 stimulatory antibody TS2/16. A distinctly weaker and activation-dependent adhesion of alpha 3A transfectants was observed on human placental laminins possessing the Am chain variant (merosin), and no adhesion occurred on bovine heart laminins and murine EHS tumor laminin. Further inactive substrates were fibronectin, nidogen, and collagen types IV and VI, indicating that the alpha 3A beta 1 integrin is a much less promiscuous receptor than thought before. By contrast, alpha 6A transfected cells adhered to all laminin isoforms when stimulated with TS2/16. Adhesion also occurred only on bovine kidney laminins in the absence of TS2/16. These results demonstrate that both alpha 3A beta 1 and alpha 6A beta 1 integrins are typical laminin receptors but that their affinity and activation dependence for binding to various laminin isoforms differ considerably.
A novel cycle involving fatty acyl-coenzyme A regulates asialoglycoprotein receptor activity in permeable hepatocytes.
Authors: Weigel P H , Medh J D , Oka J A
Published: August 2013

Asialoglycoprotein receptors (ASGP-Rs) in permeable rat hepatocytes can be inactivated in the absence of ligand. This cytosol-independent effect is relatively slow (t1/2 approximately 12 min) and is temperature and ATP dependent. Here we show that in the absence of cytosol, the addition of palmitoyl-CoA (Pal-CoA) rapidly (t1/2 < 0.4 min) and quantitatively reactivates the inactivated receptors. Receptor reactivation was half-maximal at approximately 10-12 microM free Pal-CoA at 37 degrees C. Although substantially higher total concentrations were used, much of the added Pal-CoA was cell associated and not free. The effects of Pal-CoA were eliminated by bovine serum albumin at concentrations sufficient to bind all free monomeric fatty acyl-CoA, suggesting that micellar effects are not responsible for the ability to reactivate ASGP-Rs. Also, palmitoyl-carnitine did not substitute for Pal-CoA. The initial ASGP-R inactivation is not affected by treating cells with N-ethylmaleimide or by a KCl wash but is inhibited by sodium orthovanadate or high Ca2+ levels. Myristoyl-CoA (C14) was also able to reactivate inactive ASGP-Rs about as well as Pal-CoA. Fatty acyl-CoAs with chain lengths of C12 (lauroyl) or C18 (steroyl) were < 50% as active. The ligand binding activity of these receptors can subsequently be modulated within minutes by the further addition of ATP or Pal-CoA to achieve additional rounds of ASGP-R inactivation or reactivation, respectively. These in vitro data demonstrate the occurrence of a novel asialoglycoprotein receptor inactivation-reactivation cycle that could regulate receptor activity during endocytosis and receptor recycling.
A role for autophosphorylation revealed by activated alleles of FUS3, the yeast MAP kinase homolog.
Authors: Brill J A , Elion E A , Fink G R
Published: August 2013

We have isolated dominant gain-of-function (gf) mutations in FUS3, a Saccharomyces cerevisiae mitogen-activated protein (MAP) kinase homolog, that constitutively activate the yeast mating signal transduction pathway and confer hypersensitivity to mating pheromone. Surprisingly, the phenotypes of dominant FUS3gf mutations require the two protein kinases, STE7 and STE11. FUS3gf kinases are hyperphosphorylated in yeast independently of STE7. Consistent with this, FUS3gf kinases expressed in Escherichia coli exhibit an increased ability to autophosphorylate on tyrosine in vivo. FUS3gf mutations suppress the signal transduction defect of a severely catalytically impaired allele of STE7. This finding suggests that the tyrosine-phosphorylated form of FUS3 is a better substrate for activation by STE7. Furthermore, these results imply that the degree of autophosphorylation of a MAP kinase determines its threshold of sensitivity to upstream signals.
Integrating with integrins.
Authors: Schwartz M A , Ingber D E
Published: August 2013

Our central claim is that signaling by integrins provides a mechanism by which signals generated in response to adhesion, soluble hormones, and mechanical forces can interact. Such interactions permit cells to integrate these different classes of external stimuli and hence to orchestrate an efficient response. This integrating function of integrins is likely to be essential for much of development and physiology, as well as complex pathologies such as cancer. Understanding in detail how these signals are transduced and processed is likely to be an important area of research in the near future.
Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.
Authors: Xing Z , Chen H C , Nowlen J K , Taylor S J , Shalloway D , Guan J L
Published: August 2013

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.
Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.
Authors: Wachter C , Schatz G , Glick B S
Published: August 2013

ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix is always needed for complete import of precursor proteins into this compartment, even when the precursors are presented to mitochondria in an unfolded conformation. In contrast, the requirement for external ATP is precursor-specific; depletion of external ATP strongly inhibits import of some precursors but has little or no effect with other precursors. A requirement for external ATP can often be overcome by denaturing the precursor with urea. We suggest that external ATP promotes the release of precursors from cytosolic chaperones, whereas matrix ATP drives protein translocation across the inner membrane.
Terminal differentiation and senescence in the human melanocyte: repression of tyrosine-phosphorylation of the extracellular signal-regulated kinase 2 selectively defines the two phenotypes.
Authors: Medrano E E , Yang F , Boissy R , Farooqui J , Shah V , Matsumoto K , Nordlund J J , Park H Y
Published: August 2013

Melanocytes are pigmented cells distributed in humans in several organs like the epidermis, the leptomeninges, the eye, and the inner ear. Epidermal melanocytes, whether derived from adult or neonatal skin, proliferate well in a medium supplemented with phorbol esters and other mitogens before they undergo senescence. Potent cAMP inducers like cholera toxin are also growth promoters for neonatal melanocytes but only transient growth stimulators for cells derived from adults. We used this cellular system to delineate biochemical pathways involved in proliferation and in terminal differentiation. Here we show that after a period of 4-8 wk of sustained proliferation in the presence of cholera toxin, the adult melanocytes became round, flat, and enlarged. These changes were associated with terminal growth and preceded by a five- to sixfold increase in cAMP levels and an 8- to 10-fold increase in melanin content. The simultaneous addition of phorbol esters and cholera toxin did not prevent cells from reaching terminal differentiation. Identified targets for phorbol esters are protein kinase C (PKC) and the mitogen-activated kinases (MAPKs), also called extracellular signal-regulated kinases (ERKs). PKC was found to be similarly regulated in proliferating and in terminally differentiated melanocytes. Proliferating melanocytes in early or late passage showed identical activation of the kinase ERK2. This kinase was rapidly phosphorylated upon phorbol 12-myristate 13-acetate (PMA) addition and specifically accumulated in the nucleus of the cells, whereas in unstimulated cells it had a perinuclear distribution. In contrast, senescent and terminally differentiated cells were unable to phosphorylate tyrosine residues of the ERK2 gene product in spite of presenting normal amounts of ERK2 protein. In addition, ERK2 did not show the nuclear accumulation observed in proliferating melanocytes after PMA activation and remained localized in the perinuclear area. These results demonstrate that senescent and terminally differentiated melanocytes share a common block in a critical pathway thought to integrate multiple intracellular signals transmitted by various second messengers and specifically prevent the continuation of the signal transduction cascade initiated by PMA activation of PKC.
Altered shape and cell cycle characteristics of fibroblasts expressing the E2F1 transcription factor.
Authors: Logan T J , Jordan K L , Hall D
Published: August 2013

To gain an understanding of the role the E2F1 transcription factor plays in cell physiology, the full length protein (E2F1) and an amino terminal deletion of 87 amino acids (E2F1d87) were constitutively expressed in NIH3T3 fibroblasts. Multiple cell lines were generated for each construct. These cells do not proliferate in media containing low serum and do not proliferate in soft agar, indicating that they are likely not transformed. However, both sets of cell lines show increased DNA synthesis and increased numbers of cells in S phase when cultured in media containing low serum, compared to the control cell lines. Cells expressing E2F1d87 (but not E2F1) have an extremely rounded morphology when cultured in 10% serum-containing media. These rounded cells lack detectable microfilaments, microtubules, and focal contacts. However, when these cells are cultured in low serum-containing media (0.5%), they attain the flattened morphology and cytoskeletal structure of normal NIH3T3 cells.
Multiple N-ethylmaleimide-sensitive components are required for endosomal vesicle fusion.
Authors: Rodriguez L , Stirling C J , Woodman P G
Published: August 2013

This report examines the inhibition of endosomal vesicle fusion by the alkylating agent N-ethylmaleimide (NEM). The concentration of NEM required to inhibit vesicle fusion depended upon whether membrane and cytosolic fractions were treated separately or together, enabling the resolution of at least two components to the inhibition. The first component is inactivated at low levels of NEM when cytosolic and membrane fractions are treated together. On the contrary, inhibition of the second component required higher levels of NEM but was achieved by treating cytosol and membranes separately. Reconstitution studies indicated that both components were cytosolic and that neither corresponded to the ubiquitous NEM-sensitive fusion protein (NSF). The role of NSF in this fusion reaction was further examined using salt-washed membranes depleted of NSF protein. Under these conditions the fusion reaction was fully dependent upon added NSF whose activity, in this context, was sensitive to NEM treatment. From these data we conclude that NSF activity during endosomal vesicle fusion can be dissected into several steps, only a subset of which (perhaps attachment of NSF to the membrane) are sensitive to NEM. Fusion between salt-washed endosomal membranes was also dependent on soluble NSF attachment proteins.
Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns.
Authors: Kim C W , Goldberger O A , Gallo R L , Bernfield M
Published: August 2013

The syndecans are a gene family of four transmembrane heparan sulfate proteoglycans that bind, via their HS chains, diverse components of the cellular microenvironment. To evaluate the expression of the individual syndecans, we prepared cDNA probes to compare mRNA levels in various adult mouse tissues and cultured mouse cells representing various epithelial, fibroblastic, endothelial, and neural cell types and B cells at various stages of differentiation. We also prepared antibody probes to assess whether the extracellular domains of the individual syndecans are shed into the conditioned media of cultured cells. Our results show that all cells and tissues studied, except B-stem cells, express at least one syndecan family member; most cells and tissues express multiple syndecans. However, each syndecan family member is expressed selectively in cell-, tissue-, and development-specific patterns. The extracellular domain of all syndecan family members is shed as an intact proteoglycan. Thus, most, if not all, cells acquire a distinctive repertoire of the four syndecan family members as they differentiate, resulting in selective patterns of expression that likely reflect distinct functions.
Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula polymorpha requires the cofactor flavin adenine dinucleotide.
Authors: Evers M E , Titorenko V I , van der Klei I J , Harder W , Veenhuis M
Published: August 2013

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.
Mitochondrial morphological and functional defects in yeast caused by yme1 are suppressed by mutation of a 26S protease subunit homologue.
Authors: Campbell C L , Tanaka N , White K H , Thorsness P E
Published: August 2013

The absence of functional Yme1p, a putative ATP and zinc-dependent protease localized to mitochondria of yeast, results in abnormal mitochondrial function and morphology. Yeast lacking Yme1p lose DNA from mitochondria at an accelerated rate, fail to grow on nonfermentable carbon sources at 37 degrees C, and have severely deficient growth if mitochondrial DNA suffers large deletions or is completely lost. In place of the normal reticulated mitochondrial network, strains lacking Yme1p have punctate mitochondria with some grossly swollen compartments. The growth phenotypes and morphological alterations evident in these mutant yeast can be compensated by a mutation in YNT1, an essential gene in yeast. The sequence of the YNT1 gene product indicates that it is one of a number of related regulatory subunits of the 26S protease. This proteolytic activity is necessary for progression through the cell cycle and has been implicated in the regulation of transcription. Ynt1p is more distantly related to Yme1p.
A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.
Authors: Gagnon C , White D , Huitorel P , Cosson J
Published: August 2013

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.
Expression of soybean nodulin 26 in transgenic tobacco. Targeting to the vacuolar membrane and effects on floral and seed development.
Authors: Zhang Y , Roberts D M
Published: August 2013

Nodulin 26 is an integral membrane protein of the symbiosome membrane of nitrogen-fixing soybean nodules. We expressed a nodulin 26 cDNA in transgenic tobacco (TN26 tobacco) under the control of the cauliflower mosaic virus 35S promoter to study subcellular targeting and the physiological effect(s) of its expression. Based on Northern and Western blots, the expression of nodulin 26 mRNA and protein in transgenic plants is high in apical shoot sections, flowers, and stems, low in mature leaves, and absent in roots. Western blot analysis revealed high levels of transgenic nodulin 26 protein in tonoplast membranes. In contrast, nodulin 26 protein was not found in isolated plasma membranes, the soluble fraction, nor in chloroplast and mitochondria-enriched membrane fractions. About 50-60% of the flowers and pods from TN26 tobacco plants abscised prematurely. Seed capsule size and seed fill per capsule from the remainder of surviving flowers were about 50% of that of control plants. Pollen viability was found to be normal, but flowers from TN26 tobacco plants showed shorter anther filaments compared with control plants. Normal seed production and capsule size was restored by manually crossing the stigmas from TN26 plants with isolated pollen from either transgenic or control plants. Thus, the aberrant filament growth could have resulted in the reproductive defects associated with the plants.
Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.
Authors: Schmidt W K , Moore H P
Published: August 2013

Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.
Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.
Authors: Witty J P , Wright J H , Matrisian L M
Published: August 2013

The matrix-degrading metalloproteinases stromelysin-1, stromelysin-3, and gelatinase A are expressed during ductal branching morphogenesis of the murine mammary gland. Stromelysin-1 expression in particular correlates with ductal elongation, and in situ hybridization and three-dimensional reconstruction studies revealed that stromelysin-1 mRNA was concentrated in stromal fibroblasts along the length of advancing ducts. Transgenic mice expressing an activated form of stromelysin-1 under the control of the MMTV promoter/enhancer exhibited inappropriate alveolar development in virgin females. Ultrastructural analysis demonstrated that the basement membrane underlying epithelial and myoepithelial cells was amorphous and discontinuous compared with the highly ordered basal lamina in control mammary glands. Transgenic mammary glands had at least a twofold increase in the number of cells/unit area and a 1.4-fold increase in the percent of cycling cells by 13 wk of age compared with nontransgenic littermates. In addition, transgenic glands expressed beta-casein mRNA, but not protein, and resembled the proliferative and differentiated state of an animal between 8 and 10 days pregnant. An analysis of metalloproteinase expression in the glands of normal pregnant females demonstrated that the same matrix metalloproteinase family members, including stromelysin-1, were expressed in connective tissue cells surrounding epithelial clusters during the time of lobuloalveolar development. These results suggest that metalloproteinases may assist in remodeling ECM during normal ductal and alveolar branching morphogenesis, and that disruption of the basement membrane by an activated metalloproteinase can affect basic cellular processes of proliferation and differentiation.