bluuebunny/crossref_metadata_2025_split huggingface dataset:
HP33: hepatocellular carcinoma-enriched 33-kDa protein with similarity to mitochondrial N-acyltransferase but localized in a microtubule-dependent manner at the centrosome
Authors: Nakadai Tomoyoshi , Kishimoto Toshihiko , Miyazawa Yaeko , Okada Nami , Makino Yasutaka , Obinata Takashi , Tamura Taka-aki 
Published: April 2021

ABSTRACT
Using a new subtraction method and chemically induced rat hepatocellular carcinomas, we identified a hepatocellular carcinogenesis and hepatocyte proliferation-related gene designated hp33 that encoded a 33-kDa protein. The predicted protein was similar to the bovine aralkyl N-acyltransferase and arylacetyl N-acyltransferase. HP33 was restrictively expressed in the liver and kidney, and its gene expression was stimulated in the regenerating liver as well as in hepatocellular carcinoma. Interestingly, it was demonstrated in various hepatic cells that HP33 was localized in regions surrounding the centrosome, where mitochondria were not concentrated. Moreover, its centrosomal localization was evident in the interphase but not in the mitotic phase of the cell cycle. The centrosomal localization of HP33 was dependent on microtubules, and ectopically expressed HP33 was seen at centrosomes even in fibroblasts, which do not exhibit a typical staining pattern of HP33. The centrosomal localization of HP33 became invisible by nocodazole treatment, whereas the mitochondrial staining pattern was not affected by it. In vitro cosedimentation experiments using purified microtubules indicated that HP33 bound to MTs directly and that its MT-binding ability was dependent on the C-terminal basic domain of the protein. These results suggest that, different from early predictions based on its primary structure, HP33 has a growthand carcinogenesis-related function that may be independent of mitochondrial function.
Glutathione S-conjugate transport in hepatocytes entering the cell cycle is preserved by a switch in expression from the apical MRP2 to the basolateral MRP1 transporting protein
Authors: Roelofsen Han , Hooiveld Guido J. E. J. , Koning Hans , Havinga Rick , Jansen Peter L. M. , Müller Michael 
Published: April 2021

ABSTRACT
The multidrug resistance protein MRP1 and its isoform MRP2 are involved in ATP-dependent glutathione S-conjugate transport and have similar substrate specificities. MRP2 mediates hepatic organic anion transport into bile. The physiological function of MRP1 in hepatocytes is unknown. Previous results show that MRP1 expression is low in quiescent hepatocytes but increased after SV40 large T antigen immortalization, suggesting a relationship with cell proliferation. Therefore, we determined mrp1 and mrp2 expression in rat hepatocytes in relation to the cell cycle. By varying cell density we obtained cultures that are mainly in G1 (high density) or have progressed into the S-phase or beyond (low density). In both cultures mrp1 mRNA and protein levels are increased, concomitantly with the disappearance of mrp2. This switch from mrp2 to mrp1 occurs in the G1 phase of the cell cycle and is associated with a decreased cell polarity. Mrp1 is located on lateral membranes or on intracellular vesicles, depending on whether cell-cell contact is established. In both locations mrp1 contributes to cellular glutathione S-conjugate efflux and protects against oxidative stress-inducing quinones. We conclude that a switch in expression from the apically located mrp2 to the basolaterally located mrp1 preserves glutathione S-conjugate transport in hepatocytes entering the cell cycle and protects against certain cytotoxic agents.
Plastid tubules of higher plants are tissue-specific and developmentally regulated
Authors: Köhler Rainer H. , Hanson Maureen R. 
Published: April 2021

ABSTRACT
Green fluorescent stroma filled tubules (stromules) emanating from the plastid surface were observed in transgenic plants containing plastid-localized green fluorescent protein (GFP). These transgenic tobacco plants were further investigated by epifluorescence and confocal laser scanning microscopy (CSLM) to identify developmental and/or cell type specific differences in the abundance and appearance of stromules and of plastids. Stromules are rarely seen on chlorophyll-containing plastids in cell types such as trichomes, guard cells or mesophyll cells of leaves. In contrast, they are abundant in tissues that contain chlorophyll-free plastids, such as petal and root. The morphology of plastids in roots and petals is highly dynamic, and plastids are often elongated and irregular. The shapes, size, and position of plastids vary in particular developmental zones of the root. Furthermore, suspension cells of tobacco exhibit stromules on virtually every plastid with two major forms of appearance. The majority of cells show a novel striking ‘octopusor millipede-like’ structure with plastid bodies clustered around the nucleus and with long thin stromules of up to at least 40 μm length stretching into distant areas of the cell. The remaining cells have plastid bodies distributed throughout the cell with short stromules. Photobleaching experiments indicated that GFP can flow through stromules and that the technique can be used to distinguish interconnected plastids from independent plastids.
Protein phosphatases PP1 and PP2A are located in distinct positions in the Chlamydomonas flagellar axoneme
Authors: Yang Pinfen , Fox Laura , Colbran Roger J. , Sale Winfield S. 
Published: April 2021

ABSTRACT
We postulated that microcystin-sensitive protein phosphatases are integral components of the Chlamydomonas flagellar axoneme, positioned to regulate inner arm dynein activity. To test this, we took a direct biochemical approach. Microcystin-Sepharose affinity purification revealed a prominent 35-kDa axonemal protein, predicted to be the catalytic subunit of type-1 protein phosphatase (PP1c). We cloned the Chlamydomonas PP1c and produced specific polyclonal peptide antibodies. Based on western blot analysis, the 35-kDa PP1c is anchored in the axoneme. Moreover, analysis of flagella and axonemes from mutant strains revealed that PP1c is primarily, but not exclusively, anchored in the central pair apparatus, associated with the C1 microtubule. Thus, PP1 is part of the central pair mechanism thatcontrols flagellar motility. Two additional axonemal proteins of 62 and 37 kDa were also isolated using microcystin-Sepharose affinity. Based on direct peptide sequence and western blots, these proteins are the A-and C-subunits of type 2A protein phosphatase (PP2A). The axonemal PP2A is not one of the previously identified components of the central pair apparatus, outer arm dynein, inner arm dynein, dynein regulatory complex or the radial spokes. We postulate PP2A is anchored on the doublet microtubules, possibly in position to directly control inner arm dynein activity.
Intracellular targeting and retention of the glucose transporter GLUT4 by the perinuclear storage compartment involves distinct carboxyl-tail motifs
Authors: Martinez-Arca Sonia , Lalioti Vassiliki S. , Sandoval Ignacio V. 
Published: April 2021

ABSTRACT
The mechanisms by which the insulin-sensitive glucose transporter, GLUT4, is targeted and retained in a storage compartment near to the Golgi complex are poorly understood. Here we report that removal of the carboxyl-terminal acidic Pro505AspGluAsnAsp509 sequence prevents the storage of GLUT4 in the VAMP-2 positive compartment adjacent to the Golgi complex (GSC), and results in its targeting to GLUT4-positive vesicles and Rab7-positive late endosomes. Storage of the truncated GLUT4 in the GSC is restored by substitution of Phe for the Tyr502 residue adjacent to Pro505 or by treatment of cells with the tyrosine kinase inhibitor genistein. Ablation of the Leu489Leu490-based motif prevents the targeting of GLUT4Δ5 to GLUT4-positive-vesicles and late endosomes as well as the retention of GLUT4Δ5Phe502 by the GSC.
These results are consisting with a model of GLUT4 transport in which the targeting of the protein from the TGN to the GSC is mediated by the Leu489Leu490-based motif and its release from the GSC involves Tyr502 and the adjacent carboxyl-terminal Pro505AspGluAsnAsp509 sequence.
Adhesion-dependent tyrosine phosphorylation of β-dystroglycan regulates its interaction with utrophin
Authors: James M. , Nuttall A. , Ilsley J. L. , Ottersbach K. , Tinsley J. M. , Sudol M. , Winder S. J. 
Published: April 2021

ABSTRACT
Many cell adhesion-dependent processes are regulated by tyrosine phosphorylation. In order to investigate the role of tyrosine phosphorylation of the utrophin-dystroglycan complex we treated suspended or adherent cultures of HeLa cells with peroxyvanadate and immunoprecipitated
β-dystroglycan and utrophin from cell extracts. Western blotting of β-dystroglycan and utrophin revealed adhesion- and peroxyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxy-terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and β-dystroglycan by co-immunoprecipitation with endogenous β-dystroglycan. In extracts from cells treated with peroxyvanadate, where endogenous β-dystroglycan was tyrosine phosphorylated, β-dystroglycan was no longer co-immunoprecipitated with utrophin fusion constructs. Peptide ‘SPOTs’ assays confirmed that tyrosine phosphorylation of β-dystroglycan regulated the binding of utrophin. The phosphorylated tyrosine was identified as Y892 in the β-dystroglycan WW domain binding motif PPxY thus demonstrating the physiological regulation of the β-dystroglycan/utrophin interaction by adhesion-dependent tyrosine phosphorylation.
Dictyostelium DdCP224 is a microtubule-associated protein and a permanent centrosomal resident involved in centrosome duplication
Authors: Gräf Ralph , Daunderer Christine , Schliwa Manfred 
Published: April 2021

ABSTRACT
A cDNA encoding a 224-kDa Dictyostelium discoideumcentrosomal protein (DdCP224) was isolated by immunoscreening. DdCP224 was detected at the centrosome and, more weakly, along microtubules throughout the entire cell cycle. Centrosomal localization does not require microtubules, suggesting that DdCP224 is a genuine centrosomal component. DdCP224 exhibits sequence identity to a weakly conserved class of microtubule-associated proteins including human TOGp and yeast Stu2p. Stu2p has a size of only ∼100 kDa and corresponds to the N-terminal half of DdCP224. The functions of the N- and C-terminal halves of DdCP224 were investigated in the corresponding GFP-fusion mutants. Surprisingly, the N-terminal construct showed only cytosolic localization, whereas the C-terminal construct localized exclusively to the centrosome. This is unexpected because Stu2p is localized at the spindle pole body. Full-length DdCP224-GFP was present both at centrosomes and along microtubules. Furthermore, it bound to microtubules in vitro, unlike the two truncated mutants. Thus centrosome binding is determined by the C-terminal half and microtubule binding may require the interaction of the N- and C-terminal halves. Interestingly, cells expressing full-length DdCP224-GFP exhibit supernumerary centrosomes and show a cytokinesis defect, suggesting that DdCP224 plays an important role in centrosome duplication. These features are unique among the known centrosomal proteins.
A β-catenin/engrailed chimera selectively suppresses Wnt signaling
Authors: Montross Werner T. , Ji Hong , McCrea Pierre D. 
Published: April 2021

ABSTRACT
β-catenin plays an integral role in cell-cell adhesion by linking the cadherin complex of the adherens junction to the underlying actin cytoskeleton. In addition, β-catenin transduces intracellular signals within the Wnt developmental pathway that are crucial to the proper establishment of embryonic axes and pattern formation of early mesoderm and ectoderm. For example, in the context of a defined dorsal ‘organizer’ region of early Xenopus embryos, β-catenin enters the nucleus and associates with transcription factors of the HMG (High Mobility Group) Lef/Tcf protein family. Consequently, genes such as siamois, a homeobox gene contributing to the specification of the dorsoanterior axis, are activated. To further examine the role that β-catenin plays in Wnt signaling, we generated a chimeric protein, β-Engrailed (β-Eng), in which the C-terminal trans-activation domain of β-catenin is replaced with the transcriptional repression domain of Drosophila Engrailed. Dorsal overexpression of this mRNA in early Xenopus embryos leads to suppression of organizer-specific molecular markers such as siamois, Xnr-3 and goosecoid, corresponding with the dramatic morphological ventralization of embryos. Ventralized embryos further exhibit reduced activity of the Wnt pathway, as indicated by the loss of the notochord/organizer marker, chordin. Importantly, β-Eng associates and functions normally with the known components of the cadherin complex, providing the experimental opportunity to repress β-catenin’s signaling function apart from its role in cadherin-mediated cell-cell adhesion.
Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?
Authors: Petrone Angiola , Sap Jan 
Published: April 2021

ABSTRACT
Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity of a pathway, can be controlled by multiple tyrosine phosphatases, attesting to the intricate integration of these enzymes in cellular regulation. Lastly, we are starting to appreciate the roles of intracellular topology, tyrosine phosphorylation and oligomerization among the many mechanisms regulating tyrosine phosphatase activity.
Contraction of collagen matrices mediated by α2β1A and αvβ3 integrins
Authors: Cooke Mary E. , Sakai Takao , Mosher Deane F. 
Published: April 2021

ABSTRACT
The β1-null fibroblastic cell line GD25 and its derivatives were studied to gain an understanding of the roles of β1 and β3 integrins in the initial (1-hour) contraction of collagen gels. Stable transfectants of GD25 cells expressing the β1A splice variant of β1 (β1A-GD25) did not express α2β1A and did not adhere to collagen. After transfection of α2 into β1A-GD25 cells, the α2β1A-GD25 transfectants contracted collagen gels in the presence of serum, whereas β1A-GD25 cells did not. The GD25 parental cells, however, also contracted collagen gels. Collagen gel contraction by GD25 cells was blocked by antibodies to αvβ3 or a RGD-containing peptide, indicating that αvβ3 is the integrin responsible for mediation of contraction by GD25 cells. Collagen gel contraction by α2β1A-GD25 cells was not inhibited by antibodies to αvβ3 or RGD-containing peptide, but was inhibited by anti-α2 antibody. Flow cytometry demonstrated negligible expression of αvβ3 by β1A-GD25 and α2β1A-GD25 cells when compared to GD25 cells. Platelet derived growth factor (PDGF) and sphingosine-1-phosphate (S1P) enabled gel contraction by α2β1A-GD25 and GD25 cells, respectively, in the absence of serum. PDGF-stimulated contraction by α2β1A-GD25 cells was attenuated in the presence of inhibitors of phosphatidylinositol-3-kinase whereas such inhibitors had no effect on S1P-stimulated contraction by GD25 cells. These experiments using the β1-null GD25 cells and β1A and α2β1A transfectants demonstrate that α2β1A and αvβ3 independently mediate collagen gel contraction and are regulated by different serum factors and signaling pathways.
Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis
Authors: Wong Kelvin C. Y. , Naqvi Naweed I. , Iino Yuichi , Yamamoto Masayuki , Balasubramanian Mohan K. 
Published: April 2021

ABSTRACT
Cell division in many eukaryotes, including the fission yeast Schizosaccharomyces pombe, utilizes a contractile actomyosin ring. In S. pombe, the actomyosin ring is assembled at the medial cortex upon entry into mitosis and constricts at the end of anaphase to guide the centripetal deposition of the septum. Despite identification of several structural components essential for actomyosin ring assembly, the interdependencies between these geneproducts in the process of ring assembly are unknown. This study investigates the role of Rng3p, a member of the UCS-domain containing protein family (Unc-45p, Cro1p, She4p), in actomyosin ring assembly. Null mutants in rng3 resemble deletion mutants in the type II myosin heavy chain (myo2) and rng3ts mutants show strong negative interactions with the myo2-E1 mutant, suggesting that Rng3p is involved in modulating aspects of type II myosin function. Interestingly, a green fluorescent protein (GFP) tagged Rng3p fusion is detected at the division site in the myo2- E1 mutant, but not in other myo2-alleles, wildtype cells or in 18 other cytokinesis mutants. Assembly and maintenance of Rng3p at the division site in the myo2- E1 mutant requires F-actin. Rng3p is also required for the proper assembly of Myo2p and F-actin into a functional actomyosin ring but is not necessary for their accumulation at the division site. We conclude that Rng3p is a novel component of the F-actin cytoskeleton essential for a late step in actomyosin ring assembly and that it might monitor some aspect of type II myosin assembly during actomyosin ring construction.
Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis
Authors: Hofmann Ilse , Mertens Claudia , Brettel Monika , Nimmrich Volker , Schnölzer Martina , Herrmann Harald 
Published: April 2021

ABSTRACT
Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phasebinding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several μm. The basic aminoterminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by coimmunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound – and does not bind – to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.
XCS-1, a maternally expressed gene product involved in regulating mitosis in Xenopus
Authors: Nakamura Hisashi , Wu Chuanfen , Kuang Jian , Larabell Carolyn , Etkin Laurence D. 
Published: April 2021

ABSTRACT
The regulation of the cell cycle during early development is an important and complex biological process. We have cloned a cDNA, XCS-1, that may play an important role in regulating mitosis during early embryogenesis in Xenopus laevis. XCS-1 is a maternally expressed gene product that is the Xenopus homologue of the human cleavage signal protein (CS-1). XCS-1 transcripts were detected in oocytes with the titer decreasing just prior to the MBT. During development the XCS-1 protein was detected on the membrane and in the nucleus of blastomeres. It was also detected on the mitotic spindle in mitotic cells and on the centrosomes in interphase cells. Overexpression of myc-XCS-1 in Xenopus embryos resulted in abnormal mitoses with increased numbers of centrosomes, multipolar spindles, and abnormal distribution of chromosomes. Also, we observed incomplete cytokinesis resulting in multiple nuclei residing in the same cytoplasm with the daughter nuclei in different phases of the cell cycle. The phenotype depended on the presence of the N terminus of XCS-1 (aa 1-73) and a consensus NIMA kinase phosphorylation site (aa159-167). Mutations in this site affected the ability of the overexpressed XCS-1 protein to produce the phenotype. These results suggest that XCS-1 is a maternal factor playing an important role in the regulation of the cell cycle during early embryogenesis and that its function depends on its state of phosphorylation.
Coupled transport of p24 family members
Authors: Emery Gregory , Rojo Manuel , Gruenberg Jean 
Published: April 2021

ABSTRACT
Recent studies show that small transmembrane proteins of ≈22-24 kDa (the p24 family), which are grouped into 4 subfamilies by sequence homology (p23, p24, p25 and p26), are involved in the early secretory pathway. In this study, we have investigated the mutual requirements of ectopically expressed members of the p24 family for targeting to their proper cellular destination. We find that coexpression of p23 and p24 is both necessary and sufficient for each protein to be transported to the cis-Golgi network/Golgi complex. Proteins from other subfamilies did not substitute for either p23 or p24, even after multiple coexpression. However, trafficking of the p23/p24 couple was facilitated by coexpression of proteins from other subfamilies. In addition, we find that the sequence resembling an endoplasmic reticulum retrieval signal present in the cytoplasmic domain of p23 (but not p24) is dispensable. In contrast, the conserved coiledcoil region in the lumenal domain is absolutely required in both p23 and p24 for proper targeting of the p23/p24 couple. These data demonstrate that p23 and p24 must interact with each other to reach their destination, but that this strict requirement is combined with a mutual dependence amongst p24 proteins. We speculate that p24 proteins can form different oligomeric complexes, which contribute to confer specialized sorting/trafficking properties to membranes of the early secretory pathway, perhaps serving as membrane organizers.