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V Gerke

Publications and source records attributed to V Gerke.

At least 55 records · Page 3Linked to original sources
PubMed1997

Annexin I targets S100C to early endosomes.

Immunofluorescence and subcellular fractionation localize annexin I and the EF hand protein S100C to the same membranous structures which in part correspond to transferrin receptor-positive endosomes. The association of S100C with endosomal membranes is strictly dependent on annexin I binding since a D91stop-S100C mutant protein, in which the residues essential for annexin I binding have been removed, fails to colocalize with membraneous structures. This indicates that annexin I and S100C form a complex in vivo and that the endosomal localization of this complex is mediated through an interaction of annexin I with the endosomal membrane.

Animals↗
PubMed1997

Modification of annexin II expression in PC12 cell lines does not affect Ca(2+)-dependent exocytosis.

The Ca2+/phospholipid/cytoskeletal-binding protein annexin II has been proposed to play an important role in Ca(2+)-dependent exocytosis; however, the evidence for this role is inconclusive. More direct evidence obtained by manipulating annexin II levels in cells is still required. We have attempted to do this by generating stably transfected PC12 cell lines expressing proteins which elevate or lower functional annexin II levels and using these cell lines to investigate Ca(2+)-dependent exocytosis. Three cell lines were generated: one expressing an annexin II mutant which aggregates annexin II in at least a proportion of the cells, thereby removing functional protein from the cell; a mixed clonal cell line constitutively overexpressing human annexin II; and a clonal cell line capable of over-expressing annexin II in the presence of sodium butyrate. After digitonin permeabilization, Ca(2+)-dependent dopamine release from these cell lines was compared with that from control nontransfected cells, and, in addition, release was compared in induced to uninduced cells. There were no significant differences in Ca(2+)-dependent exocytosis between any of the transfected cell lines before or after induction and the control cells. In addition, nontransfected PC12 cells treated with nerve growth factor, which elevates annexin II levels severalfold, failed to increase Ca(2+)-dependent exocytosis after digitonin permeabilization, compared with control cells. We conclude that annexin II is not an important regulator of Ca(2+)-dependent exocytosis in PC12 cells.

Animals↗
PubMed1997

Differential expression of the calpactin I subunits annexin II and p11 in cultured keratinocytes and during wound repair.

Transforming growth factor beta1 (TGF-beta1) is an important modulator of skin morphogenesis and cutaneous wound repair. To gain insight into the mechanisms of TGF-beta1 action in the skin, we used the differential display RT-PCR technique to identify genes that are regulated by this factor in cultured human keratinocytes. We obtained several partial cDNA clones. One of them was identical to the 3'-end of p11, the small and regulatory subunit of the calpactin I complex [(annexin II)2(p11)2]. RNase protection and northern blot analysis revealed specific regulation of expression of both subunits of this heterotetrameric protein (p11 and annexin II) by TGF-beta1 as well as by other growth factors, although the time course and degree of induction or suppression were different for each gene. Furthermore, we analyzed p11 and annexin II expression in normal and wounded skin. Both p11 and annexin II mRNAs were found in the dermal and epidermal compartments of normal human skin. Immunohistochemical studies demonstrated the presence of p11 at equally high levels in all layers of normal epidermis and in the hyper-proliferative epithelium at the wound edge. By contrast, annexin II expression was high in the basal layer of normal epidermis but low in the suprabasal layers and in the hyper-proliferative epithelium at the wound edge, suggesting a differentiation-specific regulation of this calpactin I subunit. The differential expression and regulation of p11 and annexin II subunits in keratinocytes suggest the existence of different ratios of monomeric versus p11-complexed annexin II that might be associated with different cellular functions.

Animals↗
PubMed1997

Localization of five annexins in J774 macrophages and on isolated phagosomes.

Annexins are a family of structurally related proteins which bind phospholipids in a calcium-dependent manner. Although the precise functions of annexins are unknown, there is an accumulating set of data arguing for a role for some of them in vesicular transport and, specifically, in membrane-membrane or membrane-cytoskeletal interactions during these processes. Here we describe our qualitative and quantitative analysis of the localization of annexins I-V in J774 macrophages that had internalized latex beads, both with and without IgG opsonization. Our results show that whereas all these annexins are present on both the plasma membrane and on phagosomes, the localization on other organelles differs. Annexins I, II, III and V were detected on early endosomes, while only annexin V was seen on late endocytic organelles and mitochondria. Annexins I and II distributed along the plasma membrane non-uniformly and co-localized with F-actin at the sites of membrane protrusions. We also investigated by western blot analysis the association of annexins with purified phagosomes isolated at different time-points after latex bead internalization. While the amounts of annexins I, II, III and V associated with phagosomes were similar at all times after their formation, the level of annexin IV was significantly higher on older phagosomes. Whereas annexins I, II, IV and V could be removed from phagosome membranes with a Ca2+ chelator they remained membrane bound under low calcium conditions. In contrast, annexin III was removed under these conditions and needed a relatively high Ca2+ concentration to remain phagosome bound. Because of their purity and ease of preparation we suggest that phagosomes are a powerful system to study the potential role of annexins in membrane traffic.

Animals↗
PubMed1997

Identification and characterization of a novel type of annexin-membrane interaction: Ca2+ is not required for the association of annexin II with early endosomes.

Annexin II, a member of a family of Ca2+ and membrane binding proteins, has been implicated in regulating membrane organization and membrane transport during endocytosis and Ca2+ regulated secretion. To characterize the mechanistic aspects of the annexin. II action we studied parameters which determine the endosomal association of annexin II. Immunoblot analysis of subcellular membrane fractions prepared from BHK cells in the presence of a Ca2+ chelating agent reveals that annexin II remains associated with endosomal membranes under such conditions. This annexin II behaviour is atypical for the Ca2+ regulated annexins and is corroborated by the finding that ectopically expressed annexin II mutants with inactivated Ca2+ binding sites continue to co-fractionate with endosomal membranes. The Ca(2+)-independent membrane association of annexin II is also not affected by introducing mutations interfering with the complex formation of annexin II with its intracellular protein ligand p11. However, a deletion of the unique N-terminal domain of annexin II, in particular the sequence spanning residues 15 to 24, abolishes the Ca(2+)-independent association of the protein with endosomes. These results describe a novel, Ca(2+)-independent type of annexin-membrane interaction and provide a first explanation for the observed preference of different annexins for different cellular membranes. In the case of annexin II this specificity could be mediated through specific membrane receptors interacting with a unique sequence in the annexin II molecule.

Amino Acid Sequence↗
PubMed1996

Annexin II modulates volume-activated chloride currents in vascular endothelial cells.

The membrane-associated, microfilament-binding protein annexin II is abundantly expressed in endothelial cells from calf pulmonary artery (CPAE cells). We have analyzed its role in the regulation of volume-activated chloride currents (ICl, vol) by loading the cells via the patch pipette with a peptide comprising the N-terminal 14 residues of annexin II. This sequence harbors the binding site for the intracellular annexin II ligand, p11, and the peptide interferes with the annexin II-p11 complex formation. Loading of a CPAE cell with this peptide caused a gradual decrease in the amplitude of ICl, vol during repetitive stimulations with a 28% hypotonic extracellular solution. This run down of the current was virtually absent in untreated cells and in cells that were loaded with a mutated 14-amino acid peptide, which has a single amino acid replacement known to result in a more than 1000 times reduced affinity for binding to p11. We conclude that annexin II-p11 complex formation is either directly or indirectly involved in the activation of ICl, vol in endothelial cells.

Animals↗
PubMed1996

Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexin II.

Annexin II is a Ca(2+)-regulated membrane- and cytoskeleton-binding protein implicated in membrane transport events along the Ca(2+)-regulated secretory and the early endocytic pathway. Biochemical properties of this annexin and its intracellular distribution are regulated by complex formation with p11 (S100A10), a member of the S100 protein family. The annexin II-p11 interaction is mediated through the unique N-terminal domain of annexin II and is inhibited by protein kinase C phosphorylation of a serine residue in annexin II. To map this regulatory serine phosphorylation site we developed a baculovirus-mediated expression system for wild-type annexin II and for a series of annexin II mutants which contained substitutions in one or more serine residues present in the N-terminal domain. The different mutant derivatives were purified and shown to display the same biochemical properties as recombinant wild-type annexin II and the authentic protein purified from porcine intestine. However, significant differences in phosphate incorporation were observed when the individual serine mutants were subjected to phosphorylation by protein kinase C. A comparison of the phosphorylation patterns obtained identified Ser-II as the protein kinase C site responsible for regulating the annexin II-p11 interaction. Ser-II lies within the sequence mediating p11 binding, i.e. amino-acid residues 1 to 14 of annexin II, and phosphorylation at this site most likely leads to a direct spatial interference with p11 binding.

Amino Acid Sequence↗
PubMed1996

Structural requirements for annexin I-S100C complex-formation.

S100C is a member of the S100 family of EF-hand-type Ca(2+)-binding proteins which are thought to bind to and thereby regulate the activity of cellular target proteins in a Ca(2+)-dependent manner. An intracellular ligand for S100C is the Ca2+/phospholipid-binding protein annexin I and we show here that complex-formation is mediated through unique domains within S100C and annexin I. Using a proteolytically truncated annexin I derivative as well as a number of N-terminal annexin I peptides in liposome co-pelleting and ligand-blotting assays we map the S100C-binding site to the N-terminal 13 residues of annexin I. Similar analyses employing recombinantly expressed S100C mutants reveal that residues D91 to 194 in the unique C-terminal extension of this S100 protein are indispensable for annexin I binding. Interaction between S100C and an N-terminal annexin I peptide containing a tryptoplan at position 11 can also be monitored by fluorescence emission spectroscopy after tryptophan excitation. This analysis indicates that the local environment of the tryptophan in annexin I becomes less aqueous on S100C binding, suggesting a hydrophobic nature of the protein-protein interaction. Thus the structural basis of the annexin 1-S100C complex-formation probably resembles to a large extent that of the well-characterized annexin II-p11 interaction.

Animals↗
PubMed1996

The crystal structure and ion channel activity of human annexin II, a peripheral membrane protein.

Annexin II binds in a calcium-dependent manner to acidic phospholipids and is a substrate of some protein kinases. An N-terminally shortened form of human annexin II was crystallized and its molecular structure determined. It is very similar to two previously described members of this protein family, annexin I and annexin V. The protein structure is nearly completely alpha-helical organized as four compact domains which consist of five alpha-helices each. The domains surround a hydrophilic pore. The calcium binding sites are located at the convex side of the structure as in annexin V. Recombinant and natural porcine annexin II are active as ion channel with characteristics similar to annexin V, while N-terminally shortened annexin II and the heterotetramer (annexin II-p11)2 are inactive. Two cysteine residues, Cys133 and Cys262, form a disulphide bridge connecting domains II and III, adding further weight to the notion that ion channel activity does not require major structural rearrangements.

Amino Acid Sequence↗
PubMed1996

Annexins in the human neuroblastoma SH-SY5Y: demonstration of relocation of annexins II and V to membranes in response to elevation of intracellular calcium by membrane depolarisation and by the calcium ionophore A23187.

The human neuroblastoma SH-SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium-dependent membrane-binding proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with Mr and Pi values similar to those of annexins I, II, III, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by K+ depolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X-100 for their solubilisation. Homogenisation of cells in calcium-containing buffers also resulted in the formation of EGTA-resistant membrane-associated annexins II and V. The results demonstrate calcium-dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium-dependent manner to non-phospholipid components of SH-SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus, Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.

Annexin A2↗
PubMed1996

The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.

Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes.

Amino Acid Sequence↗
PubMed1995

RNA1 encodes a GTPase-activating protein specific for Gsp1p, the Ran/TC4 homologue of Saccharomyces cerevisiae.

Ran/TC4 is a ras-related GTP-binding protein predominantly located in the nucleus. Ran/TC4 is essential for nuclear transport and is involved in mitotic control. In Saccharomyces cerevisiae a gene highly homologous to Ran/TC4 has been identified and named GSP1. Like all ras-related GTP-binding proteins, Gsp1p undergoes cycles of GTP hydrolysis and GDP/GTP exchange. The switching between the two different nucleotide bound states regulates the function of these GTP-binding proteins. Here we identify the product of the yeast RNA1 gene as the GTPase-activating protein (GAP) of Gsp1p. RNA1 belongs to a group of genes which are conserved in a variety of different organisms. We have expressed and purified recombinant Gsp1p and Rna1p from Escherichia coli. The GTPase activity of Gsp1p is stimulated 10(7)-fold by Rna1p. In addition, we find that the previously identified human RanGAP1 and rna1p from Schizosaccharomyces pombe are also able to induce GTPase activity of Gsp1p. The GTP hydrolysis of Ran is induced by RanGAP1 and rna1p but not by Rna1p. Implications for the suggested functions of Ran/TC4/Gsp1p in nuclear transport and mitotic control are discussed.

Base Sequence↗
PubMed1995

Recombinant expression and domain structure of the Rna1 protein from Schizosaccharomyces pombe.

The amino acid sequence of Rna1p, a yeast protein implicated in the maturation and/or nucleocytoplasmic transport of RNA, is characterised by the presence of eight leucine-rich repeats (LLRs) as well as two intervening repeats of a different type and a highly acidic C-terminal region. Limited proteolysis of purified Rna1p expressed recombinantly in bacteria reveals that the C-terminal extension but not the region containing the two types of repeats is highly accessible to proteolytic attack and that the C-terminal region most likely harbours (a) low affinity Ca(2+)-binding site(s). These results are indicative of the domain structure of the Rna1p molecule, with the repeats and the C-terminal region being accessible for different interactions.

Base Sequence↗
PubMed1995

Differential expression of calcyclin and its accessible ligands in various types of cutaneous tumors.

Calcyclin is the product of a gene that is regulated in dependence of the cell cycle in fibroblasts in vitro. It has recently been proven to be a sialic acid-binding protein in vitro and in the case of mammalian tissues to bind specifically to annexin II, annexin VI, annexin XI, and glyceraldehyde-3-phosphate dehydrogenase in a Ca(2+)-dependent manner. Since calcyclin can be labelled without impairment of its binding activity, it can be employed as a histochemical tool to localize its accessible ligands. Concomitantly, immunohistochemical localization of calcyclin with a specific antibody is warranted. By using histochemical and immunohistochemical techniques, the expression of calcyclin and its accessible binding sites are demonstrated in serial sections of normal skin and benign, pre-cancerous and malignant tumors of the skin, namely in verruca vulgaris, papillary hidradenoma, syringoma, keratoacanthoma, Bowen's disease, squamous cell carcinoma, melanocytic naevi, primary and metastatic malignant melanoma and non-Hodgkin lymphoma (NHL) of the skin. Cytoplasmic and nuclear expression of calcyclin and its ligands is unexceptionally found in normal skin, epithelial tumors and benign melanocytic tumors. Presence of calcyclin and calcyclin-binding sites is detected in more than 80% of tumor cells in the epithelial lesions. In the group of melanomas and lymphomas heterogeneity is obvious. The application of annexin-specific antibodies raises evidence that members of this protein family co-localize with calcyclin in situ to some extent. These findings suggest that calcyclin and accessible calcyclin-binding molecules, like certain annexins, may be differentially regulated in melanomas and lymphomas in contrast to epithelial lesions with presently undefined biological implications.

Annexins↗
PubMed1994

The annexin II2p11(2) complex is the major protein component of the triton X-100-insoluble low-density fraction prepared from MDCK cells in the presence of Ca2+.

Annexin II2p11(2) is present in the submembranous region of cells expressing both subunits of the complex. Most probably, this subcellular distribution is maintained through the interaction of annexin II2p11(2) with membrane phospholipids and/or elements of the cortical cytoskeleton known to occur in vitro in a Ca(2+)-dependent manner. To determine whether membrane or cytoskeleton interactions are primarily responsible for anchoring annexin II2p11(2) in the cell cortex, we subjected Madin-Darby canine kidney (MDCK) cells to serial extractions using different detergents and identified annexin II and p11 in the different fractions employing specific antibodies. The complex but not monomeric annexin II remains insoluble when the cells are extracted with Triton X-100 in the presence of Ca2+. However, treatment of the Triton X-100-insoluble cell remnants with a series of other detergents known to solubilize GPI-anchored proteins leads to a partial extraction of annexin II2p11(2) even in the presence of Ca2+. Using sucrose density gradient analysis in the presence of Ca2+ as a different means of fractionating the Triton X-100-insoluble cell remnants we show that the majority of annexin II2p11(2) copurifies with a low-density fraction which has been reported to contain GPI-anchored proteins, certain glycolipids, and VIP21/caveolin. Annexin II2p11(2) is by far the most abundant protein component in this fraction indicating that its association with the low-density material occurs via lipid binding and is not due to the interaction with a certain protein.

Animals↗
PubMed1994

The expression of different annexins in the fish embryo is developmentally regulated.

The expression of annexins, a family of Ca2+/phospholipid-binding proteins, was analyzed by biochemical and immunological criteria in the fish Misgurnus fossilis (loach), which is a good model system to study embryonic events. Five different annexins (loach annexins A to E) are present as a maternal pool in the unfertilized eggs. Only annexin E is newly synthesized in the early embryo. Its synthesis, already apparent at mid-blastula, decreases in later stages when two additional annexins (F and G) appear. They are present among the newly synthesized polypeptides of mid-gastrula and later embryonic stages and are also found in loach larvae. The developmentally controlled expression of several annexins indicates a specific role of these proteins at certain embryonic stages.

Animals↗
PubMed1994

Characterization of the macrophage migration inhibitory factor-binding site of sarcolectin and its relationship to human serum albumin.

The sialic acid-binding protein sarcolectin from human placenta specifically interacts with the lymphokine macrophage migration inhibitory factor, enabling its convenient purification and histochemical localization. After cyanogen bromide-mediated cleavage of sarcolectin one polypeptide with an apparent molecular weight of approximately 15,000 exhibited binding capacity to the labelled lymphokine, as revealed by ligand blotting. The N-terminal sequence stretch of this peptide is identical to the respective sequence of human serum albumin, following the internal methionine residue in position 298. Cleavage at a methionine moiety in position 446 can explain the size of the 15 KDa product of chemical degradation. Close similarity of circular dichroism of sarcolectin and human serum albumin added further evidence to their structural similarity, calling for further studies to rigorously define their relationship.

Amino Acid Sequence↗