1 Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
2 Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
3 Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
* Present address: Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
Present address: Argonex Inc., 2044 India Rd. Ste. 202, Charlottesville, VA 22901, USA
¶Author for correspondence (e-mail: creutz{at}virginia.edu)
Accepted May 8, 2001
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SUMMARY |
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Key words: Astrocytoma, Leptomycin B, Phosphorylation, Genistein, S100A10
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INTRODUCTION |
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AnxII has been implicated in a variety of functional contexts, usually as a docking protein mediating the formation of membrane-based protein complexes. AnxII and the AnxII2/p112 complex associate with plasma membranes, endosomes and exocytic vesicles, and may influence membrane-membrane or membrane-cytoskeletal interactions related to vesicular trafficking. Notably, AnxII is a prominent component of cholesterol-rich plasma membrane rafts, which also contain caveolins, src-related kinases, G-proteins and transmembrane receptors such as CD44. A recent study showed that a trans-dominant AnxII2/p112 chimera affects CD44 clustering and the structure of the associated actin cytoskeleton, suggesting a role for AnxII in the supramolecular organization of signal transduction-related components (Oliferenko et al., 1999). In addition, p11 has been shown to associate with and affect the activities of other signaling molecules such as cPLA2 (Wu et al., 1997), the Bcl2-family protein BAD (Hsu et al., 1997) and the cdc2 kinase-related PCTAIRE1 (Sladeczek et al., 1997), although whether these interactions also involve the AnxII2/p112 complex remains to be investigated. AnxII and/or AnxII2/p112 also appear to be present on the extracellular surface of some cell types such as endothelial cells and certain tumor cells where they can act as a receptor for plasminogen/tissue plasminogen activator (Hajjar et al., 1994; Kassam et al., 1998; Menell et al., 1999) or tenascin-C (Chung and Erickson, 1994). Finally, a function of AnxII in the nucleus that seems not to involve binding to p11 or membranes has been suggested by its purification as part of a primer recognition protein complex that enhances DNA polymerase activity in vitro (Jindal et al., 1991). Further evidence that AnxII might play a role in promoting DNA synthesis and cell proliferation was provided by subsequent immunodepletion/reconstitution experiments in Xenopus oocyte nuclear extracts (Vishwanatha and Kumble, 1993) and by antisense strategies in mammalian cell lines (Chiang et al., 1999; Kumble et al., 1992). A link between AnxII and cell transformation and neoplasia was first suggested by the identification of AnxII as a major v-src phosphorylation substrate in transformed fibroblasts (Erickson and Erickson, 1980; Radke and Martin, 1979). Subsequently, AnxII expression has been found to be upregulated in several types of spontaneous neoplasms, such as pancreatic carcinoma (Vishwanatha et al., 1993), acute promyelocytic leukemia (Menell et al., 1999) and high-grade glioma (Reeves et al., 1992); in fibroblasts transformed by viral oncogenes such as v-H-ras, v-src and v-mos (Ozaki and Sakiyama, 1993); and in cells treated with mitogenic or trophic factors such EGF, FGF, NGF or TGFß1 (Fox et al., 1991; Keutzer and Hirshhorn, 1990; Munz et al., 1997). Although AnxII and p11 are often coexpressed in cells and tissues, their relative levels vary depending on the source (Gerke, 1989; Zokas and Glenney, 1987) and detailed studies have revealed differences in AnxII and p11 expression and localization patterns within particular tissue cell types such as fibroblasts and skin keratinocytes, suggesting that AnxII monomer and AnxII2/p112 complex may have distinct and different functions (Munz et al., 1997; Zokas and Glenney, 1987).
Overall, the available evidence suggests that AnxII and p11 are docking proteins with multiple functions, whose specific roles may depend on the cell types in which they are expressed, their localization within or outside of cells, and interactions with particular binding substrates in these locations. However, the suggestions that AnxII has functions in the nucleus or at the cell surface have been controversial since, in most cases, very little of total cellular AnxII is in these locations. Thus the detected protein could be argued to represent artefactual contaminant, and the mechanisms that might regulate its localization to these compartments are unclear. Since the functions of AnxII depend in part on its localization in cells, the present study investigated mechanisms controlling AnxII localization in neoplastic and transformed cell lines. Using molecular and pharmacologic approaches, we provide evidence that: (1) AnxII monomer readily enters the nucleus but is rapidly exported due to a functional nuclear export signal (NES) sequence that closely overlaps the p11-binding region in the AnxII N-terminus; (2) p11 binding to AnxII results in sequestration of the complex in the cytoplasmic compartment; and (3) manipulation of cellular phosphorylation can affect the nucleocytoplasmic partitioning of AnxII.
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MATERIALS AND METHODS |
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A second GFP-AnxII expression vector, pEGFP-AnxII, was created by ligating the AnxII cDNA from pBS into the SalI/XbaI sites in the multiple cloning region of pEGFP-C1 (Clontech Laboratories, Palo Alto, CA). To construct pEGFP-AnxII(1-22), pBS-AnxII was digested with NdeI and BamHI, filled using Klenow fragment and religated, and digested with SalI/XbaI to yield a fragment that was ligated into the SalI/XbaI sites of pEGFP-C1. To construct pEGFP-AnxII (23-338), pBS-AnxII was digested with NdeI, filled using Klenow fragment, religated using a GGATCC linker to introduce a second BamHI site, and digested with BamHI to yield a fragment that was ligated into the BamHI site of pEGFP-C1. pEGFP-AnxII(L10A/L12A) was generated by site-directed mutagenesis of pEGFP-AnxII using PCR.
Cell lines and culture
U1242MG human astrocytoma cells (Kim-Lee et al., 1992) were provided by Alan Yates (Ohio State University, Columbus) and B31 RAT-1(v-src) transformed fibroblasts (Woodring and Garrison, 1997) by James Garrison (University of Virginia, Charlottesville). Cells were maintained in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum and glutamine at 37°C in a 5% CO2 atmosphere. Transfections were performed using Lipofectamine (Gibco BRL, Rockville, MD). Stably transfected cell populations were generated by culturing the cells in the presence of 600 µg/ml G418 for 3-4 weeks followed by fluorescence-activated cell sorting to isolate polyclonal populations of cells expressing GFP or fusion proteins. Leptomycin B (LmB) was the gift of Barbara Wolff (Novartis AG, Basel, Switzerland). Most of the experiments presented were repeated using two protocols. Either cells were rinsed with serum-free MEM, then incubated at 37°C with 100 nM LMB or 0.01% DMSO as vehicle in serum-free MEM for the indicated times; or cells were preincubated for 30 minutes with 100 nM LmB or vehicle in serum-free MEM, which was then replaced with MEM containing 10% FBS and either 200 nM LmB or vehicle, and the incubations continued for the indicated times. No significant effect of serum was seen on the localization of the examined proteins.
Immunofluorescence
Cells cultured on poly-d-lysine-coated coverslips were briefly rinsed with PBS then fixed with either 4% formaldehyde in PBS or methanol for 15-30 minutes. The cells were rinsed, blocked with 10% bovine serum albumin (BSA) in PBS for 3 hours at room temperature (RT), and incubated overnight at 4°C with primary mouse monoclonal antibodies (mAbs) directed against AnxII, p11 or AnxIV (BD Transduction Laboratories, Lexington, KY) at 1:1000 dilution in PBS/3% BSA. The AnxII mAb reacted with methanol-fixed cells but not with formalin-fixed cells. The cells were then incubated with biotinylated anti-mouse (Vector Laboratories, Burlingame, CA) at 1:500 dilution in PBS/3% BSA for 2 hours at RT followed by avidin-rhodamine (Vector Laboratories) at 1:1000 dilution for methanol-fixed cells or 1:5000 dilution for formaldehyde-fixed cells. Epifluorescence microscopy was performed using a Nikon microscope equipped with an Olympus camera and Kodak ASA 400 film. Laser scanning confocal microscopy (LSCM) was performed using a Zeiss LSM 410. Cells were imaged from top to bottom in the Z-plane; images from the midplane of the cells were captured and stored as digital images that are shown in the figures.
Immunoprecipitation
Stably transfected cells expressing GFP, GFP-AnxII, or GFP-p11 were scraped into ice-cold lysis buffer containing 100 mM NaCl, 16 mM Hepes pH 7.0, 0.5% Triton X-100, 2 mM EGTA, 1 mM DTT, 100 uM PMSF, 12 µg/ml leupeptin and 12 µg/ml aprotinin, and lysed with 10 strokes in a Dounce homogenizer. The lysates were centrifuged at 20,000 g for 15 minutes. The supernatants were incubated overnight with 4 µg/ml anti-GFP polyclonal antibody (Clontech), which was then precipitated using protein A-sepharose beads (Affi-Gel; Bio-Rad Laboratories, Hercules, CA). The beads were boiled in Laemmli sample buffer, which was then subjected to SDS-PAGE using 10% or 15% acrylamide gels and electrotransferred to nitrocellulose or PVDF membranes. Western blot analysis was performed using anti-AnxII or anti-p11 mAbs at 1:5000 dilution, followed by sheep anti-mouse antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ) at 1:1000 dilution, with visualization by enhanced chemiluminescence.
Triton X-100 extraction
Stably transfected cells expressing GFP-AnxII or GFP-p11 were scraped into ice-cold extraction buffer containing 150 mM NaCl, 20 mM Hepes pH 7.0, 0.2% Triton X-100, 2 mM MgCl2, 1 mM DTT, 200 µM PMSF, 25 µg/ml leupeptin and 15 µg/ml aprotinin, and lysed with 10 strokes in a Dounce homogenizer. Ca2+/EGTA buffers were added from 10x stocks in 20 mM Hepes pH 7.0 to give a final pCa2+ of 8.17, 6.92, 5.91, 5.02, 4.065 and 3.046 as measured by a Ca2+ electrode. The lysates were centrifuged at 20,000 g for 30 minutes. Laemmli sample buffer was added to the supernatant and pellets to give equal volumes of 1x buffer. The boiled samples were subjected to SDS-PAGE using 12% gels and electrotransferred to nitrocellulose membranes. Western blot analysis was performed using anti-AnxII mAb at 1:5000 dilution or anti-GFP polyclonal antibody (Clontech) at 1:1000 dilution, followed by anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase, with visualization by enhanced chemiluminescence.
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RESULTS |
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IP complex formation
Since the 1-22 region of AnxII contains the p11 binding region and substitutions at residues L10 and L12 have been shown to affect p11 binding (Becker et al., 1990), the above observations suggest that exclusion of GFP-AnxII from the nucleus involves formation of the GFP-AnxII/p11 complex. However, peptide binding data (Becker et al., 1990) and crystallographic structure of p11/AnxII N-tail complex (Rety et al., 1999) predict that fusion of GFP to the AnxII N-terminus would inhibit p11 binding. Therefore, the abilities of GFP-AnxII and GFP-p11 expressed in stably transfected U1242MG cells to form multimeric complexes with endogenous AnxII and p11 were examined by anti-GFP immunoprecipitation analyses of transfected cell lysates. As shown in Fig. 2, endogenous AnxII and p11 co-immunoprecipitated with GFP-p11 but not with GFP-AnxII or GFP alone. In the Coomassie Blue stains, GFP-AnxII and GFP are easily seen, whereas GFP-p11 could be identified as a faint band in the original gels but is not apparent in reproductions. Therefore, the inability to detect co-immunoprecipitation of p11 and AnxII with GFP-AnxII or GFP does not merely reflect lower levels of expression of the latter proteins versus GFP-p11 in the transfected cells. These data indicate that GFP-AnxII does not bind p11 and remains monomeric when expressed in cells, and thus p11 binding is unlikely to underlie the nuclear exclusion of GFP-AnxII. By contrast, GFP-p11 can participate in AnxII2/p112 complex formation.
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DISCUSSION |
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The ability of LmB treatment to cause detectable increases in the nuclear accumulation of endogenous AnxII appeared to be cell line-dependent, being much more evident in v-src-transformed fibroblasts than in U1242MG cells. This suggests the existence of mechanisms that sequester AnxII in the cytoplasmic compartment, preventing its entry into the nucleus. The most likely mechanism is formation of AnxII2/p112 complex, supported by our findings that LmB treatment had relatively little effect on the localizations of p11 and GFP-p11 in U1242MG cells. Cytoplasmic sequestration of AnxII2/p112 could result from the size of the complex (94 kDa) preventing passage through the nuclear diffusion barrier. The complex may also be partially bound to the plasma membrane and membraneous cytoplasmic organelles at physiological [Ca2+], since half-maximal binding of AnxII2/p112 to membranes occurs at a lower level of calcium than half-maximal binding of the AnxII monomer (Powell and Glenney, 1987; Raynal and Pollard, 1994). Previous observations indicate that the relative expression levels of AnxII and p11 can differ between tissue and cell types and may change under conditions related to cell proliferation and differentiation (Fox et al., 1991; Gerke, 1989; Harder et al., 1993; Munz et al., 1997; Puisieux et al., 1996; Zokas and Glenney, 1987). Therefore, differential expression of AnxII and p11 could represent a mechanism to regulate the amount of AnxII monomer that can enter the nucleus.
The bifunctionality of the AnxII N-terminal region in mediating nuclear export and p11 binding raises the possibility that p11 binds similarly to leucine-rich NES sequences in other proteins. However, p11 binding requires that the leucine-rich region be precisely located near the N-terminus, as indicated by the crystal structure of p11/AnxII peptide complex (Rety et al., 1999) and by the loss of p11 binding when GFP was fused to the AnxII N-terminus in the present study. Other proteins have been reported to bind p11, including cPLA2 (Wu et al., 1997), BAD (Hsu et al., 1997), and PCTAIRE1 (Sladeczek et al., 1997), but the structural bases for these interactions have not been defined.
Our experiments with genistein and pervanadate in conjunction with LmB in v-src-transformed fibroblasts suggest that tyrosine phosphorylation represents a means of promoting the nuclear entry of AnxII, consistent with the previous detection of phosphorylated AnxII in nuclear extracts of K562 and HeLa cells (Chiang et al., 1996). The effects of genistein and pervanadate on AnxII localization became obvious only when nuclear export was inhibited with LmB, which may explain why AnxII phosphorylation by v-src appeared to have little effect on its subcellular distribution in early studies (Erickson and Erickson, 1980; Radke and Martin, 1979). We have not yet elucidated the mechanism by which this occurs, but one possibility is that phosphorylation of AnxII2/p112 releases AnxII and allow its entry into the nucleus. Membrane binding of AnxII2/p112 dramatically enhances the kinetics of AnxII phosphorylation by src (Bellagamba et al., 1997). In vitro phosphorylation of Y23 or S25 does not fully dissociate the complex or prevent p11 binding, although a partial dissociation of PKC-phosphorylated complex was reported in one study (Regnouf et al., 1995). In vitro phosphorylation of AnxII on additional residues such as S11 does dissociate the complex and inhibit p11 binding (Jost and Gerke, 1996; Regnouf et al., 1995). Whether this occurs physiologically is not well-documented, although there is evidence that diphosphorylation of AnxII causes its release from the membrane in nicotine-stimulated chromaffin cells (Sagot et al., 1997). Phosphorylation of AnxII2/p112 also somewhat decreases its affinity for membrane binding (Powell and Glenney, 1987; Regnouf et al., 1995), which might result in release of the complex from the membrane in cells. However, because of its size (94 kDa), the complex probably would still need to dissociate before AnxII could enter the nucleus. It is also possible that the effects of genistein and pervanadate seen in the present study involve a mechanism other than direct AnxII phosphorylation, since the serum-stimulated accumulation of AnxV in osteosarcoma cells is also inhibited by genistein (Mohiti et al., 1997) but there is no evidence that AnxV can be phosphorylated. The present findings indicate that there may be a number of mechanisms which serve to tightly control the concentration of AnxII within the nucleus. This might be necessary if AnxII participates in a regulated nuclear process, such as its previously suggested role as an accessory protein in DNA synthesis (Jindall et al., 1991; Vishwanatha and Kumble, 1993). The early report of AnxII association with small ribonucleoprotein particles (Arrigo et al., 1983) and the more recent demonstration of AnxII binding to cytoskeleton-associated mRNA subpopulations (Vedeler and Hollas, 2000), together with the present demonstration of AnxII export, raise the possibility of another function in RNA export and localization of ribonucleoprotein particles to particular sites. Either of these proposed functions are consistent with the reported suppression of cell proliferation by AnxII down-regulation (Chiang et al., 1999; Kumble et al., 1992). It is also possible that the physiological role(s) of AnxII is restricted to the cytoplasm, and that nuclear exclusion mechanisms serve to localize the protein to the appropriate compartment.
In conclusion, nuclear export signals have been identified in a variety of molecules and may represent a basic aspect of their functions (e.g. RNA export molecules), or may direct their localizations to cellular compartments relevant to their functions, as for signal transduction molecules and cytoskeletal actin (Henderson and Eleftheriou, 2000; Wada et al., 1998). The present study identifies nuclear export as a novel mechanism controlling the localization of a member of another class of proteins, the annexins. Since these proteins move on and off membranes in the cytoplasmic compartment in response to calcium fluxes, we can now envision a role for AnxII involving a complex cycle of membrane interactions and nuclear entry. For example, the entry of calcium into a stimulated cell could promote the binding of AnxII to the plasma membrane. In this position it may readily serve as a substrate for the cellular src kinase. After the calcium transient is completed, and the AnxII is released from the membrane, it may then travel into the nucleus as a result of its phosphorylation state. The entry of the phosphorylated annexin into the nucleus thus could provide information to the nucleus concerning the prior calcium transient, even if the calcium concentration was not elevated in the nucleus per se. The annexin may then participate in a nuclear response to the initial cell stimulation and calcium transient, perhaps by regulating DNA replication or messenger RNA processing or transport. Although such a pathway is highly speculative at present, the discovery of the nuclear trafficking of AnxII is likely to lead to new insights regarding the biological roles and regulation of this ubiquitous family of molecules.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Arenzana-Seisdedos, F., Turpin, P., Rodriguez, M., Thomas, D., Hay, R. T., Virelizier, J-L. and Dargemont C. (1997). Nuclear localization of IB
promotes active transport of NF-
B from the nucleus to the cytoplasm. J. Cell Sci. 110, 369-378.
Arrigo, A.-P., Darlix, J.-L. and Spahr, P.-F. (1983). A cellular protein phosphorylated by the avian sarcoma virus transforming gene product is associated with ribonucleoprotein particles. EMBO J. 2, 309-315.[Medline]
Barwise, J. L. and Walker, J. H. (1996a). Annexins II, IV, V, and VI relocate in response to rises in intracellular calcium in human foreskin fibroblasts. J. Cell Sci. 109, 247-255.
Barwise, J. L. and Walker, J. H. (1996b). Subcellular localization of annexin V in human foreskin fibroblasts: nuclear localization depends on growth state. FEBS Lett. 394, 213-216.[Medline]
Becker, T., Weber, K. and Johnsson, N. (1990). Protein-protein recognition via short amphiphilic helices; a mutational analysis of the binding site of annexin II for p11. EMBO J. 9, 4207-4213.[Abstract]
Bellagamba, C., Hubaishy, I., Bjorge, J. D., Fitzpatrick, S. L., Fujita, D. J. and Waisman, D. M. (1997). Tyrosine phosphorylation of annexin II tetramer is stimulated by membrane binding. J. Biol. Chem. 272, 3195-3199.
Biener, Y., Feinstein, R., Mayak, M., Kaburagi, Y., Kadowaki, T. and Zick, Y. (1996). Annexin II is a novel player in insulin signal transduction. Possible association between annexin II phosphorylation and insulin receptor internalization. J. Biol Chem. 271, 29489-29496.
Brambilla, R., Zippel, R., Sturani, E., Morello, L., Peres, A. and Alberghina, L. (1991). Characterization of the tyrosine phosphorylation of calpactin I (annexin II) induced by platelet-derived growth factor. Biochem. J. 278, 447-452.[Medline]
Chiang, Y., Davis, R. G. and Vishwanatha, J. K. (1996). Altered expression of annexin II in human B-cell lymphoma cell lines. Biochim. Biophys. Acta 1313, 295-301.[Medline]
Chiang, Y., Rizzino, A., Sibenaller, Z. A., Wold, M. S. and Vishwanatha, J. K. (1999). Specific down-regulation of annexin II expression in human cells interfemres with cell proliferation. Mol. Cell Biochem. 199, 139-47.[Medline]
Chung, C. Y. and Erickson, H. P. (1994). Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C. J. Cell Biol. 126, 539-548.[Abstract]
Courtneidge, S., Ralston, R., Alitalo, K. and Bishop, J. M. (1983). Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases. Mol. Cell Biol. 3, 340-350.[Medline]
Creutz, C. E., Kambouris, N. G., Snyder, S. Y., Hamman, H. C., Nelson, M. R., Liu, W. and Rock, P. (1992). Effects of the expression of mammalian annexins in yeast secretory mutants. J. Cell Sci. 103, 1177-1192.
Erickson, E. and Erickson, R. L. (1980). Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus transforming gene product. Cell 21, 829-836.[Medline]
Fornerod, M., Ohno, M., Yoshida, M. and Mattaj, I. W. (1997). CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90, 1051-1060.[Medline]
Fox, M. T., Prentice, D. A. and Hughes, J. P. (1991) Increases in p11 and annexin II proteins correlate with differentiation in the PC12 pheochromocytoma. Biochem. Biophys. Res. Commun. 177, 1188-1193.[Medline]
Fricker, M., Hollinshead, M., White, N. and Vaux, D. (1997). Interphase nuclei of many mammalian cell types contain deep, dynamic, tubular membrane-bound invaginations of the nuclear envelope. J. Cell Biol. 136, 531-544.
Fukuda, M., Gotoh, I., Gotoh, Y. and Nishida, E. (1996). Cytoplasmic localization of mitogen-activated protein kinase kinase directed by its NH2-terminal, leucine-rich short amino acid sequence, which acts as a nuclear export signal. J. Biol. Chem. 271, 20024-20028.
Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M. and Nishida, E. (1997). CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308-311.[Medline]
Gerke, V. (1989). Tyrosine protein kinase substrate p36: a member of the annexin family of Ca2+/phospholipid-binding proteins. Cell. Motil. Cytoskel. 14, 449-454.[Medline]
Gerke, V. and Moss, S. E. (1997). Annexins and membrane dynamics. Biochim. Biophys. Acta 1357, 129-154.[Medline]
Gerke, V. and Weber, K. (1985). Calcium-dependent conformational changes in the 36-kDa subunit of intestinal protein I related to the cellular 36-kDa target of Rous sarcoma virus tyrosine kinase. J. Biol. Chem. 260, 1688-1695.[Abstract]
Hajjar, K. A., Jacovina, A. T. and Chacko, J. (1994). An endothelial cell receptor for plasminogen/tissue plasminogen activator. I. Identity with annexin II. J. Biol. Chem. 269, 21198-21203.
Harder, T., Thiel, C. and Gerke, V. (1993) Formation of the annexin AnxII2/p112 complex upon differentiation of F9 teratocarcinoma cells. J. Cell Sci. 194, 1109-1117.
Henderson, B. R. and Eleftheriou, A. (2000). A comparison of the activity, sequence specificity, and Crm1-dependence of different nuclear export signals. Exp. Cell Res. 256, 213-224.[Medline]
Hope, T. J. (1997). Viral RNA export. Chem. Biol. 4, 335-344.[Medline]
Hsu, S. Y., Kaipia, A., Zhu, L. and Hsueh, A. J. (1997). Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11. Mol. Endocrinol 11, 1858-1867.
Jiang, Y., Chan, J. L., Zong, C. S. and Wang, L. H. (1996). Effect of tyrosine mutations on the kinase activity and transforming potential of an oncogenic human insulin-like growth factor I receptor. J. Biol. Chem. 27, 160-167.
Jindal, H. K., Chaney, W. G., Anderson, C. W., Davis, R. G. and Vishwanatha, J. K. (1991). The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase . J. Biol. Chem. 266, 5169-5176.
Johnsson, N., Marriot, G. and Weber, K. (1988). p36, the major cytoplasmic substrate of src protein kinase, binds to its p11 regulatory subunit via a short amino-terminal amphipathic helix. EMBO J. 7, 2435-2442.[Abstract]
Jost, M. and Gerke, V. (1996). Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexin II. Biochim. Biophys. Acta 1313, 283-289.[Medline]
Kassam, G., Choi, K. S., Ghuman, J., Kang, H. M., Fitzpatrick, S. L., Zackson, T., Zackson, S., Toba, M., Shinomiya, A. and Waisman, D. M. (1998). The role of annexin II tetramer in the activation of plasminogen. J. Biol. Chem. 273, 4790-4799.
Katoh, N., Suzuki, T., Yuasa, A. and Miyamoto, T. (1995). Distribution of annexins I, II, and IV in bovine mammary gland. J. Dairy Sci. 78, 2382-2387.
Keutzer, J. C. and Hirschhorn, R. R. (1990). The growth-regulated gene 1B6 is identified as the heavy chain of calpactin I. Exp. Cell Res. 188, 153-159.[Medline]
Kim, T. T., Chen, C.-T. and Huang, C.-C. (1998). Expression of annexin II in middle ear cholesteatoma. Otolaryngol. Head Neck Surg. 118, 324-328.[Medline]
Kim-Lee, M. H., Stokes, B. T. and Yates, A. J. (1992). Reperfusion paradox: a novel mode of glial cell injury. Glia. 5, 56-64.[Medline]
Kudo, N., Matsumori, N., Taoka, H., Fujiwara, D., Schreiner, E. P., Wolff, B., Yoshida, M. and Horinouchi, S. (1999). Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA 96, 9112-9117.
Kumble, K. D., Iversen, P. L. and Vishwanatha, J. K. (1992). The role of primer recognition proteins in DNA replication: inhibition of cellular proliferation by antisense oligodeoxyribonucleotides. J. Cell Sci. 101, 35-41.[Abstract]
Malim, M. H., McCarn, D. F., Tiley, L. S. and Cullen, B. R. (1991). Mutational definition of the human immunodeficiency virus type 1 Rev activation domain. J. Virol. 65, 4248-4254.[Medline]
Mamiya, N., Iino, S., Mizutani, A., Kobayashi, S. and Hidaka, H. (1994). Development-related and cell-type specific nuclear localization of annexin XI: immunolocalization analysis in rat tissues. Biochem. Biophys. Res. Commun. 202, 403-409.[Medline]
Menell, J. S., Cesarman, G. M., Jacovina, A. T., McLaughlin, M. A., Lev, E. A. and Hajjar, K. A. (1999). Annexin II and bleeding in acute promyelocytic leukemia. New Engl. J. Med. 340, 994-1004.
Mizutani, A., Usuda, N., Tokumitsu, H., Minami, H., Yasui, K., Kobayashi, R. and Hidaka, H. (1992). CAP-50, a newly identified annexin, localizes in nuclei of cultured fibroblast 3Y1 cells. J. Biol. Chem. 267, 13498-13504.
Mizutani, A., Watanabe, N., Kitao, T., Tokumitsu, H. and Hidaka, H. (1995). The long amino-terminal tail domain of annexin XI is necessary for its nuclear localization. Arch. Biochem. Biophys. 318, 157-165.[Medline]
Mohiti, J., Caswell, A. M. and Walker, J. H. (1997). The nuclear location of annexin V in the human osteosarcoma cell line MG-63 depends on serum factors and tyrosine kinase signaling pathways. Exp. Cell Res. 234, 98-104.[Medline]
Munz, B., Gerke, V., Gillitzer, R. and Werner, S. (1997). Differential expression of the calpactin I subunits annexin II and p11 in cultured keratinocytes and during wound repair. J. Invest. Dermatol 108, 307-312.[Abstract]
Nigg, E. A., Cooper, J. A. and Hunter, T. (1983). Immunofluorescent localization of a 39,000 dalton substrate of tyrosine protein kinases to the cytoplasmic surface of the plasma membrane. J. Cell Biol. 96, 1601-1609.[Abstract]
Oliferenko, S., Paiha, K., Harder, T., Gerke, V., Schwarzler, C., Schwarz, H., Beug, H., Gunthert, U. and Huber, L. A. (1999). Analysis of CD44-containing lipid rafts: recruitment of annexin II and stabilization by the actin cytoskeleton. J. Cell Biol. 146, 843-854.
Ossareh-Nazari, B., Bachelerie, F. and Dargemont, C. (1997). Evidence for a role of CRM1 in signal-mediated nuclear protein export. Science 278, 141-144.
Ozaki, T. and Sakiyama, S. (1993). Molecular cloning of rat calpactin I heavy-chain cDNA whose expression is induced in v-src-transformed rat culture cell lines. Oncogene 8, 1707-1710.[Medline]
Powell, M. A. and Glenney, J. R. (1987). Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src. Biochem. J. 247, 321-328.[Medline]
Puisieux, A., Ji, J. and Ozturk, M. (1996). Annexin II up-regulates cellular levels of p11 protein by a post-translational mechanism. Biochem. J. 313, 51-55.[Medline]
Radke, K. and Martin, G. S. (1979). Transformation by Rous sarcoma virus: effects of src gene expression on the synthesis and phosphorylation of cellular polypeptides. Proc. Natl. Acad. Sci. USA 76, 5212-5216.[Abstract]
Raynal, P. and Pollard, H. B. (1994). The problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim. Biophys. Acta 1197, 63-93.[Medline]
Raynal, P., van Bergen en Henegouwen, P. M., Hullin, F., Ragab-Thomas, J. M., Fauvel, J., Verkleij, A. and Chap, H.., (1992). Morphological and biochemical evidence for partial nuclear localization of annexin 1 in endothelial cells. Biochem. Biophys. Res. Commun. 186, 432-439.[Medline]
Reeves, S. A., Chavez-Kappel, C., Davis, R., Rosenblum, M. and Israel, M. A. (1992). Developmental regulation of annexin II (lipocortin 2) in human brain and expression in high grade glioma. Cancer Res. 52, 6871-6876.[Abstract]
Regnouf, F., Sagot, I., Delouche, B., Devilliers, G., Cartaud, J., Henry, J. P., and Pradel, L. A. (1995). In vitro phosphorylation of annexin 2 heterotetramer by protein kinase C. J. Biol. Chem. 270, 27143-27150.
Rety, S., Sopkova, J., Renouard, M., Osterloh, D., Gerke, V., Tabaries, S., Russo-Marie, F. and Lewit-Bentley, A. (1999). The crystal structure of a complex of p11 with the annexin II N-terminal peptide. Nat. Struct. Biol. 6, 89-95.[Medline]
Sagot, I., Regnouf, F., Henry, J.-P. and Pradel, L. A. (1997). Translocation of cytosolic annexin 2 to a Triton-insoluble membrane subdomain upon nicotinic stimulation of chromaffin cultured cells. FEBS Lett. 410, 229-234.[Medline]
Sladeczek, F., Camonis, J. H., Burnol, A. F. and LeBouffant, F. (1997). The cdk-like protein PCTAIRE-1 from mouse brain associates with p11 and 14-3-3 proteins. Mol. Gen. Genet. 254, 571-577.[Medline]
Thiel, C., Osborn, M. and Gerke, V. (1992). The tight association of the tyrosine kinase substrate annexin II with the submembraneous cytoskeleton depends on intact p11- and Ca2+-binding sites. J. Cell Sci. 103, 733-742.
Vedeler, A. and Hollas, H. (2000). Annexin II is associated with mRNAs which may constitute a distinct subpopulation. Biochem. J. 348, 565-572.[Medline]
Vishwanatha, J. K., Jindal, H. K. and Davis, R. G. (1992). The role of primer recognition proteins in DNA replication: association with nuclear matrix in HeLa cells. J. Cell Sci. 101, 25-34.[Abstract]
Vishwanatha, J. K., Chiang, Y., Kumble, K. D., Hollingsworth, M. A. and Pour, P. M. (1993). Enhanced expression of annexin II in human pancreatic carcinoma cells and primary pancreatic cancers. Carcinogenesis. 14, 2575-2579.[Abstract]
Vishwanatha, J. K. and Kumble, S. (1993). Involvement of annexin II in DNA replication: evidence from cell-free extracts of Xenopus eggs. J. Cell Sci. 105, 533-540.
Wada, A., Fukuda, M., Mishima, M. and Nishida, E. (1998). Nuclear export of actin: a novel mechanism regulating the subcellular localization of a major cytoskeletal protein. EMBO J. 17, 1635-1641.
Waisman, D. M. (1995). Annexin II tetramer: structure and function. Mol. Cell Biochem. 149-150, 301-322.
Wen, W., Meinkoth, J. L., Tsien, R. Y. and Taylor, S. S. (1995). Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463-473.[Medline]
Woodring, P. J. and Garrison, J. C. (1997). Expression, purification, and regulation of two forms of the inositol 1,4,5-trisphophate 3-kinase. J. Biol. Chem. 272, 30447-30454.
Wu, T., Angus, C. W., Yao, X. L., Logun, C. and Shelhamer, J. H. (1997). p11, a unique member of the S100 family of calcium-binding proteins, interacts with and inhibits the activity of the 85-kDa cytosolic phospholipase A2. J. Biol. Chem. 272, 17145-17153.
Zokas, L. and Glenney, J. R., Jr (1987). The calpactin light chain is tightly linked to the cytoskeletal form of calpactin I: studies using monoclonal antibodies to calpactin subunits. J. Cell Biol. 105, 2111-2121.[Abstract]
Zolotukhin, A. S. and Felber, B. K. (1997). Mutations in the nuclear export signal of human Ran-binding protein Ran BP1 block the Rev-mediated posttranscriptional regulation of human immunodeficiency virus type 1. J. Biol. Chem. 272, 11356-11360.