Abstract
Based on the finding that the expression of some annexins varies dramatically as a function of cellular proliferation state [Schlaepfer and Haigler (1990) J. Cell Biol. 111, 229-238], it has been proposed that the cellular level of the annexins might be critical for the regulation of cell growth. To further test this hypothesis, we have studied the expression of various annexins in normal human IMR-90 fibroblasts synchronized by serum deprivation. Using immunoblotting, the cellular content of annexins (Anxs) II, V and VI was found to vary by less than 10% during the cell cycle. However, Anx IV expression increased by 50% during S-phase and the levels of Anxs I and VII were reduced by 40% in early G2/M. However, using RNase protection assays, the mRNAs of Anxs I and VII were found to be uniformly expressed throughout the cell cycle, suggesting that down-regulation of both proteins in G2/M occurred through a post-transcriptional process. In addition, cells transfected with Anx VII cDNA were shown to contain an amount of Anx VII similar to wild-type cells, despite the elevation of Anx VII mRNA content in transfected cells by approx. 2 orders of magnitude. Vector misconstruction or possible secretion of the overexpressed protein were ruled out using appropriate controls. Therefore, as with cell-cycle regulation, Anx VII expression in transfected cells is also controlled by post-transcriptional mechanisms. Furthermore, using pulse-chase analysis, we have determined that annexin VII, and other Anxs, have a slow turnover rate, consistent with the limited changes of expression throughout the cell cycle. Taken together, these results question the hypothesis that cellular expression of Anxs plays a general role in cell growth and support the concept that post-transcriptional mechanisms may control levels of Anxs I and VII.
Full Text
The Full Text of this article is available as a PDF (485.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Burns A. L., Magendzo K., Srivastava M., Rojas E., Parra C., de la Fuente M., Cultraro C., Shirvan A., Vogel T., Heldman J. Human synexin (annexin VII) polymorphisms: tissue specificity and expression in Escherichia coli. Biochem Soc Trans. 1990 Dec;18(6):1118–1121. doi: 10.1042/bst0181118. [DOI] [PubMed] [Google Scholar]
- Cardenas A. M., Kuijpers G. A., Pollard H. B. Effect of protein synthesis inhibitors on synexin levels and secretory response in bovine adrenal medullary chromaffin cells. Biochim Biophys Acta. 1995 Mar 22;1234(2):255–260. doi: 10.1016/0005-2736(94)00283-u. [DOI] [PubMed] [Google Scholar]
- Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
- Chiang Y., Schneiderman M. H., Vishwanatha J. K. Annexin II expression is regulated during mammalian cell cycle. Cancer Res. 1993 Dec 15;53(24):6017–6021. [PubMed] [Google Scholar]
- Creutz C. E. The annexins and exocytosis. Science. 1992 Nov 6;258(5084):924–931. doi: 10.1126/science.1439804. [DOI] [PubMed] [Google Scholar]
- Hermouet S., Merendino J. J., Jr, Gutkind J. S., Spiegel A. M. Activating and inactivating mutations of the alpha subunit of Gi2 protein have opposite effects on proliferation of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10455–10459. doi: 10.1073/pnas.88.23.10455. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirai S., Kawasaki H., Yaniv M., Suzuki K. Degradation of transcription factors, c-Jun and c-Fos, by calpain. FEBS Lett. 1991 Aug 5;287(1-2):57–61. doi: 10.1016/0014-5793(91)80015-u. [DOI] [PubMed] [Google Scholar]
- Hullin F., Raynal P., Ragab-Thomas J. M., Fauvel J., Chap H. Effect of dexamethasone on prostaglandin synthesis and on lipocortin status in human endothelial cells. Inhibition of prostaglandin I2 synthesis occurring without alteration of arachidonic acid liberation and of lipocortin synthesis. J Biol Chem. 1989 Feb 25;264(6):3506–3513. [PubMed] [Google Scholar]
- Jindal H. K., Chaney W. G., Anderson C. W., Davis R. G., Vishwanatha J. K. The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha. J Biol Chem. 1991 Mar 15;266(8):5169–5176. [PubMed] [Google Scholar]
- Johnston P. A., Perin M. S., Reynolds G. A., Wasserman S. A., Südhof T. C. Two novel annexins from Drosophila melanogaster. Cloning, characterization, and differential expression in development. J Biol Chem. 1990 Jul 5;265(19):11382–11388. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lozano J. J., Silberstein G. B., Hwang S., Haindl A. H., Rocha V. Developmental regulation of calcium-binding proteins (calelectrins and calpactin I) in mammary glands. J Cell Physiol. 1989 Mar;138(3):503–510. doi: 10.1002/jcp.1041380309. [DOI] [PubMed] [Google Scholar]
- Pagano M., Pepperkok R., Lukas J., Baldin V., Ansorge W., Bartek J., Draetta G. Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts. J Cell Biol. 1993 Apr;121(1):101–111. doi: 10.1083/jcb.121.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Radke K., Gilmore T., Martin G. S. Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine. Cell. 1980 Oct;21(3):821–828. doi: 10.1016/0092-8674(80)90445-6. [DOI] [PubMed] [Google Scholar]
- Raynal P., Hullin F., Ragab-Thomas J. M., Fauvel J., Chap H. Annexin 5 as a potential regulator of annexin 1 phosphorylation by protein kinase C. In vitro inhibition compared with quantitative data on annexin distribution in human endothelial cells. Biochem J. 1993 Jun 15;292(Pt 3):759–765. doi: 10.1042/bj2920759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raynal P., Pollard H. B. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta. 1994 Apr 5;1197(1):63–93. doi: 10.1016/0304-4157(94)90019-1. [DOI] [PubMed] [Google Scholar]
- Reeves S. A., Chavez-Kappel C., Davis R., Rosenblum M., Israel M. A. Developmental regulation of annexin II (Lipocortin 2) in human brain and expression in high grade glioma. Cancer Res. 1992 Dec 15;52(24):6871–6876. [PubMed] [Google Scholar]
- Sawyer S. T., Cohen S. Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells. J Biol Chem. 1985 Jul 15;260(14):8233–8236. [PubMed] [Google Scholar]
- Schlaepfer D. D., Haigler H. T. Expression of annexins as a function of cellular growth state. J Cell Biol. 1990 Jul;111(1):229–238. doi: 10.1083/jcb.111.1.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Theobald J., Smith P. D., Jacob S. M., Moss S. E. Expression of annexin VI in A431 carcinoma cells suppresses proliferation: a possible role for annexin VI in cell growth regulation. Biochim Biophys Acta. 1994 Sep 29;1223(3):383–390. doi: 10.1016/0167-4889(94)90099-x. [DOI] [PubMed] [Google Scholar]
- Wice B. M., Gordon J. I. A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin. J Cell Biol. 1992 Jan;116(2):405–422. doi: 10.1083/jcb.116.2.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- William F., Mroczkowski B., Cohen S., Kraft A. S. Differentiation of HL-60 cells is associated with an increase in the 35-kDa protein lipocortin I. J Cell Physiol. 1988 Dec;137(3):402–410. doi: 10.1002/jcp.1041370303. [DOI] [PubMed] [Google Scholar]