Abstract
In this report we analyze the protein product of a growth arrest- specific gene, gas2, by means of an affinity-purified antibody raised against the protein produced in bacteria. The regulation of Gas2 biosynthesis reflects the pattern of mRNA expression (Schneider, C., R. King, and L. Philipson. 1988. Cell. 54:787-793): its relative level is tightly associated with growth arrest. Gas2 seems to be regulated also at the posttranslational level via a phosphorylation mechanism. Gas2 is well conserved during the evolution with the same apparent molecular mass (36 kD) between mouse and human. We also demonstrate that Gas2 is a component of the microfilament system. It colocalizes with actin fiber, at the cell border and also along the stress fiber, in growth- arrested NIH 3T3 cells. The pattern of distribution, detected in arrested cells, can also be observed in growing cells when they are microinjected with the purified GST-Gas2 protein. In none of the analyzed oncogene-transformed NIH 3T3 cell lines was Gas2 expression induced under serum starvation.
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- Boschek C. B., Jockusch B. M., Friis R. R., Back R., Grundmann E., Bauer H. Early changes in the distribution and organization of microfilament proteins during cell transformation. Cell. 1981 Apr;24(1):175–184. doi: 10.1016/0092-8674(81)90513-4. [DOI] [PubMed] [Google Scholar]
- Brancolini C., Schneider C. Change in the expression of a nuclear matrix-associated protein is correlated with cellular transformation. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6936–6940. doi: 10.1073/pnas.88.16.6936. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bretscher A., Weber K. Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures. J Cell Biol. 1980 Jul;86(1):335–340. doi: 10.1083/jcb.86.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
- Field J., Vojtek A., Ballester R., Bolger G., Colicelli J., Ferguson K., Gerst J., Kataoka T., Michaeli T., Powers S. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell. 1990 Apr 20;61(2):319–327. doi: 10.1016/0092-8674(90)90812-s. [DOI] [PubMed] [Google Scholar]
- Franck Z., Footer M., Bretscher A. Microinjection of villin into cultured cells induces rapid and long-lasting changes in cell morphology but does not inhibit cytokinesis, cell motility, or membrane ruffling. J Cell Biol. 1990 Dec;111(6 Pt 1):2475–2485. doi: 10.1083/jcb.111.6.2475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Friederich E., Huet C., Arpin M., Louvard D. Villin induces microvilli growth and actin redistribution in transfected fibroblasts. Cell. 1989 Nov 3;59(3):461–475. doi: 10.1016/0092-8674(89)90030-5. [DOI] [PubMed] [Google Scholar]
- Friederich E., Pringault E., Arpin M., Louvard D. From the structure to the function of villin, an actin-binding protein of the brush border. Bioessays. 1990 Sep;12(9):403–408. doi: 10.1002/bies.950120902. [DOI] [PubMed] [Google Scholar]
- Geiger B., Tokuyasu K. T., Dutton A. H., Singer S. J. Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4127–4131. doi: 10.1073/pnas.77.7.4127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gerst J. E., Ferguson K., Vojtek A., Wigler M., Field J. CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex. Mol Cell Biol. 1991 Mar;11(3):1248–1257. doi: 10.1128/mcb.11.3.1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldschmidt-Clermont P. J., Janmey P. A. Profilin, a weak CAP for actin and RAS. Cell. 1991 Aug 9;66(3):419–421. doi: 10.1016/0092-8674(81)90002-7. [DOI] [PubMed] [Google Scholar]
- Goldschmidt-Clermont P. J., Kim J. W., Machesky L. M., Rhee S. G., Pollard T. D. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation. Science. 1991 Mar 8;251(4998):1231–1233. doi: 10.1126/science.1848725. [DOI] [PubMed] [Google Scholar]
- Hartwig J. H., Chambers K. A., Stossel T. P. Association of gelsolin with actin filaments and cell membranes of macrophages and platelets. J Cell Biol. 1989 Feb;108(2):467–479. doi: 10.1083/jcb.108.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hartwig J. H., Kwiatkowski D. J. Actin-binding proteins. Curr Opin Cell Biol. 1991 Feb;3(1):87–97. doi: 10.1016/0955-0674(91)90170-4. [DOI] [PubMed] [Google Scholar]
- Kitamura K., Suzuki M., Uyemura K. Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane. Biochim Biophys Acta. 1976 Dec 14;455(3):806–816. doi: 10.1016/0005-2736(76)90050-x. [DOI] [PubMed] [Google Scholar]
- Land H., Parada L. F., Weinberg R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983 Aug 18;304(5927):596–602. doi: 10.1038/304596a0. [DOI] [PubMed] [Google Scholar]
- Lassing I., Lindberg U. Specific interaction between phosphatidylinositol 4,5-bisphosphate and profilactin. Nature. 1985 Apr 4;314(6010):472–474. doi: 10.1038/314472a0. [DOI] [PubMed] [Google Scholar]
- Machesky L. M., Goldschmidt-Clermont P. J., Pollard T. D. The affinities of human platelet and Acanthamoeba profilin isoforms for polyphosphoinositides account for their relative abilities to inhibit phospholipase C. Cell Regul. 1990 Nov;1(12):937–950. doi: 10.1091/mbc.1.12.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manfioletti G., Ruaro M. E., Del Sal G., Philipson L., Schneider C. A growth arrest-specific (gas) gene codes for a membrane protein. Mol Cell Biol. 1990 Jun;10(6):2924–2930. doi: 10.1128/mcb.10.6.2924. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marston F. A. The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J. 1986 Nov 15;240(1):1–12. doi: 10.1042/bj2400001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsudaira P., Janmey P. Pieces in the actin-severing protein puzzle. Cell. 1988 Jul 15;54(2):139–140. doi: 10.1016/0092-8674(88)90542-9. [DOI] [PubMed] [Google Scholar]
- Matsumura F., Lin J. J., Yamashiro-Matsumura S., Thomas G. P., Topp W. C. Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells. J Biol Chem. 1983 Nov 25;258(22):13954–13964. [PubMed] [Google Scholar]
- Okabe S., Hirokawa N. Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study. J Cell Biol. 1989 Oct;109(4 Pt 1):1581–1595. doi: 10.1083/jcb.109.4.1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pavalko F. M., Burridge K. Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin. J Cell Biol. 1991 Aug;114(3):481–491. doi: 10.1083/jcb.114.3.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pepperkok R., Zanetti M., King R., Delia D., Ansorge W., Philipson L., Schneider C. Automatic microinjection system facilitates detection of growth inhibitory mRNA. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6748–6752. doi: 10.1073/pnas.85.18.6748. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pollack R., Osborn M., Weber K. Patterns of organization of actin and myosin in normal and transformed cultured cells. Proc Natl Acad Sci U S A. 1975 Mar;72(3):994–998. doi: 10.1073/pnas.72.3.994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schedl T., Burland T. G., Gull K., Dove W. F. Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum. J Cell Biol. 1984 Jul;99(1 Pt 1):155–165. doi: 10.1083/jcb.99.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schneider C., King R. M., Philipson L. Genes specifically expressed at growth arrest of mammalian cells. Cell. 1988 Sep 9;54(6):787–793. doi: 10.1016/s0092-8674(88)91065-3. [DOI] [PubMed] [Google Scholar]
- Shapland C., Lowings P., Lawson D. Identification of new actin-associated polypeptides that are modified by viral transformation and changes in cell shape. J Cell Biol. 1988 Jul;107(1):153–161. doi: 10.1083/jcb.107.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
- Stossel T. P., Chaponnier C., Ezzell R. M., Hartwig J. H., Janmey P. A., Kwiatkowski D. J., Lind S. E., Smith D. B., Southwick F. S., Yin H. L. Nonmuscle actin-binding proteins. Annu Rev Cell Biol. 1985;1:353–402. doi: 10.1146/annurev.cb.01.110185.002033. [DOI] [PubMed] [Google Scholar]
- Stossel T. P. From signal to pseudopod. How cells control cytoplasmic actin assembly. J Biol Chem. 1989 Nov 5;264(31):18261–18264. [PubMed] [Google Scholar]
- Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
- Tellam R. L., Morton D. J., Clarke F. M. A common theme in the amino acid sequences of actin and many actin-binding proteins? Trends Biochem Sci. 1989 Apr;14(4):130–133. doi: 10.1016/0968-0004(89)90142-4. [DOI] [PubMed] [Google Scholar]
- Vandekerckhove J., Bauw G., Vancompernolle K., Honoré B., Celis J. Comparative two-dimensional gel analysis and microsequencing identifies gelsolin as one of the most prominent downregulated markers of transformed human fibroblast and epithelial cells. J Cell Biol. 1990 Jul;111(1):95–102. doi: 10.1083/jcb.111.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vojtek A., Haarer B., Field J., Gerst J., Pollard T. D., Brown S., Wigler M. Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae. Cell. 1991 Aug 9;66(3):497–505. doi: 10.1016/0092-8674(81)90013-1. [DOI] [PubMed] [Google Scholar]
- Weber K., Groeschel-Stewart U. Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4561–4564. doi: 10.1073/pnas.71.11.4561. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wehland J., Osborn M., Weber K. Cell-to-substratum contacts in living cells: a direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and alpha-actinin. J Cell Sci. 1979 Jun;37:257–273. doi: 10.1242/jcs.37.1.257. [DOI] [PubMed] [Google Scholar]
- Welcher A. A., Suter U., De Leon M., Snipes G. J., Shooter E. M. A myelin protein is encoded by the homologue of a growth arrest-specific gene. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7195–7199. doi: 10.1073/pnas.88.16.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]