Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 May;12(5):1923–1927. doi: 10.1002/j.1460-2075.1993.tb05841.x

Characterization of full-length neurofibromin: tubulin inhibits Ras GAP activity.

G Bollag 1, F McCormick 1, R Clark 1
PMCID: PMC413413  PMID: 8491185

Abstract

Full-length neurofibromin is a GTPase activating protein (GAP) for the Ras proto-oncogene product. Regulation of neurofibromin activity therefore has important implications for cell growth. Neurofibromin co-purifies with tubulin when expressed in insect cells. The interaction between neurofibromin and tubulin is sensitive to the microtubule depolymerizing agent colchicine. Neurofibromin GAP activity is inhibited even at low concentrations of tubulin. However, maximal inhibition of GAP activity is only approximately 70%, suggesting that the neurofibromin-tubulin complex retains residual GAP activity. This decreased activity is reflected by a 4-fold decrease in its affinity for Ras. A truncated mutant of neurofibromin with reduced sensitivity to tubulin localizes some tubulin-binding determinants to an 80 residue segment immediately N-terminal to the GAP-related domain. Since tubulin is an abundant protein in eukaryotic cells, the tubulin-neurofibromin interaction may regulate the Ras signalling pathway.

Full text

PDF
1923

Images in this article

1924Image
on p.1924

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amann E., Ochs B., Abel K. J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988 Sep 30;69(2):301–315. doi: 10.1016/0378-1119(88)90440-4. [DOI] [PubMed] [Google Scholar]
  2. Ballester R., Marchuk D., Boguski M., Saulino A., Letcher R., Wigler M., Collins F. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell. 1990 Nov 16;63(4):851–859. doi: 10.1016/0092-8674(90)90151-4. [DOI] [PubMed] [Google Scholar]
  3. Bar-Sagi D., Feramisco J. R. Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. Science. 1986 Sep 5;233(4768):1061–1068. doi: 10.1126/science.3090687. [DOI] [PubMed] [Google Scholar]
  4. Basu T. N., Gutmann D. H., Fletcher J. A., Glover T. W., Collins F. S., Downward J. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature. 1992 Apr 23;356(6371):713–715. doi: 10.1038/356713a0. [DOI] [PubMed] [Google Scholar]
  5. Bollag G., McCormick F. Differential regulation of rasGAP and neurofibromatosis gene product activities. Nature. 1991 Jun 13;351(6327):576–579. doi: 10.1038/351576a0. [DOI] [PubMed] [Google Scholar]
  6. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
  7. Carney D. H., Crossin K. L., Ball R., Fuller G. M., Albrecht T., Thompson W. C. Changes in the extent of microtubule assembly can regulate initiation of DNA synthesis. Ann N Y Acad Sci. 1986;466:919–932. doi: 10.1111/j.1749-6632.1986.tb38477.x. [DOI] [PubMed] [Google Scholar]
  8. Cawthon R. M., Weiss R., Xu G. F., Viskochil D., Culver M., Stevens J., Robertson M., Dunn D., Gesteland R., O'Connell P. A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations. Cell. 1990 Jul 13;62(1):193–201. doi: 10.1016/0092-8674(90)90253-b. [DOI] [PubMed] [Google Scholar]
  9. Collins C. A. Reversible assembly purification of taxol-treated microtubules. Methods Enzymol. 1991;196:246–253. doi: 10.1016/0076-6879(91)96023-k. [DOI] [PubMed] [Google Scholar]
  10. DeClue J. E., Papageorge A. G., Fletcher J. A., Diehl S. R., Ratner N., Vass W. C., Lowy D. R. Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell. 1992 Apr 17;69(2):265–273. doi: 10.1016/0092-8674(92)90407-4. [DOI] [PubMed] [Google Scholar]
  11. Gelfand V. I., Bershadsky A. D. Microtubule dynamics: mechanism, regulation, and function. Annu Rev Cell Biol. 1991;7:93–116. doi: 10.1146/annurev.cb.07.110191.000521. [DOI] [PubMed] [Google Scholar]
  12. Grussenmeyer T., Scheidtmann K. H., Hutchinson M. A., Eckhart W., Walter G. Complexes of polyoma virus medium T antigen and cellular proteins. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7952–7954. doi: 10.1073/pnas.82.23.7952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Marchuk D. A., Saulino A. M., Tavakkol R., Swaroop M., Wallace M. R., Andersen L. B., Mitchell A. L., Gutmann D. H., Boguski M., Collins F. S. cDNA cloning of the type 1 neurofibromatosis gene: complete sequence of the NF1 gene product. Genomics. 1991 Dec;11(4):931–940. doi: 10.1016/0888-7543(91)90017-9. [DOI] [PubMed] [Google Scholar]
  14. Martin G. A., Viskochil D., Bollag G., McCabe P. C., Crosier W. J., Haubruck H., Conroy L., Clark R., O'Connell P., Cawthon R. M. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. doi: 10.1016/0092-8674(90)90150-d. [DOI] [PubMed] [Google Scholar]
  15. Munemitsu S., Innis M. A., Clark R., McCormick F., Ullrich A., Polakis P. Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42. Mol Cell Biol. 1990 Nov;10(11):5977–5982. doi: 10.1128/mcb.10.11.5977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Myrdal S. E., Auersperg N. p21ras. Heterogeneous localization in transformed cells. Exp Cell Res. 1985 Aug;159(2):441–450. doi: 10.1016/s0014-4827(85)80017-3. [DOI] [PubMed] [Google Scholar]
  17. Rubinfeld B., Munemitsu S., Clark R., Conroy L., Watt K., Crosier W. J., McCormick F., Polakis P. Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1. Cell. 1991 Jun 14;65(6):1033–1042. doi: 10.1016/0092-8674(91)90555-d. [DOI] [PubMed] [Google Scholar]
  18. Shacter E. Organic extraction of Pi with isobutanol/toluene. Anal Biochem. 1984 May 1;138(2):416–420. doi: 10.1016/0003-2697(84)90831-5. [DOI] [PubMed] [Google Scholar]
  19. Smith G. E., Summers M. D., Fraser M. J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1983 Dec;3(12):2156–2165. doi: 10.1128/mcb.3.12.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tsai M. H., Yu C. L., Wei F. S., Stacey D. W. The effect of GTPase activating protein upon ras is inhibited by mitogenically responsive lipids. Science. 1989 Jan 27;243(4890):522–526. doi: 10.1126/science.2536192. [DOI] [PubMed] [Google Scholar]
  21. Vallee R. B. Purification of brain microtubules and microtubule-associated protein 1 using taxol. Methods Enzymol. 1986;134:104–115. doi: 10.1016/0076-6879(86)34079-5. [DOI] [PubMed] [Google Scholar]
  22. Vallee R. B. Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments. Methods Enzymol. 1986;134:89–104. doi: 10.1016/0076-6879(86)34078-3. [DOI] [PubMed] [Google Scholar]
  23. Wallace M. R., Marchuk D. A., Andersen L. B., Letcher R., Odeh H. M., Saulino A. M., Fountain J. W., Brereton A., Nicholson J., Mitchell A. L. Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science. 1990 Jul 13;249(4965):181–186. doi: 10.1126/science.2134734. [DOI] [PubMed] [Google Scholar]
  24. Williams R. C., Jr, Detrich H. W., 3rd Separation of tubulin from microtubule-associated proteins on phosphocellulose. Accompanying alterations in concentrations of buffer components. Biochemistry. 1979 Jun 12;18(12):2499–2503. doi: 10.1021/bi00579a010. [DOI] [PubMed] [Google Scholar]
  25. Xu G. F., Lin B., Tanaka K., Dunn D., Wood D., Gesteland R., White R., Weiss R., Tamanoi F. The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell. 1990 Nov 16;63(4):835–841. doi: 10.1016/0092-8674(90)90149-9. [DOI] [PubMed] [Google Scholar]
  26. Xu G. F., O'Connell P., Viskochil D., Cawthon R., Robertson M., Culver M., Dunn D., Stevens J., Gesteland R., White R. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 1990 Aug 10;62(3):599–608. doi: 10.1016/0092-8674(90)90024-9. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES