Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Nov 15;88(22):9914–9918. doi: 10.1073/pnas.88.22.9914

Identification and characterization of the neurofibromatosis type 1 protein product.

J E DeClue 1, B D Cohen 1, D R Lowy 1
PMCID: PMC52837  PMID: 1946460

Abstract

The neurofibromatosis type 1 (NF1) gene responsible for von Recklinghausen neurofibromatosis is related to regulators of ras proteins, and a portion of NF1 that is homologous to the ras GTPase-activating protein (GAP) encodes a similar GTPase-stimulating activity. We have raised rabbit antisera to a bacterially synthesized 48-kDa peptide corresponding to the GAP-related domain of NF1 (NF1-GRD). These antisera immunoprecipitated the NF1-GRD peptide, and one of them specifically inhibited the GTPase-stimulating activity of NF1-GRD. The sera specifically detected a 280-kDa protein in lysates of mouse NIH 3T3 and human HeLa cells. This protein corresponds to the NF1 gene product, as shown by several criteria, including partial proteolysis. Subcellular fractionation revealed that while GAP is predominantly cytoplasmic, all of the NF1 was recovered in a pellet (100,000 x g) fraction. NF1 was present in a large molecular mass complex in fibroblast and Schwannoma cell lines and appears to associate with a very large (400-500 kDa) protein in both cell types. The relevance of these findings to cellular regulation of p21ras is discussed.

Full text

PDF
9915

Images in this article

9916Image
on p.9916
9916Image
on p.9916
9916Image
on p.9916
9916Image
on p.9916
9916Image
on p.9916
9917Image
on p.9917
9917Image
on p.9917
9917Image
on p.9917

Selected References

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

  1. 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]
  2. Ballester R., Michaeli T., Ferguson K., Xu H. P., McCormick F., Wigler M. Genetic analysis of mammalian GAP expressed in yeast. Cell. 1989 Nov 17;59(4):681–686. doi: 10.1016/0092-8674(89)90014-7. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  5. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  6. Buchberg A. M., Cleveland L. S., Jenkins N. A., Copeland N. G. Sequence homology shared by neurofibromatosis type-1 gene and IRA-1 and IRA-2 negative regulators of the RAS cyclic AMP pathway. Nature. 1990 Sep 20;347(6290):291–294. doi: 10.1038/347291a0. [DOI] [PubMed] [Google Scholar]
  7. DeClue J. E., Stone J. C., Blanchard R. A., Papageorge A. G., Martin P., Zhang K., Lowy D. R. A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1. Mol Cell Biol. 1991 Jun;11(6):3132–3138. doi: 10.1128/mcb.11.6.3132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Downward J., Riehl R., Wu L., Weinberg R. A. Identification of a nucleotide exchange-promoting activity for p21ras. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5998–6002. doi: 10.1073/pnas.87.15.5998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Downward J. The ras superfamily of small GTP-binding proteins. Trends Biochem Sci. 1990 Dec;15(12):469–472. doi: 10.1016/0968-0004(90)90300-z. [DOI] [PubMed] [Google Scholar]
  10. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  11. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Field J., Broek D., Kataoka T., Wigler M. Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jun;7(6):2128–2133. doi: 10.1128/mcb.7.6.2128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Glomset J. A., Gelb M. H., Farnsworth C. C. Prenyl proteins in eukaryotic cells: a new type of membrane anchor. Trends Biochem Sci. 1990 Apr;15(4):139–142. doi: 10.1016/0968-0004(90)90213-u. [DOI] [PubMed] [Google Scholar]
  14. Han J. W., McCormick F., Macara I. G. Regulation of Ras-GAP and the neurofibromatosis-1 gene product by eicosanoids. Science. 1991 Apr 26;252(5005):576–579. doi: 10.1126/science.1902323. [DOI] [PubMed] [Google Scholar]
  15. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  16. Huang Y. K., Kung H. F., Kamata T. Purification of a factor capable of stimulating the guanine nucleotide exchange reaction of ras proteins and its effect on ras-related small molecular mass G proteins. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8008–8012. doi: 10.1073/pnas.87.20.8008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marshall C. J. How does p21ras transform cells? Trends Genet. 1991 Mar;7(3):91–95. doi: 10.1016/0168-9525(91)90278-X. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Mitts M. R., Bradshaw-Rouse J., Heideman W. Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1. Mol Cell Biol. 1991 Sep;11(9):4591–4598. doi: 10.1128/mcb.11.9.4591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  21. Rine J., Kim S. H. A role for isoprenoid lipids in the localization and function of an oncoprotein. New Biol. 1990 Mar;2(3):219–226. [PubMed] [Google Scholar]
  22. Satoh T., Endo M., Nakafuku M., Nakamura S., Kaziro Y. Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5993–5997. doi: 10.1073/pnas.87.15.5993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Satoh T., Nakamura S., Kaziro Y. Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5'-O-(3-thiotriphosphate). Mol Cell Biol. 1987 Dec;7(12):4553–4556. doi: 10.1128/mcb.7.12.4553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sheela S., Riccardi V. M., Ratner N. Angiogenic and invasive properties of neurofibroma Schwann cells. J Cell Biol. 1990 Aug;111(2):645–653. doi: 10.1083/jcb.111.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tanaka K., Lin B. K., Wood D. R., Tamanoi F. IRA2, an upstream negative regulator of RAS in yeast, is a RAS GTPase-activating protein. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):468–472. doi: 10.1073/pnas.88.2.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tanaka K., Matsumoto K., Toh-E A. IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):757–768. doi: 10.1128/mcb.9.2.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tanaka K., Nakafuku M., Satoh T., Marshall M. S., Gibbs J. B., Matsumoto K., Kaziro Y., Toh-e A. S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. 1990 Mar 9;60(5):803–807. doi: 10.1016/0092-8674(90)90094-u. [DOI] [PubMed] [Google Scholar]
  28. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  29. Viskochil D., Buchberg A. M., Xu G., Cawthon R. M., Stevens J., Wolff R. K., Culver M., Carey J. C., Copeland N. G., Jenkins N. A. Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell. 1990 Jul 13;62(1):187–192. doi: 10.1016/0092-8674(90)90252-a. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Willingham M. C., Pastan I., Shih T. Y., Scolnick E. M. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1980 Apr;19(4):1005–1014. doi: 10.1016/0092-8674(80)90091-4. [DOI] [PubMed] [Google Scholar]
  32. Wolfman A., Macara I. G. A cytosolic protein catalyzes the release of GDP from p21ras. Science. 1990 Apr 6;248(4951):67–69. doi: 10.1126/science.2181667. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Zhang K., DeClue J. E., Vass W. C., Papageorge A. G., McCormick F., Lowy D. R. Suppression of c-ras transformation by GTPase-activating protein. Nature. 1990 Aug 23;346(6286):754–756. doi: 10.1038/346754a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES