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. 1991 Apr 1;113(1):13–23. doi: 10.1083/jcb.113.1.13

The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2

PMCID: PMC2288919  PMID: 2007618

Abstract

Recent evidence suggests that the conserved COOH-terminal CaaX motif of nuclear lamins may play a role in targeting newly synthesized proteins to the nuclear envelope. We have shown previously that in rabbit reticulocyte lysates the cysteine residue of the CaaX motif of chicken lamin B2 is necessary for incorporation of a derivative of mevalonic acid, the precursor of isoprenoids. Here we have analyzed the properties of normal and mutated forms of chicken lamin B2 stably expressed in mouse L cells. Mutation of the cysteine residue of the CaaX motif to alanine or introduction of a stop codon immediately after the cysteine residue was found to abolish both isoprenylation and carboxyl methylation of transfected lamin B2. Concomitantly, although nuclear import of the mutant lamin B2 proteins was preserved, their association with the inner nuclear membrane was severely impaired. From these results we conclude that the COOH-terminal CaaX motif is required for isoprenylation and carboxyl methylation of lamins in vivo, and that these modifications are important for association of B-type lamins with the nucleoplasmic surface of the inner nuclear membrane.

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Selected References

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  1. Aebi U., Cohn J., Buhle L., Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature. 1986 Oct 9;323(6088):560–564. doi: 10.1038/323560a0. [DOI] [PubMed] [Google Scholar]
  2. Beck L. A., Hosick T. J., Sinensky M. Incorporation of a product of mevalonic acid metabolism into proteins of Chinese hamster ovary cell nuclei. J Cell Biol. 1988 Oct;107(4):1307–1316. doi: 10.1083/jcb.107.4.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beck L. A., Hosick T. J., Sinensky M. Isoprenylation is required for the processing of the lamin A precursor. J Cell Biol. 1990 May;110(5):1489–1499. doi: 10.1083/jcb.110.5.1489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benavente R., Krohne G., Franke W. W. Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis. Cell. 1985 May;41(1):177–190. doi: 10.1016/0092-8674(85)90072-8. [DOI] [PubMed] [Google Scholar]
  5. Benavente R., Krohne G. Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies. J Cell Biol. 1986 Nov;103(5):1847–1854. doi: 10.1083/jcb.103.5.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borer R. A., Lehner C. F., Eppenberger H. M., Nigg E. A. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell. 1989 Feb 10;56(3):379–390. doi: 10.1016/0092-8674(89)90241-9. [DOI] [PubMed] [Google Scholar]
  7. Burke B., Gerace L. A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell. 1986 Feb 28;44(4):639–652. doi: 10.1016/0092-8674(86)90273-4. [DOI] [PubMed] [Google Scholar]
  8. Burke B. The nuclear envelope and nuclear transport. Curr Opin Cell Biol. 1990 Jun;2(3):514–520. doi: 10.1016/0955-0674(90)90136-3. [DOI] [PubMed] [Google Scholar]
  9. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chelsky D., Gutterson N. I., Koshland D. E., Jr A diffusion assay for detection and quantitation of methyl-esterified proteins on polyacrylamide gels. Anal Biochem. 1984 Aug 15;141(1):143–148. doi: 10.1016/0003-2697(84)90437-8. [DOI] [PubMed] [Google Scholar]
  11. Chelsky D., Olson J. F., Koshland D. E., Jr Cell cycle-dependent methyl esterification of lamin B. J Biol Chem. 1987 Mar 25;262(9):4303–4309. [PubMed] [Google Scholar]
  12. Chelsky D., Sobotka C., O'Neill C. L. Lamin B methylation and assembly into the nuclear envelope. J Biol Chem. 1989 May 5;264(13):7637–7643. [PubMed] [Google Scholar]
  13. Clarke S., Vogel J. P., Deschenes R. J., Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4643–4647. doi: 10.1073/pnas.85.13.4643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dagenais A., Bibor-Hardy V., Laliberté J. F., Royal A., Simard R. Detection in BHK cells of a precursor form for lamin A. Exp Cell Res. 1985 Dec;161(2):269–276. doi: 10.1016/0014-4827(85)90084-9. [DOI] [PubMed] [Google Scholar]
  15. Deschenes R. J., Stimmel J. B., Clarke S., Stock J., Broach J. R. RAS2 protein of Saccharomyces cerevisiae is methyl-esterified at its carboxyl terminus. J Biol Chem. 1989 Jul 15;264(20):11865–11873. [PubMed] [Google Scholar]
  16. Dessev G. N., Iovcheva-Dessev C., Goldman R. D. Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown. J Biol Chem. 1990 Jul 25;265(21):12636–12641. [PubMed] [Google Scholar]
  17. Enquist L. W., Vande Woude G. F., Wagner M., Smiley J. R., Summers W. C. Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus. Gene. 1979 Nov;7(3-4):335–342. doi: 10.1016/0378-1119(79)90052-0. [DOI] [PubMed] [Google Scholar]
  18. Farnsworth C. C., Wolda S. L., Gelb M. H., Glomset J. A. Human lamin B contains a farnesylated cysteine residue. J Biol Chem. 1989 Dec 5;264(34):20422–20429. [PMC free article] [PubMed] [Google Scholar]
  19. Fisher D. Z., Chaudhary N., Blobel G. cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6450–6454. doi: 10.1073/pnas.83.17.6450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Franke W. W. Nuclear lamins and cytoplasmic intermediate filament proteins: a growing multigene family. Cell. 1987 Jan 16;48(1):3–4. doi: 10.1016/0092-8674(87)90345-x. [DOI] [PubMed] [Google Scholar]
  21. Gerace L., Blobel G. The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell. 1980 Jan;19(1):277–287. doi: 10.1016/0092-8674(80)90409-2. [DOI] [PubMed] [Google Scholar]
  22. Gerace L., Burke B. Functional organization of the nuclear envelope. Annu Rev Cell Biol. 1988;4:335–374. doi: 10.1146/annurev.cb.04.110188.002003. [DOI] [PubMed] [Google Scholar]
  23. Gerace L., Comeau C., Benson M. Organization and modulation of nuclear lamina structure. J Cell Sci Suppl. 1984;1:137–160. doi: 10.1242/jcs.1984.supplement_1.10. [DOI] [PubMed] [Google Scholar]
  24. Glass J. R., Gerace L. Lamins A and C bind and assemble at the surface of mitotic chromosomes. J Cell Biol. 1990 Sep;111(3):1047–1057. doi: 10.1083/jcb.111.3.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  29. Havre P. A., Evans D. R. Disassembly and characterization of the nuclear pore complex-lamina fraction from bovine liver nuclei. Biochemistry. 1983 Jun 7;22(12):2852–2860. doi: 10.1021/bi00281a013. [DOI] [PubMed] [Google Scholar]
  30. Heald R., McKeon F. Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Cell. 1990 May 18;61(4):579–589. doi: 10.1016/0092-8674(90)90470-y. [DOI] [PubMed] [Google Scholar]
  31. Holtz D., Tanaka R. A., Hartwig J., McKeon F. The CaaX motif of lamin A functions in conjunction with the nuclear localization signal to target assembly to the nuclear envelope. Cell. 1989 Dec 22;59(6):969–977. doi: 10.1016/0092-8674(89)90753-8. [DOI] [PubMed] [Google Scholar]
  32. Höger T. H., Krohne G., Franke W. W. Amino acid sequence and molecular characterization of murine lamin B as deduced from cDNA clones. Eur J Cell Biol. 1988 Dec;47(2):283–290. [PubMed] [Google Scholar]
  33. Höger T. H., Zatloukal K., Waizenegger I., Krohne G. Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin. Chromosoma. 1990 Oct;99(6):379–390. doi: 10.1007/BF01726689. [DOI] [PubMed] [Google Scholar]
  34. Jackson J. H., Cochrane C. G., Bourne J. R., Solski P. A., Buss J. E., Der C. J. Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3042–3046. doi: 10.1073/pnas.87.8.3042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kita T., Brown M. S., Goldstein J. L. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase. J Clin Invest. 1980 Nov;66(5):1094–1100. doi: 10.1172/JCI109938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Krohne G., Waizenegger I., Höger T. H. The conserved carboxy-terminal cysteine of nuclear lamins is essential for lamin association with the nuclear envelope. J Cell Biol. 1989 Nov;109(5):2003–2011. doi: 10.1083/jcb.109.5.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Krohne G., Wolin S. L., McKeon F. D., Franke W. W., Kirschner M. W. Nuclear lamin LI of Xenopus laevis: cDNA cloning, amino acid sequence and binding specificity of a member of the lamin B subfamily. EMBO J. 1987 Dec 1;6(12):3801–3808. doi: 10.1002/j.1460-2075.1987.tb02716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  39. Laliberté J. F., Dagenais A., Filion M., Bibor-Hardy V., Simard R., Royal A. Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A. J Cell Biol. 1984 Mar;98(3):980–985. doi: 10.1083/jcb.98.3.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Langlois A. J., Lapis K., Ishizaki R., Beard J. W., Bolognesi D. P. Isolation of a transplantable cell line induced by the MC29 avian leukosis virus. Cancer Res. 1974 Jun;34(6):1457–1464. [PubMed] [Google Scholar]
  41. Lehner C. F., Fürstenberger G., Eppenberger H. M., Nigg E. A. Biogenesis of the nuclear lamina: in vivo synthesis and processing of nuclear protein precursors. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2096–2099. doi: 10.1073/pnas.83.7.2096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lehner C. F., Kurer V., Eppenberger H. M., Nigg E. A. The nuclear lamin protein family in higher vertebrates. Identification of quantitatively minor lamin proteins by monoclonal antibodies. J Biol Chem. 1986 Oct 5;261(28):13293–13301. [PubMed] [Google Scholar]
  43. Loewinger L., McKeon F. Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm. EMBO J. 1988 Aug;7(8):2301–2309. doi: 10.1002/j.1460-2075.1988.tb03073.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Magee T., Hanley M. Protein modification. Sticky fingers and CAAX boxes. Nature. 1988 Sep 8;335(6186):114–115. doi: 10.1038/335114a0. [DOI] [PubMed] [Google Scholar]
  45. McKeon F. D., Kirschner M. W., Caput D. Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature. 1986 Feb 6;319(6053):463–468. doi: 10.1038/319463a0. [DOI] [PubMed] [Google Scholar]
  46. Moir R. D., Quinlan R. A., Stewart M. Expression and characterization of human lamin C. FEBS Lett. 1990 Jul 30;268(1):301–305. doi: 10.1016/0014-5793(90)81032-j. [DOI] [PubMed] [Google Scholar]
  47. Nakagawa J., Kitten G. T., Nigg E. A. A somatic cell-derived system for studying both early and late mitotic events in vitro. J Cell Sci. 1989 Nov;94(Pt 3):449–462. doi: 10.1242/jcs.94.3.449. [DOI] [PubMed] [Google Scholar]
  48. Ngai J., Coleman T. R., Lazarides E. Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly. Cell. 1990 Feb 9;60(3):415–427. doi: 10.1016/0092-8674(90)90593-4. [DOI] [PubMed] [Google Scholar]
  49. Nigg E. A. Nuclear function and organization: the potential of immunochemical approaches. Int Rev Cytol. 1988;110:27–92. doi: 10.1016/s0074-7696(08)61847-1. [DOI] [PubMed] [Google Scholar]
  50. Nigg E. A. The nuclear envelope. Curr Opin Cell Biol. 1989 Jun;1(3):435–440. doi: 10.1016/0955-0674(89)90002-1. [DOI] [PubMed] [Google Scholar]
  51. Owen D., Kühn L. C. Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron. EMBO J. 1987 May;6(5):1287–1293. doi: 10.1002/j.1460-2075.1987.tb02366.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Padan R., Nainudel-Epszteyn S., Goitein R., Fainsod A., Gruenbaum Y. Isolation and characterization of the Drosophila nuclear envelope otefin cDNA. J Biol Chem. 1990 May 15;265(14):7808–7813. [PubMed] [Google Scholar]
  53. Peter M., Kitten G. T., Lehner C. F., Vorburger K., Bailer S. M., Maridor G., Nigg E. A. Cloning and sequencing of cDNA clones encoding chicken lamins A and B1 and comparison of the primary structures of vertebrate A- and B-type lamins. J Mol Biol. 1989 Aug 5;208(3):393–404. doi: 10.1016/0022-2836(89)90504-4. [DOI] [PubMed] [Google Scholar]
  54. Peter M., Nakagawa J., Dorée M., Labbé J. C., Nigg E. A. In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell. 1990 May 18;61(4):591–602. doi: 10.1016/0092-8674(90)90471-p. [DOI] [PubMed] [Google Scholar]
  55. Pollard K. M., Chan E. K., Grant B. J., Sullivan K. F., Tan E. M., Glass C. A. In vitro posttranslational modification of lamin B cloned from a human T-cell line. Mol Cell Biol. 1990 May;10(5):2164–2175. doi: 10.1128/mcb.10.5.2164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Reiss Y., Goldstein J. L., Seabra M. C., Casey P. J., Brown M. S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides. Cell. 1990 Jul 13;62(1):81–88. doi: 10.1016/0092-8674(90)90242-7. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Schafer W. R., Kim R., Sterne R., Thorner J., Kim S. H., Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science. 1989 Jul 28;245(4916):379–385. doi: 10.1126/science.2569235. [DOI] [PubMed] [Google Scholar]
  59. Senior A., Gerace L. Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina. J Cell Biol. 1988 Dec;107(6 Pt 1):2029–2036. doi: 10.1083/jcb.107.6.2029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Stephenson R. C., Clarke S. Identification of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate. J Biol Chem. 1990 Sep 25;265(27):16248–16254. [PubMed] [Google Scholar]
  61. Stewart M. Intermediate filaments: structure, assembly and molecular interactions. Curr Opin Cell Biol. 1990 Feb;2(1):91–100. doi: 10.1016/s0955-0674(05)80037-7. [DOI] [PubMed] [Google Scholar]
  62. Stick R., Angres B., Lehner C. F., Nigg E. A. The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum. J Cell Biol. 1988 Aug;107(2):397–406. doi: 10.1083/jcb.107.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Vorburger K., Kitten G. T., Nigg E. A. Modification of nuclear lamin proteins by a mevalonic acid derivative occurs in reticulocyte lysates and requires the cysteine residue of the C-terminal CXXM motif. EMBO J. 1989 Dec 20;8(13):4007–4013. doi: 10.1002/j.1460-2075.1989.tb08583.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Vorburger K., Lehner C. F., Kitten G. T., Eppenberger H. M., Nigg E. A. A second higher vertebrate B-type lamin. cDNA sequence determination and in vitro processing of chicken lamin B2. J Mol Biol. 1989 Aug 5;208(3):405–415. doi: 10.1016/0022-2836(89)90505-6. [DOI] [PubMed] [Google Scholar]
  65. Ward G. E., Kirschner M. W. Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C. Cell. 1990 May 18;61(4):561–577. doi: 10.1016/0092-8674(90)90469-u. [DOI] [PubMed] [Google Scholar]
  66. Weber K., Plessmann U., Traub P. Maturation of nuclear lamin A involves a specific carboxy-terminal trimming, which removes the polyisoprenylation site from the precursor; implications for the structure of the nuclear lamina. FEBS Lett. 1989 Nov 6;257(2):411–414. doi: 10.1016/0014-5793(89)81584-4. [DOI] [PubMed] [Google Scholar]
  67. Wolda S. L., Glomset J. A. Evidence for modification of lamin B by a product of mevalonic acid. J Biol Chem. 1988 May 5;263(13):5997–6000. [PubMed] [Google Scholar]
  68. Worman H. J., Yuan J., Blobel G., Georgatos S. D. A lamin B receptor in the nuclear envelope. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8531–8534. doi: 10.1073/pnas.85.22.8531. [DOI] [PMC free article] [PubMed] [Google Scholar]

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