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. 1996 Mar;16(3):990–997. doi: 10.1128/mcb.16.3.990

Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues.

W Shou 1, X Li 1, C Wu 1, T Cao 1, J Kuang 1, S Che 1, L D Etkin 1
PMCID: PMC231081  PMID: 8622702

Abstract

Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functi ons in dorsal/ventral patterning of the embryo. The xnf7 protein is stored in the oocyte nucleus germinal vesicle in a hypophosphorylated state. At oocyte maturation, xnf7 is hyperphosphorylated and released into the cytoplasm, where it is anchored until the midblastula stage, where it is dephosphorylated and enters the nucleus. We demonstrated that cytoplasmic anchoring of xnf7 was regulated by changes in the phosphorylation status of four threonines within two sites, site 1 (Thr-103) and site 2 (Thr-209, Thr-212, and Thr-218), which function in an additive manner. A mutant form of xnf7 (xnf7thr-glu) in which the threonines at sites 1 and 2 were mutated to glutamic acids to mimic a permanent state of phosphorylation was retained in the cytoplasm in oocytes and embryos through the gastrula stage. The cytoplasmic form of xnf7 was detected in a large 670-kDa protein complex probably consisting of xnf7 and several other unknown protein components. Anchoring of xnf7 was not dependent on association with either microtubule or microfilament components of the cytoskeleton, since treatment with cytochalasin B and nocodazole did not affect cytoplasmic retention. Both wild-type xnf7 and xnf7thr-glu form dimers in the yeast two-hybrid system; however, homodimerization was not required for cytoplasmic retention. We suggest that the cytoplasmic retention of xnf7 depends on the phosphorylation state of the protein whereas the cytoplasmic anchoring machinery appears to be constitutively present in oocytes and throughout development until the gastrula stage.

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Selected References

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  1. Beg A. A., Ruben S. M., Scheinman R. I., Haskill S., Rosen C. A., Baldwin A. S., Jr I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention. Genes Dev. 1992 Oct;6(10):1899–1913. doi: 10.1101/gad.6.10.1899. [DOI] [PubMed] [Google Scholar]
  2. Bellini M., Lacroix J. C., Gall J. G. A putative zinc-binding protein on lampbrush chromosome loops. EMBO J. 1993 Jan;12(1):107–114. doi: 10.1002/j.1460-2075.1993.tb05636.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blank V., Kourilsky P., Israël A. Cytoplasmic retention, DNA binding and processing of the NF-kappa B p50 precursor are controlled by a small region in its C-terminus. EMBO J. 1991 Dec;10(13):4159–4167. doi: 10.1002/j.1460-2075.1991.tb04994.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Campbell I. G., Nicolai H. M., Foulkes W. D., Senger G., Stamp G. W., Allan G., Boyer C., Jones K., Bast R. C., Jr, Solomon E. A novel gene encoding a B-box protein within the BRCA1 region at 17q21.1. Hum Mol Genet. 1994 Apr;3(4):589–594. doi: 10.1093/hmg/3.4.589. [DOI] [PubMed] [Google Scholar]
  5. Chan E. K., Hamel J. C., Buyon J. P., Tan E. M. Molecular definition and sequence motifs of the 52-kD component of human SS-A/Ro autoantigen. J Clin Invest. 1991 Jan;87(1):68–76. doi: 10.1172/JCI115003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen Y., Chen C. F., Riley D. J., Allred D. C., Chen P. L., Von Hoff D., Osborne C. K., Lee W. H. Aberrant subcellular localization of BRCA1 in breast cancer. Science. 1995 Nov 3;270(5237):789–791. doi: 10.1126/science.270.5237.789. [DOI] [PubMed] [Google Scholar]
  7. Etkin L. d., Pearman B., Roberts M., Bektesh S. L. Replication, integration and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis. Differentiation. 1984;26(3):194–202. doi: 10.1111/j.1432-0436.1984.tb01395.x. [DOI] [PubMed] [Google Scholar]
  8. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  9. Forbes D. J. Structure and function of the nuclear pore complex. Annu Rev Cell Biol. 1992;8:495–527. doi: 10.1146/annurev.cb.08.110192.002431. [DOI] [PubMed] [Google Scholar]
  10. Freemont P. S. The RING finger. A novel protein sequence motif related to the zinc finger. Ann N Y Acad Sci. 1993 Jun 11;684:174–192. doi: 10.1111/j.1749-6632.1993.tb32280.x. [DOI] [PubMed] [Google Scholar]
  11. Henkel T., Zabel U., van Zee K., Müller J. M., Fanning E., Baeuerle P. A. Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-kappa B subunit. Cell. 1992 Mar 20;68(6):1121–1133. doi: 10.1016/0092-8674(92)90083-o. [DOI] [PubMed] [Google Scholar]
  12. Hennekes H., Peter M., Weber K., Nigg E. A. Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2. J Cell Biol. 1993 Mar;120(6):1293–1304. doi: 10.1083/jcb.120.6.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunt T. Cytoplasmic anchoring proteins and the control of nuclear localization. Cell. 1989 Dec 22;59(6):949–951. doi: 10.1016/0092-8674(89)90747-2. [DOI] [PubMed] [Google Scholar]
  14. Jans D. A., Ackermann M. J., Bischoff J. R., Beach D. H., Peters R. p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins. J Cell Biol. 1991 Dec;115(5):1203–1212. doi: 10.1083/jcb.115.5.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kakizuka A., Miller W. H., Jr, Umesono K., Warrell R. P., Jr, Frankel S. R., Murty V. V., Dmitrovsky E., Evans R. M. Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell. 1991 Aug 23;66(4):663–674. doi: 10.1016/0092-8674(91)90112-c. [DOI] [PubMed] [Google Scholar]
  16. Kastner P., Perez A., Lutz Y., Rochette-Egly C., Gaub M. P., Durand B., Lanotte M., Berger R., Chambon P. Structure, localization and transcriptional properties of two classes of retinoic acid receptor alpha fusion proteins in acute promyelocytic leukemia (APL): structural similarities with a new family of oncoproteins. EMBO J. 1992 Feb;11(2):629–642. doi: 10.1002/j.1460-2075.1992.tb05095.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kerr L. D., Inoue J., Davis N., Link E., Baeuerle P. A., Bose H. R., Jr, Verma I. M. The rel-associated pp40 protein prevents DNA binding of Rel and NF-kappa B: relationship with I kappa B beta and regulation by phosphorylation. Genes Dev. 1991 Aug;5(8):1464–1476. doi: 10.1101/gad.5.8.1464. [DOI] [PubMed] [Google Scholar]
  18. Kimelman D., Kirschner M., Scherson T. The events of the midblastula transition in Xenopus are regulated by changes in the cell cycle. Cell. 1987 Feb 13;48(3):399–407. doi: 10.1016/0092-8674(87)90191-7. [DOI] [PubMed] [Google Scholar]
  19. Kloc M., Etkin L. D. Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes. Development. 1995 Feb;121(2):287–297. doi: 10.1242/dev.121.2.287. [DOI] [PubMed] [Google Scholar]
  20. Kuang J., Ashorn C. L. At least two kinases phosphorylate the MPM-2 epitope during Xenopus oocyte maturation. J Cell Biol. 1993 Nov;123(4):859–868. doi: 10.1083/jcb.123.4.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leonhardt E. A., Kapp L. N., Young B. R., Murnane J. P. Nucleotide sequence analysis of a candidate gene for ataxia-telangiectasia group D (ATDC). Genomics. 1994 Jan 1;19(1):130–136. doi: 10.1006/geno.1994.1022. [DOI] [PubMed] [Google Scholar]
  22. Li X., Etkin L. D. Xenopus nuclear factor 7 (xnf7) possesses an NLS that functions efficiently in both oocytes and embryos. J Cell Sci. 1993 Jun;105(Pt 2):389–395. doi: 10.1242/jcs.105.2.389. [DOI] [PubMed] [Google Scholar]
  23. Li X., Shou W., Kloc M., Reddy B. A., Etkin L. D. Cytoplasmic retention of Xenopus nuclear factor 7 before the mid blastula transition uses a unique anchoring mechanism involving a retention domain and several phosphorylation sites. J Cell Biol. 1994 Jan;124(1-2):7–17. doi: 10.1083/jcb.124.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li X., Shou W., Kloc M., Reddy B. A., Etkin L. D. The association of Xenopus nuclear factor 7 with subcellular structures is dependent upon phosphorylation and specific domains. Exp Cell Res. 1994 Aug;213(2):473–481. doi: 10.1006/excr.1994.1225. [DOI] [PubMed] [Google Scholar]
  25. Miki T., Fleming T. P., Crescenzi M., Molloy C. J., Blam S. B., Reynolds S. H., Aaronson S. A. Development of a highly efficient expression cDNA cloning system: application to oncogene isolation. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5167–5171. doi: 10.1073/pnas.88.12.5167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Miki Y., Swensen J., Shattuck-Eidens D., Futreal P. A., Harshman K., Tavtigian S., Liu Q., Cochran C., Bennett L. M., Ding W. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994 Oct 7;266(5182):66–71. doi: 10.1126/science.7545954. [DOI] [PubMed] [Google Scholar]
  27. Miller M., Kloc M., Reddy B., Eastman E., Dreyer C., Etkin L. xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis. Genes Dev. 1989 Apr;3(4):572–583. doi: 10.1101/gad.3.4.572. [DOI] [PubMed] [Google Scholar]
  28. Miller M., Reddy B. A., Kloc M., Li X. X., Dreyer C., Etkin L. D. The nuclear-cytoplasmic distribution of the Xenopus nuclear factor, xnf7, coincides with its state of phosphorylation during early development. Development. 1991 Oct;113(2):569–575. doi: 10.1242/dev.113.2.569. [DOI] [PubMed] [Google Scholar]
  29. Moll T., Tebb G., Surana U., Robitsch H., Nasmyth K. The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell. 1991 Aug 23;66(4):743–758. doi: 10.1016/0092-8674(91)90118-i. [DOI] [PubMed] [Google Scholar]
  30. Nigg E. A., Baeuerle P. A., Lührmann R. Nuclear import-export: in search of signals and mechanisms. Cell. 1991 Jul 12;66(1):15–22. doi: 10.1016/0092-8674(91)90135-l. [DOI] [PubMed] [Google Scholar]
  31. Patarca R., Freeman G. J., Schwartz J., Singh R. P., Kong Q. T., Murphy E., Anderson Y., Sheng F. Y., Singh P., Johnson K. A. rpt-1, an intracellular protein from helper/inducer T cells that regulates gene expression of interleukin 2 receptor and human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2733–2737. doi: 10.1073/pnas.85.8.2733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reddy B. A., Etkin L. D., Freemont P. S. A novel zinc finger coiled-coil domain in a family of nuclear proteins. Trends Biochem Sci. 1992 Sep;17(9):344–345. doi: 10.1016/0968-0004(92)90308-v. [DOI] [PubMed] [Google Scholar]
  33. Reddy B. A., Kloc M., Etkin L. The cloning and characterization of a maternally expressed novel zinc finger nuclear phosphoprotein (xnf7) in Xenopus laevis. Dev Biol. 1991 Nov;148(1):107–116. doi: 10.1016/0012-1606(91)90321-s. [DOI] [PubMed] [Google Scholar]
  34. Roth S., Stein D., Nüsslein-Volhard C. A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell. 1989 Dec 22;59(6):1189–1202. doi: 10.1016/0092-8674(89)90774-5. [DOI] [PubMed] [Google Scholar]
  35. Rushlow C. A., Han K., Manley J. L., Levine M. The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila. Cell. 1989 Dec 22;59(6):1165–1177. doi: 10.1016/0092-8674(89)90772-1. [DOI] [PubMed] [Google Scholar]
  36. Schmitz M. L., Henkel T., Baeuerle P. A. Proteins controlling the nuclear uptake of NF-kappa B, Rel and dorsal. Trends Cell Biol. 1991 Nov;1(5):130–137. doi: 10.1016/0962-8924(91)90118-s. [DOI] [PubMed] [Google Scholar]
  37. Silver P. A. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. doi: 10.1016/0092-8674(91)90233-o. [DOI] [PubMed] [Google Scholar]
  38. Steward R. Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function. Cell. 1989 Dec 22;59(6):1179–1188. doi: 10.1016/0092-8674(89)90773-3. [DOI] [PubMed] [Google Scholar]
  39. Takahashi M., Inaguma Y., Hiai H., Hirose F. Developmentally regulated expression of a human "finger"-containing gene encoded by the 5' half of the ret transforming gene. Mol Cell Biol. 1988 Apr;8(4):1853–1856. doi: 10.1128/mcb.8.4.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  41. Whiteside S. T., Goodbourn S. Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localisation. J Cell Sci. 1993 Apr;104(Pt 4):949–955. doi: 10.1242/jcs.104.4.949. [DOI] [PubMed] [Google Scholar]
  42. Wu M., Gerhart J. C. Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis. Dev Biol. 1980 Oct;79(2):465–477. doi: 10.1016/0012-1606(80)90131-1. [DOI] [PubMed] [Google Scholar]
  43. Yisraeli J. K., Sokol S., Melton D. A. A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development. 1990 Feb;108(2):289–298. doi: 10.1242/dev.108.2.289. [DOI] [PubMed] [Google Scholar]
  44. de Thé H., Lavau C., Marchio A., Chomienne C., Degos L., Dejean A. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell. 1991 Aug 23;66(4):675–684. doi: 10.1016/0092-8674(91)90113-d. [DOI] [PubMed] [Google Scholar]

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