Structure of pp32, an acidic nuclear protein which inhibits oncogene-induced formation of transformed foci (original) (raw)

Abstract

pp32 is a nuclear protein found highly expressed in normal tissues in those cells capable of self-renewal and in neoplastic cells. We report the cloning of cDNAs encoding human and murine pp32. The clones encode a 28.6-kDa protein; approximately two-thirds of the N-terminal predicts an amphipathic alpha helix containing two possible nuclear localization signals and a potential leucine zipper motif. The C-terminal third is exceptionally acidic, comprised of approximately 70% aspartic and glutamic acid residues; the predicted pI of human pp32 is 3.81. Human and murine pp32 cDNAs are 88% identical; the predicted proteins are 89% identical and 95% similar. Although the structure of pp32 is suggestive of a transcription factor, pp32 did not significantly modulate transcription of a reporter construct when fused to the Gal4 DNA-binding domain. In contrast, in cotransfection experiments, pp32 inhibited the ability of a broad assortment of oncogene pairs to transform rat embryo fibroblasts, including ras + myc, ras + jun, ras + E1a, ras + mutant p53, and E6 + E7. In related experiments, pp32 inhibited the ability of Rat 1a-myc cells to grow in soft agar, whereas it failed to affect ras-induced focus formation in NIH3T3 cells. These results suggest that pp32 may play a key role in self-renewing cell populations where it may act in the nucleus to limit their sensitivity to transformation.

2045

Images in this article

Selected References

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

  1. Allende J. E., Allende C. C. Protein kinases. 4. Protein kinase CK2: an enzyme with multiple substrates and a puzzling regulation. FASEB J. 1995 Mar;9(5):313–323. doi: 10.1096/fasebj.9.5.7896000. [DOI] [PubMed] [Google Scholar]
  2. Baker S. J., Markowitz S., Fearon E. R., Willson J. K., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990 Aug 24;249(4971):912–915. doi: 10.1126/science.2144057. [DOI] [PubMed] [Google Scholar]
  3. Barrett J. F., Lewis B. C., Hoang A. T., Alvarez R. J., Jr, Dang C. V. Cyclin A links c-Myc to adhesion-independent cell proliferation. J Biol Chem. 1995 Jul 7;270(27):15923–15925. doi: 10.1074/jbc.270.27.15923. [DOI] [PubMed] [Google Scholar]
  4. Bickenbach J. R., Mackenzie I. C. Identification and localization of label-retaining cells in hamster epithelia. J Invest Dermatol. 1984 Jun;82(6):618–622. doi: 10.1111/1523-1747.ep12261460. [DOI] [PubMed] [Google Scholar]
  5. Cairnie A. B., Lamerton L. F., Steel G. G. Cell proliferation studies in the intestinal epithelium of the rat. II. Theoretical aspects. Exp Cell Res. 1965 Sep;39(2):539–553. doi: 10.1016/0014-4827(65)90056-x. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Collins S. J., Ruscetti F. W., Gallagher R. E., Gallo R. C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci U S A. 1978 May;75(5):2458–2462. doi: 10.1073/pnas.75.5.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Earnshaw W. C., Sullivan K. F., Machlin P. S., Cooke C. A., Kaiser D. A., Pollard T. D., Rothfield N. F., Cleveland D. W. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. J Cell Biol. 1987 Apr;104(4):817–829. doi: 10.1083/jcb.104.4.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eliyahu D., Michalovitz D., Eliyahu S., Pinhasi-Kimhi O., Oren M. Wild-type p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8763–8767. doi: 10.1073/pnas.86.22.8763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eliyahu D., Raz A., Gruss P., Givol D., Oren M. Participation of p53 cellular tumour antigen in transformation of normal embryonic cells. Nature. 1984 Dec 13;312(5995):646–649. doi: 10.1038/312646a0. [DOI] [PubMed] [Google Scholar]
  11. Finlay C. A., Hinds P. W., Levine A. J. The p53 proto-oncogene can act as a suppressor of transformation. Cell. 1989 Jun 30;57(7):1083–1093. doi: 10.1016/0092-8674(89)90045-7. [DOI] [PubMed] [Google Scholar]
  12. Ginsberg D., Hirai S. I., Pinhasi-Kimhi O., Yaniv M., Oren M. Transfected mouse c-jun can inhibit transformation of primary rat embryo fibroblasts. Oncogene. 1991 Apr;6(4):669–672. [PubMed] [Google Scholar]
  13. Hayashi N., Sugimura Y., Kawamura J., Donjacour A. A., Cunha G. R. Morphological and functional heterogeneity in the rat prostatic gland. Biol Reprod. 1991 Aug;45(2):308–321. doi: 10.1095/biolreprod45.2.308. [DOI] [PubMed] [Google Scholar]
  14. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  15. Kato G. J., Barrett J., Villa-Garcia M., Dang C. V. An amino-terminal c-myc domain required for neoplastic transformation activates transcription. Mol Cell Biol. 1990 Nov;10(11):5914–5920. doi: 10.1128/mcb.10.11.5914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kessis T. D., Slebos R. J., Nelson W. G., Kastan M. B., Plunkett B. S., Han S. M., Lorincz A. T., Hedrick L., Cho K. R. Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3988–3992. doi: 10.1073/pnas.90.9.3988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim K. J., Kanellopoulos-Langevin C., Merwin R. M., Sachs D. H., Asofsky R. Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol. 1979 Feb;122(2):549–554. [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  21. Lathe R. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J Mol Biol. 1985 May 5;183(1):1–12. doi: 10.1016/0022-2836(85)90276-1. [DOI] [PubMed] [Google Scholar]
  22. Lewis S. A., Cowan N. J. Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe. J Cell Biol. 1985 Mar;100(3):843–850. doi: 10.1083/jcb.100.3.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li M., Makkinje A., Damuni Z. Molecular identification of I1PP2A, a novel potent heat-stable inhibitor protein of protein phosphatase 2A. Biochemistry. 1996 Jun 4;35(22):6998–7002. doi: 10.1021/bi960581y. [DOI] [PubMed] [Google Scholar]
  24. Malek S. N., Katumuluwa A. I., Pasternack G. R. Identification and preliminary characterization of two related proliferation-associated nuclear phosphoproteins. J Biol Chem. 1990 Aug 5;265(22):13400–13409. [PubMed] [Google Scholar]
  25. Matsuoka K., Taoka M., Satozawa N., Nakayama H., Ichimura T., Takahashi N., Yamakuni T., Song S. Y., Isobe T. A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neurons. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9670–9674. doi: 10.1073/pnas.91.21.9670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mukherjee B., Morgenbesser S. D., DePinho R. A. Myc family oncoproteins function through a common pathway to transform normal cells in culture: cross-interference by Max and trans-acting dominant mutants. Genes Dev. 1992 Aug;6(8):1480–1492. doi: 10.1101/gad.6.8.1480. [DOI] [PubMed] [Google Scholar]
  27. Mäkelä T. P., Koskinen P. J., Västrik I., Alitalo K. Alternative forms of Max as enhancers or suppressors of Myc-ras cotransformation. Science. 1992 Apr 17;256(5055):373–377. doi: 10.1126/science.256.5055.373. [DOI] [PubMed] [Google Scholar]
  28. Pinna L. A. Casein kinase 2: an 'eminence grise' in cellular regulation? Biochim Biophys Acta. 1990 Sep 24;1054(3):267–284. doi: 10.1016/0167-4889(90)90098-x. [DOI] [PubMed] [Google Scholar]
  29. Resar L. M., Dolde C., Barrett J. F., Dang C. V. B-myc inhibits neoplastic transformation and transcriptional activation by c-myc. Mol Cell Biol. 1993 Feb;13(2):1130–1136. doi: 10.1128/mcb.13.2.1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rimm D. L., Pollard T. D. New plasmid vectors for high level synthesis of eukaryotic fusion proteins in Escherichia coli. Gene. 1989 Feb 20;75(2):323–327. doi: 10.1016/0378-1119(89)90278-3. [DOI] [PubMed] [Google Scholar]
  31. Ruley H. E. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature. 1983 Aug 18;304(5927):602–606. doi: 10.1038/304602a0. [DOI] [PubMed] [Google Scholar]
  32. Ruppert J. M., Vogelstein B., Kinzler K. W. The zinc finger protein GLI transforms primary cells in cooperation with adenovirus E1A. Mol Cell Biol. 1991 Mar;11(3):1724–1728. doi: 10.1128/mcb.11.3.1724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schütte J., Minna J. D., Birrer M. J. Deregulated expression of human c-jun transforms primary rat embryo cells in cooperation with an activated c-Ha-ras gene and transforms rat-1a cells as a single gene. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2257–2261. doi: 10.1073/pnas.86.7.2257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Slebos R. J., Lee M. H., Plunkett B. S., Kessis T. D., Williams B. O., Jacks T., Hedrick L., Kastan M. B., Cho K. R. p53-dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5320–5324. doi: 10.1073/pnas.91.12.5320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sugden B., Marsh K., Yates J. A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus. Mol Cell Biol. 1985 Feb;5(2):410–413. doi: 10.1128/mcb.5.2.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vaesen M., Barnikol-Watanabe S., Götz H., Awni L. A., Cole T., Zimmermann B., Kratzin H. D., Hilschmann N. Purification and characterization of two putative HLA class II associated proteins: PHAPI and PHAPII. Biol Chem Hoppe Seyler. 1994 Feb;375(2):113–126. doi: 10.1515/bchm3.1994.375.2.113. [DOI] [PubMed] [Google Scholar]
  37. Walensky L. D., Coffey D. S., Chen T. H., Wu T. C., Pasternack G. R. A novel M(r) 32,000 nuclear phosphoprotein is selectively expressed in cells competent for self-renewal. Cancer Res. 1993 Oct 1;53(19):4720–4726. [PubMed] [Google Scholar]
  38. Wisdom R., Verma I. M. Revertants of v-fos-transformed rat fibroblasts: suppression of transformation is dominant. Mol Cell Biol. 1990 Nov;10(11):5626–5633. doi: 10.1128/mcb.10.11.5626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yehiely F., Oren M. The gene for the rat heat-shock cognate, hsc70, can suppress oncogene-mediated transformation. Cell Growth Differ. 1992 Nov;3(11):803–809. [PubMed] [Google Scholar]
  40. van den Heuvel S. J., van Laar T., The I., van der Eb A. J. Large E1B proteins of adenovirus types 5 and 12 have different effects on p53 and distinct roles in cell transformation. J Virol. 1993 Sep;67(9):5226–5234. doi: 10.1128/jvi.67.9.5226-5234.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]