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Nuclear localization signal in insulin-like growth factor-binding protein type 3
PROTEINSEQUENCEMOTIF
TIBS 1 9 - J U L Y 1 9 9 4
and Deppert, W. (1993) EMBO 3.12, 4739-4746 11 Montano,X. et ai. (1990) Proc. Natl Acad. Sci. USA 87, 7448-7452 12 Walter, G., Carbone, A. and Welch, W. J. (1987) Virology 61, 405-410 13 Sawai, E. T. and Butel, J. S. (1989) J. Virol. 63, 3961-3973 14 Sawai, E. 1., Rasmussen, G. and Butel, J. S. (1994) Virus Res. 31, 367-378
15 May, E. Breugnot, C., Duthu, A. and May, P. (1991) Virology 180, 285-293 16 Fanning, E. (1992) J. Virol. 66, 1289-1293 17 Adamzewski, J. P., Gannon, J. V. and Hunt, T. (1993) J. Virol. 67, 6551-6557 18 Georgopoulos, C. and Welch, W. J. (1993) Annu. Rev. Cell Biol. 9, 601-634
Nuclear localization signal in insulin-like growth iactor-binding protein type 3
gene expression and cell proliferation. The precise mechanisms involved in the nuclear translocation of insulin and IGF-I have not yet been fully elucidated. The uptake of protein by the nucleus is extremely selective, so nuclear proteins must contain within their final structure a signal that specifies selective accumulation in the nucleus 7,8. Studies on some nuclear proteins, such as the large T antigen of SV40, have indicated which part of the sequence is required for nuclear translocation. The known nuclear targeting sequences are generally basic, but there seems to be no clear common denominator among all the known sequences. Although some consensus sequence patterns have been proposed (see, for example, Ref. 9), the current best strategy to detect an NTS is based l° on the following definition of a bipartite nuclear targeting sequence: (1) two adjacent basic amino acids (Arg or Lys); (2) a spacer region of any ten residues; and (3) at least three basic residues (Arg or Lys) in the five positions after the spacer region. Fifty-six percent of known nuclear proteins have previously been found to contain this distinctive amino acid pattern l°. By coatrast, only 4.2% of the amino acid sequences of non-nuclear proteins deposited in the SWISS-PROT database harboured this motiU °. The amino acid sequences of human and porcine IGFBP-3, as communicated previously u, were screened for the presence of a bipartite NLS as defined above. A bipartite NLS was detected in both human and porcine IGFBP-3 (Fig. 1). This finding supports the initial hypothesis of a putative nuclear localization of IGFBP-3 and suggests that this may be relevant to the negative growth regulatory action of this molecule. Among the six known IGFBPs lz, only IGFBP-5 was also found to contain a bipartite NLS at positions corresponding to those present in the IGFBP-3 NLS (data not shown). Previous studies have indicated that IGFBPs bind IGFs in a region of IGF equivalent to the insulin B chain 12.
Recently, it has been shown that expression of the cDNA encoding human insulin-like growth factor-binding protein type 3 (IGFBP-3) in a murine fibroblast cell line had an inhibitory effect on cell growthL The authors of this study concluded that IGFBP-3 might be a tumour suppressor gene product, and suggested an intracellular mode of action as one possible mechanism. Structural considerations have previously led me to predict that insulin and the insulin-like growth factors (IGFs) IGF.I and IGF-IImay partly exert their growth regulatory effects through complex formation with the nuclear anti-oncogene product retinoblastoma protein (RB) 2,3. The present study addressed the more specific question of whether RB, insulin, IGFs and IGFBP-3 might all be located in the cell nucleus. This has already been extensiveiy validated for RB; in particular, it has been demonstrated that RB contains a bipartite nuclear localization signal (NLS) or a bipartite nuclear targeting sequence (NTS), respectively, that is critical for the biological activity of this turnout suppressor 4. Furthermore, insulin s and IGF-!6 have also been shown to translocate to the nucleus, suggesting that nuclear accumulation might be crucial for their regulation of
I KK GFYKKKQCRP l
SKGRK
Rgure 1 Proposed nuclear localization signal (NLS) in IGFBP-3(residues 215-232 in human IGFBP-3 and residues 217-234 in porcine IGFBP-3). Amino acids are designated by the singleletter code. The basic amino acids, K and R, located in the two clusters of the bipartite NLS, are highlighted in bold.
278
WILLIAM L. KELLEY Departement de Biochimie M~dicale, Centre M~dical Universitaire, Universit~ de Gen~ve, 1211 Gen~ve 4, Switzerland.
SAMUEL J. LANDRY Department of Biochemistry, Tulane University School of Medicine, N~w Orleans, LA 70112, USA.
Moreover, the insulin B chain and its corresponding domain in the IGFs harbour a sequence motif that is identical, or highly related, respectively, to the LxCxE RB-binding motif in RB-binding proteins (RBPs) 1 and 2 (Refs 2, 3). It is therefore tempting to speculate that the above-mentioned IGFBPs and RBPs may interact with one another and thus form a regulatory network of binding proteins. Such a network could contribute an additional level of control in the homeostasis of growth. Taken together, the evidence described here suggests that IGFBP-3 may develop its negative growth regulatory actions in the cell nucleus, possibly through physical association with IGFs and/or RBPs. As such, IGFBP-3 may protect RB from the postulated inactivation by IGFs 2,3.
References I Cohen, P,, Lamson, G., Okajlma, T. and Rosenfeld, R. G. (1993) Mol. Endocrinol. 7, 380-386 2 Radulescu, R. T. and Wendtner, C. M. (1992) J. Mol. Recognit. 5, 133-137 3 Radulescu, R. T. and Wendtr~er,C. M. (1993) J. Endocrinol. 139, 1-7 4 Zacksenhaus, E., Bremner, R., Phillips, R. A. and Gallie, B. L. (1993) MoL Cell. Biol. 13, 4588-4599 5 Harada, S., Smith, R. M., Smith, J. A. and Jarett, L. (1993) Endocrinology 132, 2293-2298 6 Peralta Soler, A. et al. (1990) Endocrinology 127, 595-603 7 Dingwall, C. and Laskey, R. A. (1986) Annu. Rev. Cell Biol. 2, 367-390 8 Garcia-Bustos,J., Heitman, J. and Hall, M. N. (1991) Biochim. Biophys. Acta 1071, 83-101 9 Gomez-Marquez,J. and Segade,F. (1988) FEBS Lett. 226, 217-219 10 Dingwall, C. and Laskey, R. A. (1991) Trends BiocheT}. Sci. 16, 478-481 11 Shirnasaki, S. et al. (1990) J. Biol. Chem. 265, 2198-2202 12 Drop, S. L. S. et al. (1992) Growth Regul. 2, 69-79
RAZVAN 1". RADULESCU Molecular Concepts Research, Kunigundenstrasse 34, 80805 Munich, Germany.
© 1994, Elsevier Science Ltd 0968-0004/94/$07.00
TIBS 1 9 - J U L Y 1 9 9 4
and Deppert, W. (1993) EMBO 3.12, 4739-4746 11 Montano,X. et ai. (1990) Proc. Natl Acad. Sci. USA 87, 7448-7452 12 Walter, G., Carbone, A. and Welch, W. J. (1987) Virology 61, 405-410 13 Sawai, E. T. and Butel, J. S. (1989) J. Virol. 63, 3961-3973 14 Sawai, E. 1., Rasmussen, G. and Butel, J. S. (1994) Virus Res. 31, 367-378
15 May, E. Breugnot, C., Duthu, A. and May, P. (1991) Virology 180, 285-293 16 Fanning, E. (1992) J. Virol. 66, 1289-1293 17 Adamzewski, J. P., Gannon, J. V. and Hunt, T. (1993) J. Virol. 67, 6551-6557 18 Georgopoulos, C. and Welch, W. J. (1993) Annu. Rev. Cell Biol. 9, 601-634
Nuclear localization signal in insulin-like growth iactor-binding protein type 3
gene expression and cell proliferation. The precise mechanisms involved in the nuclear translocation of insulin and IGF-I have not yet been fully elucidated. The uptake of protein by the nucleus is extremely selective, so nuclear proteins must contain within their final structure a signal that specifies selective accumulation in the nucleus 7,8. Studies on some nuclear proteins, such as the large T antigen of SV40, have indicated which part of the sequence is required for nuclear translocation. The known nuclear targeting sequences are generally basic, but there seems to be no clear common denominator among all the known sequences. Although some consensus sequence patterns have been proposed (see, for example, Ref. 9), the current best strategy to detect an NTS is based l° on the following definition of a bipartite nuclear targeting sequence: (1) two adjacent basic amino acids (Arg or Lys); (2) a spacer region of any ten residues; and (3) at least three basic residues (Arg or Lys) in the five positions after the spacer region. Fifty-six percent of known nuclear proteins have previously been found to contain this distinctive amino acid pattern l°. By coatrast, only 4.2% of the amino acid sequences of non-nuclear proteins deposited in the SWISS-PROT database harboured this motiU °. The amino acid sequences of human and porcine IGFBP-3, as communicated previously u, were screened for the presence of a bipartite NLS as defined above. A bipartite NLS was detected in both human and porcine IGFBP-3 (Fig. 1). This finding supports the initial hypothesis of a putative nuclear localization of IGFBP-3 and suggests that this may be relevant to the negative growth regulatory action of this molecule. Among the six known IGFBPs lz, only IGFBP-5 was also found to contain a bipartite NLS at positions corresponding to those present in the IGFBP-3 NLS (data not shown). Previous studies have indicated that IGFBPs bind IGFs in a region of IGF equivalent to the insulin B chain 12.
Recently, it has been shown that expression of the cDNA encoding human insulin-like growth factor-binding protein type 3 (IGFBP-3) in a murine fibroblast cell line had an inhibitory effect on cell growthL The authors of this study concluded that IGFBP-3 might be a tumour suppressor gene product, and suggested an intracellular mode of action as one possible mechanism. Structural considerations have previously led me to predict that insulin and the insulin-like growth factors (IGFs) IGF.I and IGF-IImay partly exert their growth regulatory effects through complex formation with the nuclear anti-oncogene product retinoblastoma protein (RB) 2,3. The present study addressed the more specific question of whether RB, insulin, IGFs and IGFBP-3 might all be located in the cell nucleus. This has already been extensiveiy validated for RB; in particular, it has been demonstrated that RB contains a bipartite nuclear localization signal (NLS) or a bipartite nuclear targeting sequence (NTS), respectively, that is critical for the biological activity of this turnout suppressor 4. Furthermore, insulin s and IGF-!6 have also been shown to translocate to the nucleus, suggesting that nuclear accumulation might be crucial for their regulation of
I KK GFYKKKQCRP l
SKGRK
Rgure 1 Proposed nuclear localization signal (NLS) in IGFBP-3(residues 215-232 in human IGFBP-3 and residues 217-234 in porcine IGFBP-3). Amino acids are designated by the singleletter code. The basic amino acids, K and R, located in the two clusters of the bipartite NLS, are highlighted in bold.
278
WILLIAM L. KELLEY Departement de Biochimie M~dicale, Centre M~dical Universitaire, Universit~ de Gen~ve, 1211 Gen~ve 4, Switzerland.
SAMUEL J. LANDRY Department of Biochemistry, Tulane University School of Medicine, N~w Orleans, LA 70112, USA.
Moreover, the insulin B chain and its corresponding domain in the IGFs harbour a sequence motif that is identical, or highly related, respectively, to the LxCxE RB-binding motif in RB-binding proteins (RBPs) 1 and 2 (Refs 2, 3). It is therefore tempting to speculate that the above-mentioned IGFBPs and RBPs may interact with one another and thus form a regulatory network of binding proteins. Such a network could contribute an additional level of control in the homeostasis of growth. Taken together, the evidence described here suggests that IGFBP-3 may develop its negative growth regulatory actions in the cell nucleus, possibly through physical association with IGFs and/or RBPs. As such, IGFBP-3 may protect RB from the postulated inactivation by IGFs 2,3.
References I Cohen, P,, Lamson, G., Okajlma, T. and Rosenfeld, R. G. (1993) Mol. Endocrinol. 7, 380-386 2 Radulescu, R. T. and Wendtner, C. M. (1992) J. Mol. Recognit. 5, 133-137 3 Radulescu, R. T. and Wendtr~er,C. M. (1993) J. Endocrinol. 139, 1-7 4 Zacksenhaus, E., Bremner, R., Phillips, R. A. and Gallie, B. L. (1993) MoL Cell. Biol. 13, 4588-4599 5 Harada, S., Smith, R. M., Smith, J. A. and Jarett, L. (1993) Endocrinology 132, 2293-2298 6 Peralta Soler, A. et al. (1990) Endocrinology 127, 595-603 7 Dingwall, C. and Laskey, R. A. (1986) Annu. Rev. Cell Biol. 2, 367-390 8 Garcia-Bustos,J., Heitman, J. and Hall, M. N. (1991) Biochim. Biophys. Acta 1071, 83-101 9 Gomez-Marquez,J. and Segade,F. (1988) FEBS Lett. 226, 217-219 10 Dingwall, C. and Laskey, R. A. (1991) Trends BiocheT}. Sci. 16, 478-481 11 Shirnasaki, S. et al. (1990) J. Biol. Chem. 265, 2198-2202 12 Drop, S. L. S. et al. (1992) Growth Regul. 2, 69-79
RAZVAN 1". RADULESCU Molecular Concepts Research, Kunigundenstrasse 34, 80805 Munich, Germany.
© 1994, Elsevier Science Ltd 0968-0004/94/$07.00