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• W2034020444 abstract "Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transcription factor, has been shown to function extracellularly, implying that some Tat molecules escape nuclear import and are secreted. This raises the question of what regulates, in HIV-1-infected cells, the nuclear targeting of the polypeptide. Here we show that cytosolic components activated by Ca2+ ions are required to reveal the karyophilic properties of Tat: in vitro translated Tat molecules do not associate with isolated nuclei unless preincubated with Ca2+. Moreover, Ca2+ ions induce karyophilicity of chemically synthesized Tat molecules only upon addition of cytosolic extracts. The Ca2+-induced karyophilicity is prevented by inhibitors of either tyrosine kinases (herbimycin A and genistein) or tyrosine phosphatases (vanadate), suggesting the involvement of Ca2+-dependent phosphorylation/dephosphorylation events. In line with these observations, the transcriptional activity of Tat is inhibited by treatment with either vanadate or genistein. The same occurs with Tat mutants lacking either one or both the two tyrosine residues (positions 26 and 47). Hence, Ca2+-dependent tyrosine kinase(s) and phosphatase(s) act on accessory cellular protein(s), which in turn are responsible of Tat karyophilicity. Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transcription factor, has been shown to function extracellularly, implying that some Tat molecules escape nuclear import and are secreted. This raises the question of what regulates, in HIV-1-infected cells, the nuclear targeting of the polypeptide. Here we show that cytosolic components activated by Ca2+ ions are required to reveal the karyophilic properties of Tat: in vitro translated Tat molecules do not associate with isolated nuclei unless preincubated with Ca2+. Moreover, Ca2+ ions induce karyophilicity of chemically synthesized Tat molecules only upon addition of cytosolic extracts. The Ca2+-induced karyophilicity is prevented by inhibitors of either tyrosine kinases (herbimycin A and genistein) or tyrosine phosphatases (vanadate), suggesting the involvement of Ca2+-dependent phosphorylation/dephosphorylation events. In line with these observations, the transcriptional activity of Tat is inhibited by treatment with either vanadate or genistein. The same occurs with Tat mutants lacking either one or both the two tyrosine residues (positions 26 and 47). Hence, Ca2+-dependent tyrosine kinase(s) and phosphatase(s) act on accessory cellular protein(s), which in turn are responsible of Tat karyophilicity. INTRODUCTIONIn eukaryotic cells, gene expression is often regulated by post-translational modifications such as phosphorylation (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar) of the relevant transcription factors or associated proteins. These modifications may modulate the cellular localization of the transcription factors, their DNA binding capacity, or their transactivating activity. Examples of transcription factors able to undergo inducible nuclear import include steroid receptors (2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar), the v-jun oncogenic counterpart of the AP-1 complex member c-jun (3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar), the yeast SW15 (4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar), and NF-κB, which is imported to the nucleus following dissociation from the cytosolic anchoring protein IκB-a (5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar).In addition to this group of transcription factors, which have a different intracellular localization depending on their functional state, many examples of proteins with dual intracellular targeting have been reported (7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar). Among HIV 1The abbreviations used are: HIVhuman immunodeficiency virusHIV-1human immunodeficiency virus-1CsAcyclosporin ALTRlong terminal repeatCMVcytomegalovirusβ-galβ-galactosidasePBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisONPGo-nitrophenyl-β-D-galactopyranosideTKtyrosine kinasePTPaseprotein tyrosine phosphatasePMAphorbol 12-myristate 13-acetate. products, the matrix protein MA contains two subcellular localization signals with competing effects: a myristoylated N terminus that targets the protein to the plasma membrane and a nuclear localization sequence. Myristoylation is the dominant signal. However, a small subset of MA molecules undergo tyrosine phosphorylation, and this modification is sufficient to reveal their karyophilic properties (8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar). The case of human immunodeficiency virus-1 (HIV-1) Tat is still more complex, as in addition to its transactivating function, necessary for viral replication (9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar), it can also be secreted by transfected or virus-infected cells (11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar) and exert several extracellular activities that interfere with growth regulation of different cells (12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar). While much effort has been spent on elucidating the molecular details of transactivation of HIV-1, the mechanisms regulating nuclear import versus secretion of Tat protein are largely unknown.In a previous study, we have shown that Tat molecules, synthesized in vitro in wheat germ extracts, do not associate with nuclei when added to lysed cells (14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar); however, we noted that after a period of incubation in culture medium, some Tat molecules became capable of associating postlytically with the nuclear fraction, suggesting that some components of the culture medium might induce modification(s) on Tat molecules that reveal its karyophilicity.Here we exploit in vitro and in vivo assays to investigate the effects of Ca2+ and of cytosolic factors on the nuclear targeting of Tat.DISCUSSIONIn this paper we describe a novel mechanism of control of HIV-1 Tat activity, involving tyrosine phosphorylation/dephosphorylation of cytosolic component(s), whose activation, dependent on Ca2+, unveils the karyophilicity of Tat.The kinetics and the irreversibility of the Ca2+-induced karyophilicity (Fig. 1) suggest that Ca2+ ions do not act directly on Tat itself; this notion is further supported by the observation that chemically synthesized Tat does not associate to nuclei after treatement with Ca2+ unless cytosolic factors are added. Ca2+ may thus activate cellular protein(s) which in turn confer karyophilicity to Tat. As drugs blocking either TK or PTPase inhibit karyophilicity, Ca2+-dependent tyrosine phosphorylation/dephosphorylation events seem to be involved in controlling the nuclear association of Tat. The similar inhibitory effects of drugs acting on targets with opposite functions (TK and PTPase) is not surprising as kinases may be activated by dephosphorylation and vice versa (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar, 2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar, 3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar, 4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar, 5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar, 7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar, 8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar, 9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar, 11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar, 12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar, 14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar, 15Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Google Scholar, 16MacGregor G.R. Caskey C.T. Nucleic Acids Res. 1989; 17: 2365Google Scholar, 17Zappavigna V. Renucci A. Izpisua-Belmonte J.C. Urrier G. Peschle C. Duboule D. EMBO J. 1991; 10: 4177-4187Google Scholar, 18Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar, 19Ensoli B. Bonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Google Scholar, 20Charbonneau H. Tonks N.K. Annu. Rev. Cell Biol. 1992; 8: 463-493Google Scholar). The nature of the TK(s) and PTPase(s) involved, as well as the order of their activation, remain to be investigated. Whatever their nature, the factors are highly conserved through evolution, as demonstrated by the similar activity of wheat germ and HeLa cell cytosolic extracts. Several examples of Ca2+-induced serine/threonine protein kinases or phosphatases involved in activation of transcription factors have been provided (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar, 2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar, 3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar, 4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar, 5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar, 7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar, 8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar, 9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar, 11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar, 12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar, 14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar, 15Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Google Scholar, 16MacGregor G.R. Caskey C.T. Nucleic Acids Res. 1989; 17: 2365Google Scholar, 17Zappavigna V. Renucci A. Izpisua-Belmonte J.C. Urrier G. Peschle C. Duboule D. EMBO J. 1991; 10: 4177-4187Google Scholar, 18Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar, 19Ensoli B. Bonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Google Scholar, 20Charbonneau H. Tonks N.K. Annu. Rev. Cell Biol. 1992; 8: 463-493Google Scholar, 21Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Google Scholar); in contrast, while the importance of TKs and PTPases in the early steps of signal transduction is well established, their direct involvement in regulating the activity of transcription factors is less defined. Tyrosine phosphorylation of the three subunits of the interferon-stimulated gene factor 3 appears to be responsible for their translocation into the nucleus and hence their activation (22Fu X.-Y. Cell. 1992; 70: 323-335Google Scholar); the regulatory enzyme involved is TYK-2, a cytoplasmic, Ca2+-independent TK induced by interferon-α (23Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Google Scholar). Another example of differential targeting modulated by tyrosine phosphorylation is HIV-1 MA protein (8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar).In the case of Tat, substitution of either one or both Tyr-26 or Tyr-47 results in mutant Tat molecules that undergo the same control of nuclear association as wild type Tat. These observations confirm that the Ca2+-activated TK/PTPases involved do not act directly on Tat molecules. Rather, these data suggest that the target of the phosphorylation is accessory protein(s), perhaps involved in chaperoning Tat molecules to the nucleus. In agreement with this, the functional activity is only slightly inhibited when Tyr-26 and/or Tyr-47 are replaced: these mutants are able to transactivate a reporter gene at approximately the same extent as wild type Tat. Three Tat mutants bearing single substitution of Tyr-26 with Ala or Tyr-47 with Ala or histidine have been reported previously (24Kuppuswamy M. Subramanian T. Srinivasan A. Chinnadurai G. Nucleic Acids Res. 1989; 6: 331-357Google Scholar, 25Rice A.P. Carlotti F. J. Virol. 1990; 64: 1864-1868Google Scholar); in these mutants substitution of either tyrosine did not abolish Tat transcriptional activity, although Ala-26 had a weaker activity in HeLa cells. The maintainance of the full activity by the Tat mutants described here may be due to the more conservative substitution of Tyr with Phe.The TK/PTPases-mediated induction of Tat karyophilicity has a functional correspondence in the finding that genistein and vanadate ihibit HIV-1 LTR transactivation in transfected cells, of either lymphoid or non-lymphoid origin. Genistein and vanadate also inhibit transactivating activity of the three Tyr mutants, confirming that the TK/PTPases involved in Tat nuclear targeting and transcriptional activity act on accessory protein(s).Our observations of a more effective transactivation of LTR-β galactosidase in stimulated rather than in resting Jurkat T cells are in line with previous reports (26Siekevitz M. Josephs S.F. Ducovich M. Peffer N. Wong-Staal F. Greene W.C. Science. 1987; 238: 1575-1578Google Scholar, 27Tong-Starksen S.E. Luciw P.A. Peterlin B.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6845-6849Google Scholar) and support the hypothesis of a role for intracellular [Ca2+] in regulating Tat activity. The immunosuppressive drug CsA was shown to block the Tat-mediated increase of IL-2 promoter activity in activated T cells (28Westendorp M.O. Li-Weber M. Frank R.W. Krammer P.H. J. Virol. 1994; 67: 4177-4185Google Scholar). Similarly, we show that CsA inhibits LTR transactivation in stimulated, but not resting, T cells, suggesting that, in activated cells, additional, CsA-sensitive, cellular factors act in concert with Tat with resulting activation of HIV-1 gene expression.Altogether, these findings suggest that, depending on their activation state, HIV-1-infected cells have the possibility of modifying the intracellular fate and the transcriptional activity of Tat by modulating its karyophilicity through activation of TK/PTPase, with obvious influence on viral replication and on the development of HIV-associated syndromes. Compounds that block the activity of these enzymes may lead to novel strategies to slow the spread of the virus in HIV-infected individuals. INTRODUCTIONIn eukaryotic cells, gene expression is often regulated by post-translational modifications such as phosphorylation (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar) of the relevant transcription factors or associated proteins. These modifications may modulate the cellular localization of the transcription factors, their DNA binding capacity, or their transactivating activity. Examples of transcription factors able to undergo inducible nuclear import include steroid receptors (2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar), the v-jun oncogenic counterpart of the AP-1 complex member c-jun (3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar), the yeast SW15 (4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar), and NF-κB, which is imported to the nucleus following dissociation from the cytosolic anchoring protein IκB-a (5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar).In addition to this group of transcription factors, which have a different intracellular localization depending on their functional state, many examples of proteins with dual intracellular targeting have been reported (7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar). Among HIV 1The abbreviations used are: HIVhuman immunodeficiency virusHIV-1human immunodeficiency virus-1CsAcyclosporin ALTRlong terminal repeatCMVcytomegalovirusβ-galβ-galactosidasePBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisONPGo-nitrophenyl-β-D-galactopyranosideTKtyrosine kinasePTPaseprotein tyrosine phosphatasePMAphorbol 12-myristate 13-acetate. products, the matrix protein MA contains two subcellular localization signals with competing effects: a myristoylated N terminus that targets the protein to the plasma membrane and a nuclear localization sequence. Myristoylation is the dominant signal. However, a small subset of MA molecules undergo tyrosine phosphorylation, and this modification is sufficient to reveal their karyophilic properties (8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar). The case of human immunodeficiency virus-1 (HIV-1) Tat is still more complex, as in addition to its transactivating function, necessary for viral replication (9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar), it can also be secreted by transfected or virus-infected cells (11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar) and exert several extracellular activities that interfere with growth regulation of different cells (12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar). While much effort has been spent on elucidating the molecular details of transactivation of HIV-1, the mechanisms regulating nuclear import versus secretion of Tat protein are largely unknown.In a previous study, we have shown that Tat molecules, synthesized in vitro in wheat germ extracts, do not associate with nuclei when added to lysed cells (14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar); however, we noted that after a period of incubation in culture medium, some Tat molecules became capable of associating postlytically with the nuclear fraction, suggesting that some components of the culture medium might induce modification(s) on Tat molecules that reveal its karyophilicity.Here we exploit in vitro and in vivo assays to investigate the effects of Ca2+ and of cytosolic factors on the nuclear targeting of Tat. In eukaryotic cells, gene expression is often regulated by post-translational modifications such as phosphorylation (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar) of the relevant transcription factors or associated proteins. These modifications may modulate the cellular localization of the transcription factors, their DNA binding capacity, or their transactivating activity. Examples of transcription factors able to undergo inducible nuclear import include steroid receptors (2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar), the v-jun oncogenic counterpart of the AP-1 complex member c-jun (3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar), the yeast SW15 (4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar), and NF-κB, which is imported to the nucleus following dissociation from the cytosolic anchoring protein IκB-a (5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar). In addition to this group of transcription factors, which have a different intracellular localization depending on their functional state, many examples of proteins with dual intracellular targeting have been reported (7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar). Among HIV 1The abbreviations used are: HIVhuman immunodeficiency virusHIV-1human immunodeficiency virus-1CsAcyclosporin ALTRlong terminal repeatCMVcytomegalovirusβ-galβ-galactosidasePBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisONPGo-nitrophenyl-β-D-galactopyranosideTKtyrosine kinasePTPaseprotein tyrosine phosphatasePMAphorbol 12-myristate 13-acetate. products, the matrix protein MA contains two subcellular localization signals with competing effects: a myristoylated N terminus that targets the protein to the plasma membrane and a nuclear localization sequence. Myristoylation is the dominant signal. However, a small subset of MA molecules undergo tyrosine phosphorylation, and this modification is sufficient to reveal their karyophilic properties (8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar). The case of human immunodeficiency virus-1 (HIV-1) Tat is still more complex, as in addition to its transactivating function, necessary for viral replication (9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar), it can also be secreted by transfected or virus-infected cells (11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar) and exert several extracellular activities that interfere with growth regulation of different cells (12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar). While much effort has been spent on elucidating the molecular details of transactivation of HIV-1, the mechanisms regulating nuclear import versus secretion of Tat protein are largely unknown. human immunodeficiency virus human immunodeficiency virus-1 cyclosporin A long terminal repeat cytomegalovirus β-galactosidase phosphate-buffered saline polyacrylamide gel electrophoresis o-nitrophenyl-β-D-galactopyranoside tyrosine kinase protein tyrosine phosphatase phorbol 12-myristate 13-acetate. In a previous study, we have shown that Tat molecules, synthesized in vitro in wheat germ extracts, do not associate with nuclei when added to lysed cells (14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar); however, we noted that after a period of incubation in culture medium, some Tat molecules became capable of associating postlytically with the nuclear fraction, suggesting that some components of the culture medium might induce modification(s) on Tat molecules that reveal its karyophilicity. Here we exploit in vitro and in vivo assays to investigate the effects of Ca2+ and of cytosolic factors on the nuclear targeting of Tat. DISCUSSIONIn this paper we describe a novel mechanism of control of HIV-1 Tat activity, involving tyrosine phosphorylation/dephosphorylation of cytosolic component(s), whose activation, dependent on Ca2+, unveils the karyophilicity of Tat.The kinetics and the irreversibility of the Ca2+-induced karyophilicity (Fig. 1) suggest that Ca2+ ions do not act directly on Tat itself; this notion is further supported by the observation that chemically synthesized Tat does not associate to nuclei after treatement with Ca2+ unless cytosolic factors are added. Ca2+ may thus activate cellular protein(s) which in turn confer karyophilicity to Tat. As drugs blocking either TK or PTPase inhibit karyophilicity, Ca2+-dependent tyrosine phosphorylation/dephosphorylation events seem to be involved in controlling the nuclear association of Tat. The similar inhibitory effects of drugs acting on targets with opposite functions (TK and PTPase) is not surprising as kinases may be activated by dephosphorylation and vice versa (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar, 2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar, 3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar, 4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar, 5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar, 7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar, 8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar, 9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar, 11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar, 12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar, 14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar, 15Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Google Scholar, 16MacGregor G.R. Caskey C.T. Nucleic Acids Res. 1989; 17: 2365Google Scholar, 17Zappavigna V. Renucci A. Izpisua-Belmonte J.C. Urrier G. Peschle C. Duboule D. EMBO J. 1991; 10: 4177-4187Google Scholar, 18Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar, 19Ensoli B. Bonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Google Scholar, 20Charbonneau H. Tonks N.K. Annu. Rev. Cell Biol. 1992; 8: 463-493Google Scholar). The nature of the TK(s) and PTPase(s) involved, as well as the order of their activation, remain to be investigated. Whatever their nature, the factors are highly conserved through evolution, as demonstrated by the similar activity of wheat germ and HeLa cell cytosolic extracts. Several examples of Ca2+-induced serine/threonine protein kinases or phosphatases involved in activation of transcription factors have been provided (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar, 2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar, 3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar, 4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar, 5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar, 7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar, 8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar, 9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar, 11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar, 12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar, 14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar, 15Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Google Scholar, 16MacGregor G.R. Caskey C.T. Nucleic Acids Res. 1989; 17: 2365Google Scholar, 17Zappavigna V. Renucci A. Izpisua-Belmonte J.C. Urrier G. Peschle C. Duboule D. EMBO J. 1991; 10: 4177-4187Google Scholar, 18Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar, 19Ensoli B. Bonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Google Scholar, 20Charbonneau H. Tonks N.K. Annu. Rev. Cell Biol. 1992; 8: 463-493Google Scholar, 21Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Google Scholar); in contrast, while the importance of TKs and PTPases in the early steps of signal transduction is well established, their direct involvement in regulating the activity of transcription factors is less defined. Tyrosine phosphorylation of the three subunits of the interferon-stimulated gene factor 3 appears to be responsible for their translocation into the nucleus and hence their activation (22Fu X.-Y. Cell. 1992; 70: 323-335Google Scholar); the regulatory enzyme involved is TYK-2, a cytoplasmic, Ca2+-independent TK induced by interferon-α (23Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Google Scholar). Another example of differential targeting modulated by tyrosine phosphorylation is HIV-1 MA protein (8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar).In the case of Tat, substitution of either one or both Tyr-26 or Tyr-47 results in mutant Tat molecules that undergo the same control of nuclear association as wild type Tat. These observations confirm that the Ca2+-activated TK/PTPases involved do not act directly on Tat molecules. Rather, these data suggest that the target of the phosphorylation is accessory protein(s), perhaps involved in chaperoning Tat molecules to the nucleus. In agreement with this, the functional activity is only slightly inhibited when Tyr-26 and/or Tyr-47 are replaced: these mutants are able to transactivate a reporter gene at approximately the same extent as wild type Tat. Three Tat mutants bearing single substitution of Tyr-26 with Ala or Tyr-47 with Ala or histidine have been reported previously (24Kuppuswamy M. Subramanian T. Srinivasan A. Chinnadurai G. Nucleic Acids Res. 1989; 6: 331-357Google Scholar, 25Rice A.P. Carlotti F. J. Virol. 1990; 64: 1864-1868Google Scholar); in these mutants substitution of either tyrosine did not abolish Tat transcriptional activity, although Ala-26 had a weaker activity in HeLa cells. The maintainance of the full activity by the Tat mutants described here may be due to the more conservative substitution of Tyr with Phe.The TK/PTPases-mediated induction of Tat karyophilicity has a functional correspondence in the finding that genistein and vanadate ihibit HIV-1 LTR transactivation in transfected cells, of either lymphoid or non-lymphoid origin. Genistein and vanadate also inhibit transactivating activity of the three Tyr mutants, confirming that the TK/PTPases involved in Tat nuclear targeting and transcriptional activity act on accessory protein(s).Our observations of a more effective transactivation of LTR-β galactosidase in stimulated rather than in resting Jurkat T cells are in line with previous reports (26Siekevitz M. Josephs S.F. Ducovich M. Peffer N. Wong-Staal F. Greene W.C. Science. 1987; 238: 1575-1578Google Scholar, 27Tong-Starksen S.E. Luciw P.A. Peterlin B.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6845-6849Google Scholar) and support the hypothesis of a role for intracellular [Ca2+] in regulating Tat activity. The immunosuppressive drug CsA was shown to block the Tat-mediated increase of IL-2 promoter activity in activated T cells (28Westendorp M.O. Li-Weber M. Frank R.W. Krammer P.H. J. Virol. 1994; 67: 4177-4185Google Scholar). Similarly, we show that CsA inhibits LTR transactivation in stimulated, but not resting, T cells, suggesting that, in activated cells, additional, CsA-sensitive, cellular factors act in concert with Tat with resulting activation of HIV-1 gene expression.Altogether, these findings suggest that, depending on their activation state, HIV-1-infected cells have the possibility of modifying the intracellular fate and the transcriptional activity of Tat by modulating its karyophilicity through activation of TK/PTPase, with obvious influence on viral replication and on the development of HIV-associated syndromes. Compounds that block the activity of these enzymes may lead to novel strategies to slow the spread of the virus in HIV-infected individuals. In this paper we describe a novel mechanism of control of HIV-1 Tat activity, involving tyrosine phosphorylation/dephosphorylation of cytosolic component(s), whose activation, dependent on Ca2+, unveils the karyophilicity of Tat. The kinetics and the irreversibility of the Ca2+-induced karyophilicity (Fig. 1) suggest that Ca2+ ions do not act directly on Tat itself; this notion is further supported by the observation that chemically synthesized Tat does not associate to nuclei after treatement with Ca2+ unless cytosolic factors are added. Ca2+ may thus activate cellular protein(s) which in turn confer karyophilicity to Tat. As drugs blocking either TK or PTPase inhibit karyophilicity, Ca2+-dependent tyrosine phosphorylation/dephosphorylation events seem to be involved in controlling the nuclear association of Tat. The similar inhibitory effects of drugs acting on targets with opposite functions (TK and PTPase) is not surprising as kinases may be activated by dephosphorylation and vice versa (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar, 2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar, 3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar, 4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar, 5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar, 7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar, 8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar, 9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar, 11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar, 12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar, 14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar, 15Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Google Scholar, 16MacGregor G.R. Caskey C.T. Nucleic Acids Res. 1989; 17: 2365Google Scholar, 17Zappavigna V. Renucci A. Izpisua-Belmonte J.C. Urrier G. Peschle C. Duboule D. EMBO J. 1991; 10: 4177-4187Google Scholar, 18Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar, 19Ensoli B. Bonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Google Scholar, 20Charbonneau H. Tonks N.K. Annu. Rev. Cell Biol. 1992; 8: 463-493Google Scholar). The nature of the TK(s) and PTPase(s) involved, as well as the order of their activation, remain to be investigated. Whatever their nature, the factors are highly conserved through evolution, as demonstrated by the similar activity of wheat germ and HeLa cell cytosolic extracts. Several examples of Ca2+-induced serine/threonine protein kinases or phosphatases involved in activation of transcription factors have been provided (1Hunter T. Karin M. Cell. 1992; 70: 375-387Google Scholar, 2Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Google Scholar, 3Tagawa T. Kuroki T. Vogt P.K. Chida K. J. Cell Biol. 1995; 130: 255-263Google Scholar, 4Moll T. Tebb G. Surana U. Robitsch H. Nasmyth K. Cell. 1991; 66: 743-758Google Scholar, 5Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Google Scholar, 6Gosh S. Baltimore D. Nature. 1990; 344: 678-682Google Scholar, 7Danpure C.J. Trends Cell Biol. 1995; 5: 230-238Google Scholar, 8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar, 9Wright C.M. Felber B.K. Paskalis H. Pavlakis G.N. Science. 1986; 234: 988-992Google Scholar, 10Jones K.A. Curr. Opin. Cell Biol. 1993; 5: 461-468Google Scholar, 11Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Google Scholar, 12Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297Google Scholar, 13Westendorp M.O. Rainer F. Ochsenbauer C. Stricker K. Dehin J. Walczak H. Debatin K.-M. Krammer P.H. Nature. 1995; 375: 497-500Google Scholar, 14Bonifaci N. Sitia R. Rubartelli A. AIDS. 1995; 9: 995-1000Google Scholar, 15Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Google Scholar, 16MacGregor G.R. Caskey C.T. Nucleic Acids Res. 1989; 17: 2365Google Scholar, 17Zappavigna V. Renucci A. Izpisua-Belmonte J.C. Urrier G. Peschle C. Duboule D. EMBO J. 1991; 10: 4177-4187Google Scholar, 18Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar, 19Ensoli B. Bonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Google Scholar, 20Charbonneau H. Tonks N.K. Annu. Rev. Cell Biol. 1992; 8: 463-493Google Scholar, 21Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Google Scholar); in contrast, while the importance of TKs and PTPases in the early steps of signal transduction is well established, their direct involvement in regulating the activity of transcription factors is less defined. Tyrosine phosphorylation of the three subunits of the interferon-stimulated gene factor 3 appears to be responsible for their translocation into the nucleus and hence their activation (22Fu X.-Y. Cell. 1992; 70: 323-335Google Scholar); the regulatory enzyme involved is TYK-2, a cytoplasmic, Ca2+-independent TK induced by interferon-α (23Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Google Scholar). Another example of differential targeting modulated by tyrosine phosphorylation is HIV-1 MA protein (8Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar). In the case of Tat, substitution of either one or both Tyr-26 or Tyr-47 results in mutant Tat molecules that undergo the same control of nuclear association as wild type Tat. These observations confirm that the Ca2+-activated TK/PTPases involved do not act directly on Tat molecules. Rather, these data suggest that the target of the phosphorylation is accessory protein(s), perhaps involved in chaperoning Tat molecules to the nucleus. In agreement with this, the functional activity is only slightly inhibited when Tyr-26 and/or Tyr-47 are replaced: these mutants are able to transactivate a reporter gene at approximately the same extent as wild type Tat. Three Tat mutants bearing single substitution of Tyr-26 with Ala or Tyr-47 with Ala or histidine have been reported previously (24Kuppuswamy M. Subramanian T. Srinivasan A. Chinnadurai G. Nucleic Acids Res. 1989; 6: 331-357Google Scholar, 25Rice A.P. Carlotti F. J. Virol. 1990; 64: 1864-1868Google Scholar); in these mutants substitution of either tyrosine did not abolish Tat transcriptional activity, although Ala-26 had a weaker activity in HeLa cells. The maintainance of the full activity by the Tat mutants described here may be due to the more conservative substitution of Tyr with Phe. The TK/PTPases-mediated induction of Tat karyophilicity has a functional correspondence in the finding that genistein and vanadate ihibit HIV-1 LTR transactivation in transfected cells, of either lymphoid or non-lymphoid origin. Genistein and vanadate also inhibit transactivating activity of the three Tyr mutants, confirming that the TK/PTPases involved in Tat nuclear targeting and transcriptional activity act on accessory protein(s). Our observations of a more effective transactivation of LTR-β galactosidase in stimulated rather than in resting Jurkat T cells are in line with previous reports (26Siekevitz M. Josephs S.F. Ducovich M. Peffer N. Wong-Staal F. Greene W.C. Science. 1987; 238: 1575-1578Google Scholar, 27Tong-Starksen S.E. Luciw P.A. Peterlin B.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6845-6849Google Scholar) and support the hypothesis of a role for intracellular [Ca2+] in regulating Tat activity. The immunosuppressive drug CsA was shown to block the Tat-mediated increase of IL-2 promoter activity in activated T cells (28Westendorp M.O. Li-Weber M. Frank R.W. Krammer P.H. J. Virol. 1994; 67: 4177-4185Google Scholar). Similarly, we show that CsA inhibits LTR transactivation in stimulated, but not resting, T cells, suggesting that, in activated cells, additional, CsA-sensitive, cellular factors act in concert with Tat with resulting activation of HIV-1 gene expression. Altogether, these findings suggest that, depending on their activation state, HIV-1-infected cells have the possibility of modifying the intracellular fate and the transcriptional activity of Tat by modulating its karyophilicity through activation of TK/PTPase, with obvious influence on viral replication and on the development of HIV-associated syndromes. Compounds that block the activity of these enzymes may lead to novel strategies to slow the spread of the virus in HIV-infected individuals. We thank Drs. F. Blasi, B. Ensoli, M. Emerman, and V. Zappavigna for reagents and advice and Dr. G. Fassina for generously supplying chemically synthesized Tat and biotinylated Tat. We also thank Dr. C. E. Grossi for support and suggestions." @default.
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