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Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription.

Ammosova T, Berro R, Jerebtsova M, Jackson A, Charles S, Klase Z, Southerland W, Gordeuk VR, Kashanchi F, Nekhai S - Retrovirology (2006)

Bottom Line: CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat.Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells.Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Sickle Cell Disease, Howard University College of Medicine, Washington, DC 20059, USA. tammosova@mail.ru

ABSTRACT

Background: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.

Results: We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease.

Conclusion: Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

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Tat and CDK2 co-migrate on glycerol gradient. 293T cell lysated from the cells infected with Adeno-Tat were fractionated on 10%–30% glycerol gradients by centrifugation and analyzed with indicated antibodies by Immunoblotting.
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Figure 6: Tat and CDK2 co-migrate on glycerol gradient. 293T cell lysated from the cells infected with Adeno-Tat were fractionated on 10%–30% glycerol gradients by centrifugation and analyzed with indicated antibodies by Immunoblotting.

Mentions: We next investigated whether Tat phosphorylation was mediated by CDK2 in vivo using CDK2-directed RNA interference [33]. HeLa cells were infected with Adeno-Tat and subsequently transfected with siRNAs against CDK2. Transfection of HeLa cells with siRNAs against CDK2 decreased the level of expression of CDK2 by 2.5-fold (Figs. 4A and 4B, lane 3). A control non-targeting siRNA pool did not affect expression of CDK2 (Figs. 4A and 4B, lane 2). The non-targeting siRNA control was used to ensure that transfection itself did not affect CDK2 expression. Western blot analysis of tubulin and CDK9 was used as control for the specificity of siRNAs. As shown in Fig. 4A transfection with both siRNAs did not affect the level of α-tubulin expression. To ensure that CDK2-directed siRNA decreased the enzymatic activity of cellular CDK2, CDK2 was immunoprecipitated from cells transfected with non-targeting or CDK2-directed siRNA and assayed for its enzymatic activity using recombinant Tat as a substrate. The activity of CDK2 was decreased in the cells transfected with CDK2-directed siRNA (Fig. 4C, lane 2) as compared to the cells transfected with non-targeting siRNA (Fig. 4C, lane 1). Next the cells were infected with Adeno-Tat, transfected with non-targeting or CDK2-directed siRNAs and pulse-labeled with (32P). In this experiment no okadaic acid was used. Inhibition of CDK2 by siRNA reduced the level of Tat phosphorylation by 3-fold (Figs. 5A,B and 5C, compare lanes 2 and 3). Thus CDK2 is likely to mediate Tat phosphorylation in cultured cells. To analyze whether Tat and CDK2 might be present in the same molecular weight complex, we analyzed sedimentation of Tat and CDK2 by ultracentrifugation on a glycerol gradient (Fig. 6). Both Tat and CDK2 co-migrated in fractions 1–8 (Fig. 6). The CDK9 was present in most of the fractions with the peak in fractions 4–5 and 9–10 (Fig. 6), which is likely to correspond to low and high molecular weight P-TEFb complexes. HEXIM1, and Brd4 were mostly present in fractions 2–6, although HEXIM1 was also detected in higher molecular weight fractions 10 and 11 (Fig. 6). Neither Tat nor CDK2 co-migrated with RNAPII which was present in fractions 1–13 (Fig. 6). We further analyzed association of Tat with CDK2 by immunoprecipitation. Flag-Tat was expressed in HeLa cells by infection with Adeno-Tat and precipitated from cellular extracts with anti-Flag antibodies (Fig. 7A). CDK2 co-precipitated with Tat (Fig. 7A, lane 4). CDK2 was not precipitated with anti-Tat antiserum from non-infected cells (Fig. 7A, lane 3) or not with non-specific preimmune serum from adeno-Tat infected cells (Fig. 7A, lane 5). Inhibition of CDK2 expression by CDK2-specific RNAi significantly reduced CDK2 co-precipitated to Tat apparently due reduction of the expressed CDK2 (Fig. 7B, compare lane 4 to lane 2). Association of Tat with CDK9 was slightly reduced (Fig. 6B) but this reduction correlated to the decreased amount of Tat precipitated by anti-Flag antibodies. Binding of Tat-cyclin T1 was not reduced (Fig. 7C, lanes 3 and 5). The cyclin T2 did not precipitate with Tat (Fig. 7C), which indicated a specificity of the immunoprecipitation. Taking together, these results suggest that CDK2 associates with Tat in cultured cells, and that inhibition of CDK2 expression prevents Tat phosphorylation. Thus, CDK2 is likely to phosphorylate Tat directly in cultured cells.


Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription.

Ammosova T, Berro R, Jerebtsova M, Jackson A, Charles S, Klase Z, Southerland W, Gordeuk VR, Kashanchi F, Nekhai S - Retrovirology (2006)

Tat and CDK2 co-migrate on glycerol gradient. 293T cell lysated from the cells infected with Adeno-Tat were fractionated on 10%–30% glycerol gradients by centrifugation and analyzed with indicated antibodies by Immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1636661&req=5

Figure 6: Tat and CDK2 co-migrate on glycerol gradient. 293T cell lysated from the cells infected with Adeno-Tat were fractionated on 10%–30% glycerol gradients by centrifugation and analyzed with indicated antibodies by Immunoblotting.
Mentions: We next investigated whether Tat phosphorylation was mediated by CDK2 in vivo using CDK2-directed RNA interference [33]. HeLa cells were infected with Adeno-Tat and subsequently transfected with siRNAs against CDK2. Transfection of HeLa cells with siRNAs against CDK2 decreased the level of expression of CDK2 by 2.5-fold (Figs. 4A and 4B, lane 3). A control non-targeting siRNA pool did not affect expression of CDK2 (Figs. 4A and 4B, lane 2). The non-targeting siRNA control was used to ensure that transfection itself did not affect CDK2 expression. Western blot analysis of tubulin and CDK9 was used as control for the specificity of siRNAs. As shown in Fig. 4A transfection with both siRNAs did not affect the level of α-tubulin expression. To ensure that CDK2-directed siRNA decreased the enzymatic activity of cellular CDK2, CDK2 was immunoprecipitated from cells transfected with non-targeting or CDK2-directed siRNA and assayed for its enzymatic activity using recombinant Tat as a substrate. The activity of CDK2 was decreased in the cells transfected with CDK2-directed siRNA (Fig. 4C, lane 2) as compared to the cells transfected with non-targeting siRNA (Fig. 4C, lane 1). Next the cells were infected with Adeno-Tat, transfected with non-targeting or CDK2-directed siRNAs and pulse-labeled with (32P). In this experiment no okadaic acid was used. Inhibition of CDK2 by siRNA reduced the level of Tat phosphorylation by 3-fold (Figs. 5A,B and 5C, compare lanes 2 and 3). Thus CDK2 is likely to mediate Tat phosphorylation in cultured cells. To analyze whether Tat and CDK2 might be present in the same molecular weight complex, we analyzed sedimentation of Tat and CDK2 by ultracentrifugation on a glycerol gradient (Fig. 6). Both Tat and CDK2 co-migrated in fractions 1–8 (Fig. 6). The CDK9 was present in most of the fractions with the peak in fractions 4–5 and 9–10 (Fig. 6), which is likely to correspond to low and high molecular weight P-TEFb complexes. HEXIM1, and Brd4 were mostly present in fractions 2–6, although HEXIM1 was also detected in higher molecular weight fractions 10 and 11 (Fig. 6). Neither Tat nor CDK2 co-migrated with RNAPII which was present in fractions 1–13 (Fig. 6). We further analyzed association of Tat with CDK2 by immunoprecipitation. Flag-Tat was expressed in HeLa cells by infection with Adeno-Tat and precipitated from cellular extracts with anti-Flag antibodies (Fig. 7A). CDK2 co-precipitated with Tat (Fig. 7A, lane 4). CDK2 was not precipitated with anti-Tat antiserum from non-infected cells (Fig. 7A, lane 3) or not with non-specific preimmune serum from adeno-Tat infected cells (Fig. 7A, lane 5). Inhibition of CDK2 expression by CDK2-specific RNAi significantly reduced CDK2 co-precipitated to Tat apparently due reduction of the expressed CDK2 (Fig. 7B, compare lane 4 to lane 2). Association of Tat with CDK9 was slightly reduced (Fig. 6B) but this reduction correlated to the decreased amount of Tat precipitated by anti-Flag antibodies. Binding of Tat-cyclin T1 was not reduced (Fig. 7C, lanes 3 and 5). The cyclin T2 did not precipitate with Tat (Fig. 7C), which indicated a specificity of the immunoprecipitation. Taking together, these results suggest that CDK2 associates with Tat in cultured cells, and that inhibition of CDK2 expression prevents Tat phosphorylation. Thus, CDK2 is likely to phosphorylate Tat directly in cultured cells.

Bottom Line: CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat.Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells.Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Sickle Cell Disease, Howard University College of Medicine, Washington, DC 20059, USA. tammosova@mail.ru

ABSTRACT

Background: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.

Results: We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease.

Conclusion: Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

Show MeSH
Related in: MedlinePlus