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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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HIV-1 Tat is phosphorylated in cultured cells. HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 3, 4, 6 and 7). Lanes 1, 2, and 5 – control uninfected cells. At 48 hours post infection cells were labeled with (32P)-orthophosphate for 2 hours without (lanes 1, 3 and 6) or with (lanes 2, 4, 5 and 7) 1 μM okadaic acid (OA). Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Tat rabbit polyclonal antibodies (lanes 1–4) or anti-Flag monoclonal murine antibodies (lanes 5–7). Immunoprecipitated Tat was resolved by 15% Tris-Tricine SDS-PAGE, and transferred to polyvinylidene fluoride membrane. A, immunoblot of the membrane with anti-Tat monoclonal antibodies using the 3,3'-diaminobenzidine enhancer system. B, autoradiography of the membrane on Phosphor Imager. C, quantification of panel B. The position of light chain of IgG recognized in anti-Flag immunoprecipitates by anti-mouse HRP-conjugated secondary antibodies is indicated by asterisk.
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Figure 3: HIV-1 Tat is phosphorylated in cultured cells. HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 3, 4, 6 and 7). Lanes 1, 2, and 5 – control uninfected cells. At 48 hours post infection cells were labeled with (32P)-orthophosphate for 2 hours without (lanes 1, 3 and 6) or with (lanes 2, 4, 5 and 7) 1 μM okadaic acid (OA). Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Tat rabbit polyclonal antibodies (lanes 1–4) or anti-Flag monoclonal murine antibodies (lanes 5–7). Immunoprecipitated Tat was resolved by 15% Tris-Tricine SDS-PAGE, and transferred to polyvinylidene fluoride membrane. A, immunoblot of the membrane with anti-Tat monoclonal antibodies using the 3,3'-diaminobenzidine enhancer system. B, autoradiography of the membrane on Phosphor Imager. C, quantification of panel B. The position of light chain of IgG recognized in anti-Flag immunoprecipitates by anti-mouse HRP-conjugated secondary antibodies is indicated by asterisk.

Mentions: Previous attempts to detect Tat phosphorylation in vivo were unsuccessful [25]. We hypothesized that low level of Tat expression after transfection and/or rapid de-phosphorylation by cellular phosphatases might prevent detection of Tat phosphorylation in vivo. To overcome these difficulties, we expressed Flag-tagged Tat which we found to be expressed to a higher level in COS-7 cells than untagged Tat (Fig. 2, compare lanes 3 and 4). To facilitate expression of Flag-Tat in HeLa cells we used adenovirus-mediated expression of Tat [31]. HeLa cells were infected with Adeno-Tat and incubated 48 hours post infection to allow expression of Tat. Then cells were pulsed with (32P)-labeled orthophosphate, and Tat was immunoprecipitated from cellular lysates using monoclonal anti-Flag or polyclonal anti-Tat antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE on 15% Tris-Tricine gel [32] and transferred to PVDF membrane. The membrane was probed with monoclonal anti-Tat antibodies (Fig. 3A) using 3,3'-Diaminobenzidine enhancer system (DABM, Sigma), and also exposed to a PhosphoImager screen (Fig. 3B). Both antibodies precipitated a well detectable amount of Tat protein (Fig. 3A, lanes 3, 4, 6 and 7). Under these experimental conditions, Tat was phosphorylated (Figs. 3B and 3C, compare lane 3 to lane 1 and lane 6 to lane 5). Next we treated cells with okadaic acid, an inhibitor of PPP-type phosphatases, to prevent rapid dephosphorylation of Tat in the cells and during the lysis procedure. Treatment with okadaic acid did not change the amount of precipitated Tat (Fig. 3A, lanes 3, 4, 6 and 7). In contrast, Tat phosphorylation was significantly enhanced when cells were treated with okadaic acid (Figs.3B and 3C, lanes 4 and 7). Taken together, these results indicate that Tat is phosphorylated in vivo and that Tat phosphorylation is enhanced when cells are treated with the inhibitor of PPP-phosphatases.


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)

HIV-1 Tat is phosphorylated in cultured cells. HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 3, 4, 6 and 7). Lanes 1, 2, and 5 – control uninfected cells. At 48 hours post infection cells were labeled with (32P)-orthophosphate for 2 hours without (lanes 1, 3 and 6) or with (lanes 2, 4, 5 and 7) 1 μM okadaic acid (OA). Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Tat rabbit polyclonal antibodies (lanes 1–4) or anti-Flag monoclonal murine antibodies (lanes 5–7). Immunoprecipitated Tat was resolved by 15% Tris-Tricine SDS-PAGE, and transferred to polyvinylidene fluoride membrane. A, immunoblot of the membrane with anti-Tat monoclonal antibodies using the 3,3'-diaminobenzidine enhancer system. B, autoradiography of the membrane on Phosphor Imager. C, quantification of panel B. The position of light chain of IgG recognized in anti-Flag immunoprecipitates by anti-mouse HRP-conjugated secondary antibodies is indicated by asterisk.
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Related In: Results  -  Collection

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Figure 3: HIV-1 Tat is phosphorylated in cultured cells. HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods (lanes 3, 4, 6 and 7). Lanes 1, 2, and 5 – control uninfected cells. At 48 hours post infection cells were labeled with (32P)-orthophosphate for 2 hours without (lanes 1, 3 and 6) or with (lanes 2, 4, 5 and 7) 1 μM okadaic acid (OA). Whole cell extracts were prepared and Tat was immunoprecipitated with anti-Tat rabbit polyclonal antibodies (lanes 1–4) or anti-Flag monoclonal murine antibodies (lanes 5–7). Immunoprecipitated Tat was resolved by 15% Tris-Tricine SDS-PAGE, and transferred to polyvinylidene fluoride membrane. A, immunoblot of the membrane with anti-Tat monoclonal antibodies using the 3,3'-diaminobenzidine enhancer system. B, autoradiography of the membrane on Phosphor Imager. C, quantification of panel B. The position of light chain of IgG recognized in anti-Flag immunoprecipitates by anti-mouse HRP-conjugated secondary antibodies is indicated by asterisk.
Mentions: Previous attempts to detect Tat phosphorylation in vivo were unsuccessful [25]. We hypothesized that low level of Tat expression after transfection and/or rapid de-phosphorylation by cellular phosphatases might prevent detection of Tat phosphorylation in vivo. To overcome these difficulties, we expressed Flag-tagged Tat which we found to be expressed to a higher level in COS-7 cells than untagged Tat (Fig. 2, compare lanes 3 and 4). To facilitate expression of Flag-Tat in HeLa cells we used adenovirus-mediated expression of Tat [31]. HeLa cells were infected with Adeno-Tat and incubated 48 hours post infection to allow expression of Tat. Then cells were pulsed with (32P)-labeled orthophosphate, and Tat was immunoprecipitated from cellular lysates using monoclonal anti-Flag or polyclonal anti-Tat antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE on 15% Tris-Tricine gel [32] and transferred to PVDF membrane. The membrane was probed with monoclonal anti-Tat antibodies (Fig. 3A) using 3,3'-Diaminobenzidine enhancer system (DABM, Sigma), and also exposed to a PhosphoImager screen (Fig. 3B). Both antibodies precipitated a well detectable amount of Tat protein (Fig. 3A, lanes 3, 4, 6 and 7). Under these experimental conditions, Tat was phosphorylated (Figs. 3B and 3C, compare lane 3 to lane 1 and lane 6 to lane 5). Next we treated cells with okadaic acid, an inhibitor of PPP-type phosphatases, to prevent rapid dephosphorylation of Tat in the cells and during the lysis procedure. Treatment with okadaic acid did not change the amount of precipitated Tat (Fig. 3A, lanes 3, 4, 6 and 7). In contrast, Tat phosphorylation was significantly enhanced when cells were treated with okadaic acid (Figs.3B and 3C, lanes 4 and 7). Taken together, these results indicate that Tat is phosphorylated in vivo and that Tat phosphorylation is enhanced when cells are treated with the inhibitor of PPP-phosphatases.

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