Characterization of Cdk9 T-loop phosphorylation in resting and activated CD4+ T lymphocytes

Ramakrishnan, Rajesh; Dow, Eugene C.; Rice, Andrew P.

doi:10.1189/jlb.0509309

Cited by 56 publications

(89 citation statements)

References 28 publications

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“…As a positive control for CycT1 and pCDK9 expression, we examined the levels of both proteins in Jurkat cells with or without TNF-␣ stimulation. In agreement with previous findings (33,38,53,70), Jurkat cells had high endogenous levels of CycT1 and pCDK9 that were not affected by TNF-␣ stimulation, while resting memory T cells had low levels of nuclear CycT1 and pCDK9 that increased dramatically following overnight incubation with anti-CD3/anti-CD28 beads (Fig. 6).…”

Section: Tnf-␣ Reactivates Latent Virus In Hpcs Through An Nf-bdependsupporting

confidence: 81%

Latent HIV-1 Infection Occurs in Multiple Subsets of Hematopoietic Progenitor Cells and Is Reversed by NF-κB Activation

McNamara

Ganesh

Collins

2012

J Virol

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The ability of HIV-1 to establish a latent infection presents a barrier to curing HIV. The best-studied reservoir of latent virus in vivo is resting memory CD4 + T cells, but it has recently been shown that CD34 + hematopoietic progenitor cells (HPCs) can also become latently infected by HIV-1 in vitro and in vivo . CD34 + cells are not homogenous, however, and it is not yet known which types of CD34 + cells support a latent infection. Furthermore, the mechanisms through which latency is established in this cell type are not yet known. Here we report the development of a primary cell model for latent HIV-1 infection in HPCs. We demonstrate that in this model, latent infection can be established in all subsets of HPCs examined, including HPCs with cell surface markers consistent with immature hematopoietic stem and progenitor cells. We further show that the establishment of latent infection in these cells can be reversed by tumor necrosis factor alpha (TNF-α) through an NF-κB-dependent mechanism. In contrast, we do not find evidence for a role of positive transcription elongation factor b (P-TEFb) in the establishment of latent infection in HPCs. Finally, we demonstrate that prostratin and suberoylanilide hydroxamic acid (SAHA), but not hexamethylene bisacetamide (HMBA) or 5-aza-2′-deoxycytidine (Aza-CdR), reactivate latent HIV-1 in HPCs. These findings illuminate the mechanisms through which latent infection can be established in HPCs and suggest common pathways through which latent virus could be reactivated in both HPCs and resting memory T cells to eliminate latent reservoirs of HIV-1.

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Section: Tnf-␣ Reactivates Latent Virus In Hpcs Through An Nf-bdependsupporting

confidence: 81%

Latent HIV-1 Infection Occurs in Multiple Subsets of Hematopoietic Progenitor Cells and Is Reversed by NF-κB Activation

McNamara

Ganesh

Collins

2012

J Virol

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“…In resting memory CD4 + T cells, which form the bulk of the latent HIV reservoir (32), P-TEFb is not yet formed (33,34). In these quiescent cells, CycT1 is largely absent and CDK9 is found in the cytoplasm (35,36), where it is thought to be in an immature, inactive state. Here we demonstrated that in resting CD4 + T cells Hsp90/Cdc37 stabilizes the basal expression of CDK9 prior to the elevation of CycT1 protein levels.…”

Section: Introductionmentioning

confidence: 99%

Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation

Mbonye

Wang

Gokulrangan

et al. 2018

Journal of Biological Chemistry

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The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr186 on the activation loop (T-loop) of cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved Tloop site, Ser175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr186 by an arginine triad and Glu96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr186-dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser175 phosphorylation. Mutation of Ser175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.http://www.jbc.org/cgi

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“…Activation of these cells leads to significant induction of cyclin T1 protein levels, primarily by a posttranscriptional mechanism (33,38). Similarly, pCDK9 levels are considerably upregulated upon activation (30). In this study, we wished to determine if P-TEFb shows a similar pattern of regulation in naïve and quiescent memory CD4 ϩ T cells that were either uninfected or latently infected with HIV-1.…”

Section: Expression Of P-tefb In Naïve and Central Memory Cd4mentioning

confidence: 99%

“…While the total levels of CDK9 remain relatively constant through resting versus activated states, the levels of T-loop-phosphorylated CDK9 (pCDK9) are very low in resting CD4 ϩ T cells due to the action of the phosphatase PPM1A (29,30). However, activation of these cells induces phosphorylation of the CDK9 T loop by an activating kinase that has recently been shown to be CDK7 (31).…”

mentioning

confidence: 99%

Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4⁺T Cells

Budhiraja

Famiglietti

Bosque

et al. 2013

J Virol

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111

110

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c P-TEFb, a cellular kinase composed of Cyclin T1 and CDK9, is essential for processive HIV-1 transcription. P-TEFb activity is dependent on phosphorylation of Thr186 in the CDK9 T loop. In resting CD4؉ T cells which are nonpermissive for HIV-1 replication, the levels of Cyclin T1 and T-loop-phosphorylated CDK9 are very low but increase significantly upon cellular activation. Little is known about how P-TEFb activity and expression are regulated in resting central memory CD4؉ T cells, one of the main reservoirs of latent HIV-1. We used an in vitro primary cell model of HIV-1 latency to show that P-TEFb availability in resting memory CD4؉ T cells is governed by the differential expression and phosphorylation of its subunits. This is in contrast to previous observations in dividing cells, where P-TEFb can be regulated by its sequestration in the 7SK RNP complex. We find that resting CD4؉ T cells, whether naïve or memory and independent of their infection status, have low levels of Cyclin T1 and Tloop-phosphorylated CDK9, which increase upon activation. We also show that the decrease in Cyclin T1 protein upon the acquisition of a memory phenotype is in part due to proteasome-mediated proteolysis and likely also to posttranscriptional downregulation by miR-150. We also found that HEXIM1 levels are very low in ex vivo-and in vitro-generated resting memory CD4 ؉ T cells, thus limiting the sequestration of P-TEFb in the 7SK RNP complex, indicating that this mechanism is unlikely to be a driver of viral latency in this cell type.

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Characterization of Cdk9 T-loop phosphorylation in resting and activated CD4+ T lymphocytes

Cited by 56 publications

References 28 publications

Latent HIV-1 Infection Occurs in Multiple Subsets of Hematopoietic Progenitor Cells and Is Reversed by NF-κB Activation

Latent HIV-1 Infection Occurs in Multiple Subsets of Hematopoietic Progenitor Cells and Is Reversed by NF-κB Activation

Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation

Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4⁺T Cells

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Characterization of Cdk9 T-loop phosphorylation in resting and activated CD4+ T lymphocytes

Cited by 56 publications

References 28 publications

Latent HIV-1 Infection Occurs in Multiple Subsets of Hematopoietic Progenitor Cells and Is Reversed by NF-κB Activation

Latent HIV-1 Infection Occurs in Multiple Subsets of Hematopoietic Progenitor Cells and Is Reversed by NF-κB Activation

Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation

Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4+T Cells

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Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4⁺T Cells