scholarly journals Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways

2021 ◽  
Vol 17 (9) ◽  
pp. e1009581
Author(s):  
Uri Mbonye ◽  
Konstantin Leskov ◽  
Meenakshi Shukla ◽  
Saba Valadkhan ◽  
Jonathan Karn
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
T Cell ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Map Kinases ◽  
Cell Model ◽  
Hiv Latency ◽  
Kinase C ◽  
Latent Hiv

The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4+ T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4+ T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.

scholarly journals Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways

2021 ◽  
Author(s):  
Uri Mbonye ◽  
Konstantin Leskov ◽  
Meenakshi Shukla ◽  
Saba Valadkhan ◽  
Jonathan Karn
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
T Cell ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Cd4 T Cells ◽  
Hiv Latency ◽  
Kinase C ◽  
Latent Hiv ◽  
Memory Cd4 T Cells

The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4 + T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4 + T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4 + T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.

scholarly journals Identification of Combinations of Protein Kinase C Activators and Histone Deacetylase Inhibitors that Potently Reactivate Latent HIV

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 609
Author(s):  
Francesca Curreli ◽  
Shahad Ahmed ◽  
Sofia M. Benedict Victor ◽  
Asim K. Debnath
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Synergistic Effect ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Latent Virus ◽  
Hiv Latency ◽  
Kinase C ◽  
Protein Kinase C Activators ◽  
Latent Hiv

Combination antiretroviral therapy (cART) is successful in maintaining undetectable levels of HIV in the blood; however, the persistence of latent HIV reservoirs has become the major barrier for a HIV cure. Substantial efforts are underway in finding the best latency-reversing agents (LRAs) to purge the latent viruses from the reservoirs. We hypothesize that identifying the right combination of LRAs will be the key to accomplishing that goal. In this study, we evaluated the effect of combinations of three protein kinase C activators (prostratin, (-)-indolactam V, and TPPB) with four histone deacetylase inhibitors (AR-42, PCI-24781, givinostat, and belinostat) on reversing HIV latency in different cell lines including in a primary CD4+ T-cell model. Combinations including indolactam and TPPB with AR-42 and PCI produced a strong synergistic effect in reactivating latent virus as indicated by higher p24 production and envelope gp120 expression. Furthermore, treatment with TPPB and indolactam greatly downregulated the cellular receptor CD4. Indolactam/AR-42 combination emerged from this study as the best combination that showed a strong synergistic effect in reactivating latent virus. Although AR-42 alone did not downregulate CD4 expression, indolactam/AR-42 showed the most efficient downregulation. Our results suggest that indolactam/AR-42 is the most effective combination, showing a strong synergistic effect in reversing HIV latency combined with the most efficient CD4 downregulation.

scholarly journals A Model System for Activation-Induced Alternative Splicing of CD45 Pre-mRNA in T Cells Implicates Protein Kinase C and Ras

2000 ◽  
Vol 20 (1) ◽  
pp. 70-80 ◽  
Author(s):  
Kristen W. Lynch ◽  
Arthur Weiss
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
Alternative Splicing ◽  
T Cell ◽  
Protein Kinase ◽  
Cell Line ◽  
T Cell Activation ◽  
Cell Activation ◽  
Model System ◽  
Kinase C

ABSTRACT Multiple isoforms of the protein tyrosine phosphatase CD45 are expressed on the surface of human T cells. Interestingly, the expression of these isoforms has been shown to vary significantly upon T-cell activation. In this report, we describe a novel cell line-based model system in which we can mimic the activation-induced alternative splicing of CD45 observed in primary T cells. Of the many proximal signaling events induced by T-cell stimulation, we show that activation of protein kinase C and activation of Ras are important for the switch toward the exclusion of CD45 variable exons, whereas events related to Ca2+ flux are not. In addition, the ability of cycloheximide to block the activation-induced alternative splicing of CD45 suggests a requirement for de novo protein synthesis. We further demonstrate that sequences which have previously been implicated in the tissue-specific regulation of CD45 variable exons are likewise necessary and sufficient for activation-induced splicing. These results provide an initial understanding of the requirements for CD45 alternative splicing upon T-cell activation, and they confirm the importance of this novel cell line in facilitating a more detailed analysis of the activation-induced regulation of CD45 than has been previously possible.

scholarly journals Dual modes of control of c-fos mRNA induction by intracellular calcium in T cells.

1994 ◽  
Vol 14 (7) ◽  
pp. 4579-4587 ◽  
Author(s):  
G Lee ◽  
M Gilman
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
T Cell ◽  
Protein Kinase ◽  
Intracellular Calcium ◽  
Molecular Mechanisms ◽  
Calcium Ionophore ◽  
Cytoplasmic Calcium ◽  
Mrna Induction ◽  
Kinase C

Cytoplasmic calcium is a nearly universal second messenger in eukaryotes. In many cell types, elevated intracellular calcium interacts synergistically with inducers of protein kinase C to elicit activation of complete biological programs normally induced by extracellular signals. In T cells, elevated cytoplasmic calcium is a critical mediator of activation in response to stimulation of the antigen receptor, and in some T-cell lines, treatment with a combination of calcium ionophore and protein kinase C activator mimics authentic antigen treatment. The synergistic interaction of calcium and protein kinase C in T cells is also observed at the level of gene expression. Here we examine the molecular mechanisms through which these agents exert synergistic control over the expression of the c-fos proto-oncogene in a T-cell hybridoma. We find that the principal effect of calcium is on the elongation of c-fos transcripts. This step constitutes the major control of c-fos mRNA accumulation in these cells. In addition, calcium regulates the initiation of c-fos transcription. This effect requires the serum response element of the c-fos gene and an additional sequence immediately 3' to this element. Thus, calcium regulates c-fos expression through at least two distinct molecular pathways.

scholarly journals The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter.

1992 ◽  
Vol 175 (3) ◽  
pp. 853-862 ◽  
Author(s):  
J Jain ◽  
V E Valge-Archer ◽  
A J Sinskey ◽  
A Rao
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
T Cell ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Interleukin 2 ◽  
Prolonged Treatment ◽  
Nf Kappa B ◽  
Kinase C ◽  
T Cell Clone

Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter.

scholarly journals Protein Kinase C-Theta Interacts with mTORC2 and Vimentin to Limit Regulatory T-Cell Function

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 849-849
Author(s):  
Cameron McDonald-Hyman ◽  
Govindarajan Thangavelu ◽  
James Muller ◽  
Guoan Zhang ◽  
Sudha Kumari ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
T Cell ◽  
Protein Kinase ◽  
Acute Gvhd ◽  
Hematopoietic Stem ◽  
Phosphorylated Proteins ◽  
Kinase C ◽  
Treg Function

Abstract Regulatory T-cells (Tregs) play a critical role in preventing autoimmune and alloimmune reactions, including graft-versus-host disease (GVHD). Two recent clinical trials demonstrated that in patients undergoing hematopoietic stem cell transplantation, adoptive transfer of Tregs significantly reduced the incidence of grades II-IV GVHD. While Tregs significantly reduced GVHD severity, they did not eliminate GVHD. One potential way to augment Treg-mediated inhibition of GVHD is to increase Treg suppressive potency. We showed previously that Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function (Science 328:372, 2010). However, it is unclear whether PKC-θ inhibition can boost Treg function in a systemic inflammatory condition like GVHD. Furthermore, the mechanism by which PKC-θ inhibition augments Treg function is unknown. In this study, we address these unanswered questions. Using a mouse MHC class I/II disparate acute GVHD model, we found that freshly isolated Tregs treated for 30 minutes with 10uM of the clinically available PKC-θ inhibitor AEB071 suppressed GVHD mortality (Fig 1A) and severity significantly better than DMSO treated Tregs. As Tregs exert much of their protective effect against GVHD early in the course of the disease, we analyzed proliferation of GVHD-causing conventional T-cells (Tcon) on D4 after transplant. We observed a significant reduction in Tcon proliferation in mice given AEB071 treated Tregs compared to DMSO treated Tregs. We then performed multi-photon microscopy on D4 after transplant using TEα-GFP Tcon, CD11c-eYFP antigen presenting cells (APCs) and wild-type Tregs. Compared to DMSO, AEB071 treated Tregs significantly increased Tcon velocity and displacement from APCs. Increased velocity and displacement are indicative of decreased Tcon-APC interactions, suggesting reduced priming when AEB071 Tregs are present. Mechanistically, AEB071 vs DMSO treatment of Tregs resulted in augmented expression of the suppressive molecules Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3) after in vitro activation (Fig 1B, C) and in Tregs isolated from acute GVHD mice. Antibody blockade of Nrp1 and Lag3 in in vitro transwell suppression assays reduced the effect of AEB071 treatment, suggesting that these molecules may play a role in enhancing Treg function after PKC-θ inhibition. Flow cytometry analysis of phosphorylated proteins in activated Tregs revealed that PKC-θ inhibition resulted in reduced phosphorylation of the mTORC2 target FoxO3a, but not mTORC1 targets S6 and 4E-BP1. In addition, the mTORC2-specific phosphorylation site on Akt, serine 473, was reduced, whereas the mTORC1-specific site, threonine 308, was unaltered. Together, these data suggest reduced mTORC2 activity. Reduced phosphorylation increases Foxo3a nuclear translocation, which may result in increased Nrp1 and Lag3 expression, since Foxo3a has binding sites in both gene promoters. As both mTORC1 and 2 are involved in T-cell metabolism, we investigated the effect of AEB071 treatment on Treg oxygen consumption rate (OCR). Compared to DMSO, AEB071 treatment significantly increased Treg baseline and maximal OCRs after activation (Fig 1D). Increased OCR has been associated with increased Treg function. To identify additional alterations in phosphorylated proteins after PKC-θ inhibition, we performed a phosphoproteomic screen using in vitro expanded human Tregs treated with AEB701 or DMSO. We identified significant alterations in phosphorylation sites on 72 proteins, including reduced phosphorylation of an adaptor molecule that links PKC-θ to the intermediate filament vimentin. We found that vimentin is highly upregulated in Tregs compared to Tcon and that in Tregs, vimentin interacts with PKC-θ after activation. AEB071 treatment reduced the interaction between vimentin and PKC-θ. As with AEB071 treatment, Vimentin siRNA significantly increased Treg suppression in vitro compared to control transfected Tregs (Fig 1E), and augmented expression of Nrp1 and Lag3. AEB071 treatment of vimentin siRNA transfected Tregs did not further augment Treg function, suggesting an overlapping mechanism. In summary, our data demonstrate that PKC-θ interacts with mTORC2 and vimentin to modulate multiple aspects of Treg function, and that a brief incubation of Tregs with a PKC-θ inhibitor may be a viable method to enhance the efficacy of Treg therapeutics. Disclosures No relevant conflicts of interest to declare.

Protein kinase C: a regulator of cytoskeleton remodelling and T-cell migration

2014 ◽  
Vol 42 (6) ◽  
pp. 1490-1497 ◽  
Author(s):  
Aideen Long ◽  
Michael Freeley
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
Cell Migration ◽  
T Cell ◽  
Protein Kinase ◽  
Immune Responses ◽  
Chemokine Receptors ◽  
T Cell Subsets ◽  
T Cell Migration ◽  
Kinase C

Protein kinase C (PKC) is a family of ten serine/threonine kinases that have diverse roles in the signalling pathways regulating cellular proliferation, differentiation, apoptosis and immune responses. Elucidating roles for individual PKC isoforms in the immune responses of T-cells have long been a challenging prospect, because these cells are known to express nine of these isoforms. A variety of approaches including the use of knockout mice, overexpression of kinase-inactive mutants, cell-permeable peptides, pharmacological inhibitors and siRNAs have shown that PKCs regulate the production of inflammatory cytokines and the cytotoxic responses of various T-cell subsets. Central to the T-cell immune response is a requirement to migrate to various organs and tissues in search of pathogens and micro-organisms. T-cell migration is guided by specific sets of chemokines and integrin ligands that activate their cognate chemokine receptors and integrins on T-cells, resulting in remodelling of the cytoskeleton and the dynamic protrusive/contractile forces necessary for cell adhesion and motility. In the present article, we review the role of PKC in T-cell migration, with an emphasis on studies that have defined their roles in cytoskeletal remodelling, cell polarity and intracellular trafficking downstream of chemokine receptors and integrins.

scholarly journals Integrin-mediated Tyrosine Phosphorylation of Shc in T Cells Is Regulated by Protein Kinase C-dependent Phosphorylations of Lck

2003 ◽  
Vol 14 (2) ◽  
pp. 349-360 ◽  
Author(s):  
Shi Niu ◽  
Haichun Xie ◽  
Eugene E. Marcantonio
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
T Cell ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Receptor ◽  
Serine Phosphorylation ◽  
Kinase C

Integrin receptor signals are costimulatory for mitogenesis with the T-cell receptor during T-cell activation. A subset of integrin receptors can link to the adapter protein Shc and provide a mitogenic stimulus. Using a combination of genetic and pharmacological approaches, we show herein that integrin signaling to Shc in T cells requires the receptor tyrosine phosphatase CD45, the Src family kinase member Lck, and protein kinase C. Our results suggest a model in which integrin-dependent serine phosphorylation of Lck is the critical step that determines the efficiency of Shc tyrosine phosphorylation in T cells. Serine phosphorylation of Lck is dependent on PKC and is also linked to CD45 dephosphorylation. Mutants of Lck that cannot be phosphorylated on the critical serine residues do not signal efficiently to Shc and have greatly reduced kinase activity. This signaling from integrins to Lck may be an important step in the costimulation with the T-cell receptor during lymphocyte activation.

Sign in / Sign up

Export Citation Format

Share Document