scholarly journals Phosphoinositide-dependent kinase 1 targets protein kinase A in a pathway that regulates interleukin 4

2006 ◽  
Vol 203 (7) ◽  
pp. 1733-1744 ◽  
Author(s):  
Ajay Nirula ◽  
Mary Ho ◽  
Hyewon Phee ◽  
Jeroen Roose ◽  
Arthur Weiss
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Critical Role ◽  
Interleukin 4 ◽  
Dependent Manner ◽  
Activated T Cells ◽  
Kinase A ◽  
Phosphoinositide Dependent Kinase

CD28 plays a critical role in T cell immune responses. Although the kinase Akt has been shown to act downstream of CD28 in T helper (Th)1 cytokine induction, it does not induce Th2 cytokines such as interleukin 4 (IL-4). We recently reported that phosphoinositide-dependent kinase 1 (PDK1) partially corrects the defect in IL-4 production present in CD28-deficient T cells, suggesting that PDK1 regulates IL-4 independently of Akt. We now describe a signaling pathway in which PDK1 targets IL-4 in the murine Th2 cell line D10. PDK1-mediated activation of this pathway is dependent on protein kinase A (PKA) and the nuclear factor of activated T cells (NFAT) P1 transcriptional element in the IL-4 promoter. PDK1 localizes to the immune synapse in a phosphatidylinositol 3-kinase–dependent manner, partially colocalizes with PKA at the synapse, and physically interacts with PKA. In RNA interference knockdown experiments, PDK1 is necessary for phosphorylation of PKA in T cells, as well as for activation of the IL-4 NFAT P1 element by the T cell receptor (TCR) and CD28. Phosphorylation of the critical PKA threonine residue is stimulated by engagement of TCR/CD28 via a PDK1-dependent mechanism. These findings together define a pathway linking the kinases PDK1 and PKA in the induction of the Th2 cytokine IL-4.

scholarly journals Phosphoinositide (3,4,5)-Triphosphate Binding to Phosphoinositide-Dependent Kinase 1 Regulates a Protein Kinase B/Akt Signaling Threshold That Dictates T-Cell Migration, Not Proliferation

2009 ◽  
Vol 29 (21) ◽  
pp. 5952-5962 ◽  
Author(s):  
Caryll Waugh ◽  
Linda Sinclair ◽  
David Finlay ◽  
Jose R. Bayascas ◽  
Doreen Cantrell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Effector T Cells ◽  
T Cell Response ◽  
Cell Trafficking ◽  
Ph Domain ◽  
Activated T Cells ◽  
Phosphoinositide Dependent Kinase

ABSTRACT The present study explored the consequences of phosphoinositide (3,4,5)-triphosphate [PI(3,4,5)P3] binding to the pleckstrin homology (PH) domain of the serine/threonine kinase 3-phosphoinositide-dependent kinase 1 (PDK1). The salient finding is that PDK1 directly transduces the PI(3,4,5)P3 signaling that determines T-cell trafficking programs but not T-cell growth and proliferation. The integrity of the PDK1 PH domain thus is not required for PDK1 catalytic activity or to support cell survival and the proliferation of thymic and peripheral T cells. However, a PDK1 mutant that cannot bind PI(3,4,5)P3 cannot trigger the signals that terminate the expression of the transcription factor KLF2 in activated T cells and cannot switch the chemokine and adhesion receptor profile of naïve T cells to the profile of effector T cells. The PDK1 PH domain also is required for the maximal activation of Akt/protein kinase B (PKB) and for the maximal phosphorylation and inactivation of Foxo family transcription factors in T cells. PI(3,4,5)P3 binding to PDK1 and the strength of PKB activity thus can dictate the nature of the T-cell response. Low levels of PKB activity can be sufficient for T-cell proliferation but insufficient to initiate the migratory program of effector T cells.

scholarly journals P06.04 Enhancing T cell function for cancer immunotherapy by microRNA mediated knockdown of PRKAR1A

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A43.1-A43
Author(s):  
A Poudel ◽  
SM Rad ◽  
G Tan ◽  
AD McLellan
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cancer Immunotherapy ◽  
Cell Function ◽  
Luciferase Assay ◽  
T Cell Function ◽  
Car T ◽  
Kinase A

BackgroundProtein Kinase A (PKA) is a heterotetramer holoenzyme that consists of two regulatory and catalytic subunits. During T cell activation, one of the regulatory subunits (PRKAR1A) localizes to the immune synapse, inhibiting several central proteins in the T-cell signalling cascade and leading to T cell inactivation. Previously, the disruption of localisation of PKA type I R1α (PRKAR1A) to the immune synapse using disruptor peptides has been shown to improve chimeric antigen receptor (CAR) T cell function.1 2 However, the effect of PRKAR1A knockdown in T cells (including CAR T cells) has not been studied yet. In this study, we have utilized microRNAs (miR); miR96/183 or miR155 to knockdown PRKAR1A and explored the advantages of PRKAR1A knockdown on T cell activation and function.Materials and MethodsMicroRNAs (miR); miR96/183 or miR155 were cloned from human genomic DNA into a sleeping beauty system under a doxycycline inducible promoter (TCE). Overexpression of miRNA and target knockdown was assessed at both transcript level (by real time RT-PCR) and/or protein level (by western blot) respectively while target validation was done by luciferase assay. The fate of PRKAR1A knockdown on Jurkat T cells activated with anti-CD3 and anti-CD28 antibodies were determined by measuring IL-2 production (ELISA) and CD69 surface expression (flow cytometry). The effect of miR96/183 or miR155 overexpression in primary T cells expressing HER2-CAR were also compared.ResultsWe efficiently overexpressed both miRNAs and downregulated PRKAR1A expression in HEK293 cells at both mRNA and protein level. Luciferase assay confirmed miRNA mediated specific knockdown of PRKAR1A; mutated 3’UTR of PRKAR1A was used as negative control. Overexpression of miRNAs also downregulated PRKAR1A expression in Jurkat cells which resulted in enhanced activation (CD69 expression) and IL-2 production following anti-CD3/CD28 stimulation compared to untransfected controls (with normal PRKAR1A expression). Additionally, miRNA 96/183 and miRNA155 were found to target inhibitory proteins of TCR signalling such as CTLA4, Foxo3 and ptpn2 and resulted in superior T cell function. A third-generation lentiviral system has been optimised to express either miR96/183 or miR155 and HER2-CAR in the same vector and currently we are assessing the effect of PRKAR1A knockdown on primary CAR T cells.ConclusionsOverexpressing miRNA for knockdown of inhibitory proteins could be an efficient way of enhancing T cell function against solid tumours. Additionally, co-expressing CAR and miRNAs using lentiviral system would benefit such approaches for cancer immunotherapy.ReferencesNewick K, O’ Brien S, Sun J, Kappor V, Maceyko S, Lo A, Pure E, Moon E, Albelda SM. Augmentation of CAR T cell trafficking and antitumour efficacy by blocking protein kinase A (PKA) localization. Cancer Immunol Res 2016; 4(6): 541–551Hussain M, Shah Z, Abbas N, Javeed A, Mukhtar MM, Zhang J. Targeting tumour-associated immune suppression with selective protein kinase A type I (PKAI) inhibitors may enhance cancer immunotherapy. Medical Hypotheses 2016;86: 56–59Disclosure InformationA. Poudel: None. S.M. Rad: None. G. Tan: None. A.D. McLellan: None.

Commitment of Activated T Cells to Secondary Responsiveness Is Enhanced by Signals Mediated by cAMP-Dependent Protein Kinase A-I

2002 ◽  
Vol 62 (6) ◽  
pp. 1471-1481 ◽  
Author(s):  
Monika Vig ◽  
Anna George ◽  
Ranjan Sen ◽  
Jeannine Durdik ◽  
Satyajit Rath ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Dependent Protein Kinase ◽  
Activated T Cells ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Kinase A

The adaptor protein EBP50 is important for localization of the protein kinase A–Ezrin complex in T-cells and the immunomodulating effect of cAMP

2009 ◽  
Vol 425 (2) ◽  
pp. 381-388 ◽  
Author(s):  
Anne Jorunn Stokka ◽  
Randi Mosenden ◽  
Anja Ruppelt ◽  
Birgitte Lygren ◽  
Kjetil Taskén
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
T Cell Activation ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Membrane Microdomains ◽  
Type I ◽  
Kinase A

We recently reported that the dual-specificity AKAP (A-kinaseanchoring protein) Ezrin targets type I PKA (protein kinase A) to the vicinity of the TCR (T-cell receptor) in T-cells and, together with PAG (phosphoprotein associated with glycosphingolipid-enriched membrane microdomains) and EBP50 [ERM (Ezrin/Radixin/Moesin)-binding phosphoprotein 50], forms a scaffold that positions PKA close to its substrate, Csk (C-terminal Src kinase). This complex is important for controlling the activation state of T-cells. Ezrin binds the adaptor protein EBP50, which again contacts PAG. In the present study, we show that Ezrin and EBP50 interact with high affinity (KD=58±7 nM). A peptide corresponding to the EB (Ezrin-binding) region in EBP50 (EBP50pep) was used to further characterize the binding kinetics and compete the Ezrin–EBP50 interaction by various methods in vitro. Importantly, loading T-cells with EBP50pep delocalized Ezrin, but not EBP50. Furthermore, disruption of this complex interfered with cAMP modulation of T-cell activation, which is seen as a reversal of cAMP-mediated inhibition of IL-2 (interleukin 2) production, demonstrating an important role of EBP50 in this complex. In summary, both the biochemical and functional data indicate that targeting the Ezrin–EBP interaction could be a novel and potent strategy for immunomodulation.

scholarly journals Adenylyl Cyclase-Associated Protein Aca1 Regulates Virulence and Differentiation of Cryptococcus neoformans via the Cyclic AMP-Protein Kinase A Cascade

2004 ◽  
Vol 3 (6) ◽  
pp. 1476-1491 ◽  
Author(s):  
Yong-Sun Bahn ◽  
Julie K. Hicks ◽  
Steven S. Giles ◽  
Gary M. Cox ◽  
Joseph Heitman
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Adenylyl Cyclase ◽  
Cryptococcus Neoformans ◽  
Critical Role ◽  
Camp Signaling ◽  
Dependent Manner ◽  
Cell Functions ◽  
Kinase A

ABSTRACT The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Gα subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.

Critical Role for CCR1:CCL5 (RANTES) Receptor Ligand Interactions in Modulating Allogeneic T Cell Responses Following Bone Marrow Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3107-3107
Author(s):  
Sung Won Choi ◽  
Gerhard C. Hildebrandt ◽  
Ines Silva ◽  
Krystyna M. Olkiewicz ◽  
Stephen W. Chensue ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Critical Role ◽  
T Cell Responses ◽  
Dependent Manner ◽  
Activated T Cells ◽  
Cell Responses

Abstract Acute graft versus host disease (GVHD) and leukemic relapse are the most serious complications of allogeneic (allo) stem cell transplantation (SCT), and separating desirable graft-versus-leukemia (GVL) effects from GVHD remains the ultimate challenge to successful outcomes. The recruitment of activated T cells to host target tissues (GVHD) or sites of leukemic infiltration (GVL) is likely mediated by chemokine receptor:ligand interactions. CCR1 is a chemokine receptor that binds to CC chemokines including RANTES (CCL5), and is expressed on a variety of cells including activated T cells, monocytes, and macrophages. We have previously shown that mRNA expression of both CCR1 and RANTES is increased in GVHD target tissues following allo-SCT. Using a well established murine SCT model (B6->B6D2F1) and mice deficient in CCR1, we examined the contribution of CCR1 expression to allo T cell responses in vitro and to GVH and GVL effects in vivo. Lethally (1100cGy) irradiated B6D2F1 mice received SCT either from syngeneic (B6D2F1) or allogeneic (B6) CCR1+/+ or CCR1−/− donors. The severity of GVHD was assessed by survival and a well described clinical scoring system. Syngeneic SCT recipients all survived and were indistinguishable from naïve, untransplanted controls, whereas animals receiving allo-SCT from CCR1+/+ donors developed significant GVHD. By contrast, allo-SCT with CCR1−/− donor cells resulted in significantly improved survival (92% vs. 50%) and less severe clinical GVHD (p<0.01) by day 35 compared to allo-CCR1+/+ controls. GVL effects were next assessed by adding 500 P815 tumor cells (H-2d and syngeneic to host) to the bone marrow inoculum on day 0. F1 recipients of syngeneic BMT all died from tumor infiltration by day +15. Although all allo-SCT recipients effectively rejected their tumor, mice receiving CCR1-/− SCT had significantly improved leukemia free survival (45% vs. 5%) by day 60 compared to allo controls. At higher tumor doses, significant GVL activity remained in CCR1−/− SCT recipients, but the survival advantage was lost. Further examination of allo T cell responses in vivo revealed that day 7 splenic T cell expansion and serum IFNγ levels were significantly lower following CCR1−/− SCT (p < 0.01). Surprisingly, proliferation and IFNγ secretion were also reduced by ~70% when CCR1−/− T cells were stimulated with host antigens in vitro, whereas CTL activity remained equivalent to CCR1+/+ controls. The reduction in proliferation was not secondary to a migration defect, but was dependent on interactions between CCR1 and RANTES; neutralization of RANTES with a monoclonal antibody significantly reduced proliferation of CCR1+/+ T cells in a dose dependent manner. Finally, we found that GVHD mortality was also less when RANTES−/− mice were used as recipients in a second, MHC-disparate, SCT model (p = 0.03). Collectively these data demonstrate a critical role for CCR1 in donor T cell alloreactivity following SCT. These responses contribute to both GVHD and GVL effects in vivo and are likely dependent upon interactions between CCR1 and the chemokine ligand RANTES.

patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets

Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 4161-4170 ◽  
Author(s):  
R.L. Johnson ◽  
J.K. Grenier ◽  
M.P. Scott
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Target Genes ◽  
Critical Role ◽  
Wing Disc ◽  
Wing Vein ◽  
Anterior Compartment ◽  
Protein Levels ◽  
Anterior Posterior ◽  
Kinase A

The membrane protein, Patched, plays a critical role in patterning embryonic and imaginal tissues in Drosophila. patched constitutively inactivates the transcription of target genes such as wingless, decapentaplegic, and patched itself. The secreted protein, Hedgehog, induces transcription of target genes by opposing the Patched signaling pathway. Using the Gal4 UAS system we have overexpressed patched in wing imaginal discs and found that high Patched levels, expressed in either normal or ectopic patterns, result in loss of wing vein patterning in both compartments centering at the anterior/posterior border. In addition, patched inhibits the formation of the mechanosensory neurons, the campaniform sensilla, in the wing blade. The patched wing vein phenotype is modulated by mutations in hedgehog and cubitus interruptus (ci). Patched overexpression inhibits transcription of patched and decapentaplegic and post-transcriptionally decreases the amount of Ci protein at the anterior/posterior boundary. In hedgehogMrt wing discs, which express ectopic hedgehog, Ci levels are correspondingly elevated, suggesting that hedgehog relieves patched repression of Ci accumulation. Protein kinase A also regulates Ci; protein kinase A mutant clones in the anterior compartment have increased levels of Ci protein. Thus patched influences wing disc patterning by decreasing Ci protein levels and inactivating hedgehog target genes in the anterior compartment.

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