The T-Cell Receptor Regulates Akt (Protein Kinase B) via a Pathway Involving Rac1 and Phosphatidylinositide 3-Kinase

ABSTRACT The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.

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Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B

Biochemical Journal ◽

10.1042/bj3560143 ◽

2001 ◽

Vol 356 (1) ◽

pp. 143-149 ◽

Cited By ~ 11

Author(s):

Mireille CORMONT ◽

Nadine GAUTIER ◽

Karine ILC ◽

Yannick Le MARCHAND-BRUSTEL

Keyword(s):

Protein Kinase ◽

Protein Kinase B ◽

Active Form ◽

Small Gtpase ◽

Dominant Negative ◽

Glut4 Translocation ◽

Phosphatidylinositol 3 Kinase ◽

Isolated Adipocytes ◽

Phosphatidylinositol 3

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 ΔCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 ΔCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 ΔCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 ΔCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.

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Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801340115 ◽

2018 ◽

Vol 115 (17) ◽

pp. E4051-E4060 ◽

Cited By ~ 30

Author(s):

Wanjing Shang ◽

Yong Jiang ◽

Michael Boettcher ◽

Kang Ding ◽

Marianne Mollenauer ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Cell Activation ◽

Active Form ◽

Cell Receptor ◽

Small Gtpase ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B–Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.

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Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00414.2002 ◽

2003 ◽

Vol 285 (2) ◽

pp. F219-F229 ◽

Cited By ~ 195

Author(s):

Yves Gorin ◽

Jill M. Ricono ◽

Nam-Ho Kim ◽

Basant Bhandari ◽

Goutam Ghosh Choudhury ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Nadph Oxidase ◽

Mesangial Cells ◽

Protein Kinase B ◽

Small Gtpase ◽

Dominant Negative ◽

Ros Generation ◽

Ang Ii ◽

Phagocyte Nadph Oxidase

ANG II induces protein synthesis through the serine-threonine kinase Akt/protein kinase B (PKB) in mesangial cells (MCs). The mechanism(s) of activation of Akt/PKB particularly by G protein-coupled receptors, however, is not well characterized. We explored the role of the small GTPase Rac1, a component of the phagocyte NADPH oxidase, and the gp91 phox homologue Nox4/Renox in this signaling pathway. ANG II causes rapid activation of Rac1, an effect abrogated by phospholipase A2 inhibition and mimicked by arachidonic acid (AA). Northern blot analysis revealed high levels of Nox4 transcript in MCs and transfection with antisense (AS) oligonucleotides for Nox4 markedly decreased NADPH-dependent reactive oxygen species (ROS)-producing activity. Dominant negative Rac1 (N17Rac1) as well as AS Nox4 inhibited ROS generation in response to ANG II and AA, whereas constitutively active Rac1 stimulated ROS formation. Moreover, N17Rac1 blocked stimulation of NADPH oxidase activity by AA. N17Rac1 or AS Nox4 abolished ANG II- or AA-induced activation of the hypertrophic kinase Akt/PKB. In addition, AS Nox4 inhibited ANG II-induced protein synthesis. These data provide the first evidence that activation by AA of a Rac1-regulated, Nox4-based NAD(P)H oxidase and subsequent generation of ROS mediate the effect of ANG II on Akt/PKB activation and protein synthesis in MCs.

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Combinatorial Effect of T-Cell Receptor Ligation and CD45 Isoform Expression on the Signaling Contribution of the Small GTPases Ras and Rap1

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8740-8747.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8740-8747 ◽

Cited By ~ 22

Author(s):

Jan Czyzyk ◽

David Leitenberg ◽

Tom Taylor ◽

Kim Bottomly

Keyword(s):

Signal Transduction ◽

Molecular Weight ◽

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Small Gtpases ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Negative Mutant

ABSTRACT By using ligands with various affinities for the T-cell receptor (TCR) and by altering the contribution of the CD45 tyrosine phosphatase, the effects of the potency of TCR-induced signals on the function of small GTPases Ras and Rap1 were studied. T cells expressing low-molecular-weight CD45 isoforms (e.g., CD45RO) exhibited the strongest activation of the Ras-dependent Elk-1 transcription factor and the highest sensitivity to the inhibitory action of dominant negative mutant Ras compared to T cells expressing high-molecular-weight CD45 isoforms (ABC). Moreover, stimulation of CD45RO+, but not CD45ABC+, T cells with a high-affinity TCR ligand induced suboptimal Elk-1 activation compared with the stimulation induced by an intermediate-affinity TCR-ligand interaction. This observation suggested that the Ras-dependent signaling pathway is safeguarded in CD45RO+ expressors by a negative regulatory mechanism(s) which prohibits maximal activation of the Ras-dependent signaling events following high-avidity TCR-ligand engagement. Interestingly, the biochemical activity of another small GTPase, the Ras-like protein Rap1, which has been implicated in the functional suppression of Ras signaling, was inversely correlated with the extent of Elk-1 activation induced by different-affinity TCR ligands. Consistently, overexpression of putative Rap dominant negative mutant RapN17 or the physiologic inhibitor of Rap1, the Rap GTPase-activating protein RapGAP, augmented the Elk-1 response in CD45RO+ T cells. This is in contrast to the suppressive effect of RapN17 and RapGAP on CD45ABC+ T cells, underscoring the possibility that Rap1 can act as either a repressor or a potentiator of Ras effector signals, depending on CD45 isoform expression. These observations suggest that cells expressing distinct isoforms of CD45 employ different signal transduction schemes to optimize Ras-mediated signal transduction in activated T lymphocytes.

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Protein Kinase B/Akt Regulates Coxsackievirus B3 Replication through a Mechanism Which Is Not Caspase Dependent

Journal of Virology ◽

10.1128/jvi.78.8.4289-4298.2004 ◽

2004 ◽

Vol 78 (8) ◽

pp. 4289-4298 ◽

Cited By ~ 81

Author(s):

Mitra Esfandiarei ◽

Honglin Luo ◽

Bobby Yanagawa ◽

Agripina Suarez ◽

Darya Dabiri ◽

...

Keyword(s):

Protein Kinase ◽

Protein Expression ◽

Hela Cells ◽

Protein Kinase B ◽

Coxsackievirus B3 ◽

Dominant Negative ◽

Viral Rna ◽

Dominant Negative Mutant ◽

Cvb3 Infection

ABSTRACT The role of signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) during viral infection has gained much recent attention. Our laboratory reported on an important regulatory role for extracellular signal-regulated kinases (ERK1/2), subfamily members of the MAPKs, during coxsackievirus B3 (CVB3) infection. However, the role of the PI3K pathway in CVB3 infection has not been well characterized. CVB3 is the most common known viral infectant of heart muscle that directly injures and kills infected cardiac myocytes during the myocarditic process. In the present study, we investigated the role of protein kinase B (PKB) (also known as Akt), a general downstream mediator of survival signals through the PI3K cascade, in regulating CVB3 replication and virus-induced apoptosis in a well-established HeLa cell model. We have demonstrated that CVB3 infection leads to phosphorylation of PKB/Akt on both Ser-473 and Thr-308 residues through a PI3K-dependent mechanism. Transfection of HeLa cells with a dominant negative mutant of Akt1 or pretreatment of wild-type HeLa cells with the specific PI3K inhibitor LY294002 significantly suppresses viral RNA expression, as reflected in diminished viral capsid protein expression and viral release. Dominant negative Akt1 and LY294002 also increase apoptosis in infected cells, which can be reversed by addition of the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Interestingly, blocking of apoptosis by zVAD.fmk does not reverse the viral RNA translation blockade, indicating that the inhibitory effect of dominant negative Akt1 on viral protein expression is not caspase dependent. In addition, we showed that the attachment of virus to its receptor-coreceptor complex is not sufficient for PKB/Akt activation and that postentry viral replication is required for Akt phosphorylation. Taken together, these data illustrate a new and imperative role for Akt in CVB3 infection in HeLa cells and show that the PI3K/Akt signaling is beneficial to CVB3 replication.

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CD28-dependent Activation of Protein Kinase B/Akt Blocks Fas-mediated Apoptosis by Preventing Death-inducing Signaling Complex Assembly

Journal of Experimental Medicine ◽

10.1084/jem.20020307 ◽

2002 ◽

Vol 196 (3) ◽

pp. 335-348 ◽

Cited By ~ 104

Author(s):

Russell G. Jones ◽

Alisha R. Elford ◽

Michael J. Parsons ◽

Linda Wu ◽

Connie M. Krawczyk ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Protein Kinase ◽

Cell Survival ◽

Protein Kinase B ◽

Active Form ◽

Apoptosis Pathway ◽

Signaling Complex ◽

T Cell Survival

The T cell costimulatory molecule CD28 is important for T cell survival, yet both the signaling pathways downstream of CD28 and the apoptotic pathways they antagonize remain poorly understood. Here we demonstrate that CD4+ T cells from CD28-deficient mice show increased susceptibility to Fas-mediated apoptosis via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. Protein kinase B (PKBα/Akt1) is an important serine/threonine kinase that promotes survival downstream of PI3K signals. To understand how PI3K-mediated signals downstream of CD28 contribute to T cell survival, we examined Fas-mediated apoptosis in T cells expressing an active form of PKBα. Our data demonstrate that T cells expressing active PKB are resistant to Fas-mediated apoptosis in vivo and in vitro. PKB transgenic T cells show reduced activation of caspase-8, BID, and caspase-3 due to impaired recruitment of procaspase-8 to the death-inducing signaling complex (DISC). Similar alterations are seen in T cells from mice which are haploinsufficient for PTEN, a lipid phosphatase that regulates phosphatidylinositol-3,4,5-trisphosphate (PIP3) and influences PKBα activity. These findings provide a novel link between CD28 and an important apoptosis pathway in vivo, and demonstrate that PI3K/PKB signaling prevents apoptosis by inhibiting DISC assembly.

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Faculty Opinions recommendation of Protein kinase D2 is a digital amplifier of T cell receptor-stimulated diacylglycerol signaling in naïve CD8⁺ T cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.723554877.793537595 ◽

2017 ◽

Author(s):

Leslie Berg

Keyword(s):

T Cells ◽

T Cell ◽

Protein Kinase ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Cell Receptor

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Ca2+/Calmodulin-Dependent Protein Kinase II Is a Modulator of CARMA1-Mediated NF-κB Activation

Molecular and Cellular Biology ◽

10.1128/mcb.02469-05 ◽

2006 ◽

Vol 26 (14) ◽

pp. 5497-5508 ◽

Cited By ~ 72

Author(s):

Kazuhiro Ishiguro ◽

Todd Green ◽

Joseph Rapley ◽

Heather Wachtel ◽

Cosmas Giallourakis ◽

...

Keyword(s):

T Cell ◽

Protein Kinase ◽

T Cell Activation ◽

Cell Activation ◽

Cell Receptor ◽

Tcr Signaling ◽

Dependent Protein Kinase ◽

Immune Synapse ◽

Protein Kinase Ii ◽

Dependent Protein

ABSTRACT CARMA1 is a central regulator of NF-κB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-κB signaling downstream of the T-cell receptor (TCR). Computational analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca2+/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-κB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-κB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-κB activation.

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