Lawrence P. Kane

Induction of NF-κB by the Akt/PKB kinase

Abstract The serine/threonine kinase Akt (also known as protein kinase B, PKB) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival [1]. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16 [2–4]. Here, we show that Akt can regulate signaling pathways that lead to induction of the NF-κB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-κB inhibitor IκB, and is specific for NF-κB, as other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-κB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-κB induction that could have implications for the control of T-cell growth and survival.

Signal transduction by the TCR for antigen.

The past several years have seen the beginning of a shift in the way that TCR signal transduction is studied. Although many investigators continue to identify new molecules, particularly adaptor proteins, others have attempted to look at signaling events in a larger cellular context. Thus the identification of distinct formations of signaling molecules at junctions between T cells and antigen-presenting cells, the role of the cytoskeleton and the partitioning of molecules into specialized lipid subdomains have been the subjects of many publications. Such concepts are helping to assemble a blueprint of how the myriad adaptors and kinases fit together to effect T cell activation.

Akt provides the CD28 costimulatory signal for up-regulation of IL-2 and IFN-gamma but not TH2 cytokines.

A region of the interleukin-2 (IL-2) promoter known as the RE/AP element is activated in concert by signals that originate from the T cell antigen receptor and the CD28 coreceptor. We show here that the serine-threonine kinase Akt can provide a costimulatory signal for RE/AP activation that is indistinguishable from the signal provided by CD28. This includes the ability of Akt, like antibodies to CD28, to synergize with protein kinase C θ (PKC-θ) in the induction of RE/AP. Retrovirus-mediated expression of activated Akt in primary T cells from CD28-deficient mice is capable of selectively restoring production of IL-2 and interferon γ, but not IL-4 or IL-5. Our results provide evidence that CD28 costimulation of different cytokines is mediated by discrete signaling pathways, one of which includes Akt.

The PI‐3 kinase/Akt pathway and T cell activation: pleiotropic pathways downstream of PIP3

Summary Ligation of the T cell receptor for antigen (TCR) and/or costimulatory receptor CD28 results in rapid activation of phosphoinositide‐3u2003kinase (PI‐3u2003kinase). It remains unclear, however, precisely how this activation occurs and also how the newly generated phospholipid products trigger the various events associated with T cell activation. Here we discuss the current understanding of how PI‐3u2003kinase is activated by the TCR and CD28 and what roles its products play in T cell activation. We also review recent advances in understanding the function of Akt in particular, especially its role in CD28 costimulation. Several functional targets of Akt are discussed in this regard: inducible transcription, cell survival, glucose metabolism, and the cellular translational machinery. These pathways have been associated with TCR/CD28 costimulation, and they have also been implicated as targets of Akt.

It's all Rel-ative: NF-κB and CD28 costimulation of T-cell activation

Costimulatory signals complement or modify the signals provided to a lymphocyte through antigen receptors. For productive T-cell activation, the CD28 molecule is apparently the most important, although not the only, costimulatory receptor. CD28 can provide a signal that is at least partially distinct from that delivered by the T cell receptor (TCR)-CD3 complex. Several lines of evidence indicate that the nuclear factor (NF)-kappaB pathway is perhaps the most relevant biochemical or transcriptional target for the costimulatory activity of CD28. Although many questions remain, recent years have witnessed significant progress in understanding the signal transduction pathways leading from the TCR and CD28 to Rel/NF-kappaB-dependent transcription.

Radioprotection of tumor and normal tissues by thiophosphate compounds

The efficacy of the radioprotective thiophosphate compounds WR638 (sodium hydrogen S‐[2 aminoethyl] phosphorothioate) and WR2721 (S‐2‐[3‐amino‐propylamino] ethyl phosphorothioate) was determined for a wide range of normal tissue and tumor endpoints. Dose‐modifying factors (D.M.F.) were obtained for mouse skin, small intestine, bone marrow, esophagus, lung, and kidney. D.M.F. values were also obtained for the P‐388 leukemia, the EMT‐6 mouse mammary carcinoma assayed in vitro, and the EMT‐6 mammary carcinoma assaysed for cure. The D.M.F. values obtained for normal tissue ranged from 1.2 for lung to 3 for bone marrow CFUs. The D.M.F. values for tumor ranged from 1.3 for cure of the EMT‐6 carcinoma to 2.2 for mean survival time in the P‐388 leukemia. There appears to be a decreasing dose‐modifying factor as a function of increasing dose in both tumor and normal tissue systems. The degree of protection afforded tumors appears to relate to their vascularization and their anoxic cell component. Although the D.M.F. obtained for cure of the solid EMT‐6 carcinoma would appear to be lower than for normal tissues with single doses, the data presently available do not allow one to predict that such a beneficial effect would occur with the multiple small doses used clinically.

A NCK-PAK1 SIGNALING MODULE IS REQUIRED FOR T-CELL RECEPTOR-MEDIATED ACTIVATION OF NFAT, BUT NOT OF JNK

The T‐cell antigen receptor (TCR) triggers a signaling cascade initiated by the tyrosine kinase Lck and requiring the proto‐oncogene p95vav. Vav is activated by Lck and can function as a guanine nucleotide exchange factor for the Rho‐family GTPases, Rac1 and Cdc42. To investigate the involvement of these GTPases in TCR signaling, we focused on their well characterized effector, Pak1. This serine/threonine kinase is activated by GTP‐bound Rac1 or Cdc42. However, its role in mediating downstream signaling events is controversial. We observed rapid, TCR‐dependent activation of Pak1 and TCR‐inducible association of Pak1 with Nck, which was tyrosine phosphorylated following stimulation. Pak1 activation occurred independently of Ras activation or calcium flux, but was dependent on the Lck tyrosine kinase, and was downstream of Vav and Cdc42. Dominant negative Pak1 or Nck specifically inhibited TCR‐mediated activation of the nuclear factor of activated T cells (NFAT) transcription factor. TCR‐mediated activation of Erk2 was also inhibited by dominant negative Pak. However, Pak1 activation was neither necessary nor sufficient for TCR‐dependent c‐Jun N‐terminal kinase (JNK) activation. Therefore, Pak1 acts downstream of Vav and is required for activation of Erk2 and NFAT by a JNK‐independent pathway. This is the first demonstration of a requirement for Pak to mediate the regulation of gene expression by an extracellular ligand.

Disruption of Akt kinase activation is important for immunosuppression induced by measles virus

Surface-contact–mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2–dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

Distinct regions in the CD28 cytoplasmic domain are required for T helper type 2 differentiation.

CD28 costimulation is essential for CD4+ T cell proliferation, survival, interleukin 2 (IL-2) production and T helper type 2 development. To define the nature of the signals that may drive different T cell responses, we have done a structure-function analysis of the CD28 cytoplasmic tail in primary T cells. CD28-mediated T cell proliferation and IL-2 production did not require a particular cytoplasmic domain. In contrast, IL-4 production was driven by the cooperative activity of specific motifs within the CD28 cytoplasmic tail. Using a gene-complementation approach, we provide evidence that one component of this T helper type 2 differentiation signal was mediated by 3-phosphoinositide-dependent protein kinase 1. Thus, different mechanisms underlie the induction of distinct T cell functional responses by CD28.

Response of a Murine Solid Tumor to In Vivo Combined Chemotherapy and Irradiation

The EMTG murine solid tumor was employed to examine the combined effect of gamma irradiation and chemotherapy. A dose of cyclophosphamide, actinomycin D, BCNU, and bleomycin A2, selected to yield a 10% survival fraction when given singly, was administered 2 hours before single graded doses of whole-body irradiation and the results compared to a radiation-only dose-response curve. Bleomycin A2 and BCNU modified the shoulder region of the curve. Actinomycin D was less than additive in the hypoxic region of the curve, while cyclophosphamide was simply additive throughout the curve, suggesting that it killed both well-oxygenated and poorly-oxygenated tumor cells in similar proportions.