A potential therapeutic strategy based on acute oxidative stress induction for wild-type NRF2/KEAP1 lung squamous cell carcinoma.

Extensive efforts have been conducted in the search for new targetable drivers of lung squamous cell carcinoma (LUSC); to date, however, candidates remain mostly unsuccessful. One of the oncogenic pathways frequently found to be active in LUSC is NFE2L2 (NRF2 transcription factor), the levels of which are regulated by KEAP1. Mutations in NFE2L2 or KEAP1 trigger NRF2 activation, an essential protector against reactive oxygen species (ROS). We hypothesized that the frequency of NRF2 activation in LUSC (∼35 %) may reflect a sensitivity of LUSC to ROS. Results from this study reveal that whereas tumors containing active forms of NRF2 were protected, ROS induction in wild-type NFE2L2/KEAP1 LUSC cells triggered ferroptosis. The mechanism of ROS action in normal-NRF2 LUSC cells involved transient NRF2 activation, miR-126-3p/miR-126-5p upregulation, and reduction of p85ß and SETD5 levels. SETD5 levels reduction triggered pentose pathway gene levels increase to toxic values. Simultaneous depletion of p85ßPI3K and SETD5 triggered LUSC cell death, while p85ßPI3K and SETD5 overexpression rescued survival of ROS-treated normal-NRF2 LUSC cells. This shows that the cascade involving NRF2 > miR-126-3p, miR-126-5p > p85ßPI3K and SETD5 is responsible for ROS-induced cell death in normal-NRF2 LUSC. Transient ROS-induced cell death is shown in 3D spheroids, patient-derived organoids, and in xenografts of wild-type NFE2L2/KEAP1 LUSC cells, supporting the potential of acute local ROS induction as a therapeutic strategy for LUSC patients with normal-NRF2.

Role of the PI3K regulatory subunit in the control of actin organization and cell migration.

Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85alpha regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85alpha in controlling PDGF receptor-induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.

Linomide prevents the lethal effect of anti-Fas antibody and reduces Fas-mediated ceramide production in mouse hepatocytes.

Fas is an apoptosis-signaling receptor molecule expressed in vivo on thymocytes, liver, heart, and ovary. In vivo administration of the anti-Fas Jo2 antibody in mice induces severe apoptotic liver damage leading to fulminant hepatitis and death. Linomide, a quinoline 3-carboxamide, inhibits apoptosis of B and T cells induced by various stimuli including viruses, superantigens, and glucocorticoids. Mice treated with linomide survived the lethal effect of anti-Fas antibody, did not accumulate ceramide in hepatocytes, and recovered liver structure and function within 96 h of anti-Fas injection, as confirmed by histology and glutamic oxalacetic transaminase, glutamic pyruvic transaminase, and lactate dehydrogenase levels. Surviving mice showed severe depletion of cortical thymocytes, but medullar thymic cells expressing high CD3 and Fas levels also survived the treatment with anti-Fas in the presence of linomide. Heart, lung, and ovary showed no signs of apoptosis promoted by Fas ligation. These results suggest that linomide prevents cell death triggered by Fas ligation and can be useful for therapeutic intervention in fulminant hepatitis.

Apoptosis, Fas and systemic autoimmunity: the MRL-lpr/lpr model.

Proteins encoded by the fas and fas ligand (fasL) genes are involved in apoptotic cell death in lymphocytes. In this article we review the recent elucidation of the role of the Fas-FasL interactions in the maintenance of tolerance to self antigens and in the homeostatic regulation of lymphocyte clonal expansion, and discuss the mechanisms of autoimmunity in Fas- and FasL-deficient mutant mouse strains.

Role of the autophosphorylation site on the biological function of pp56lck.

Src-family tyrosine kinases act as signaling molecules in a wide array of cellular activation processes. The existence of the various src-family members reflects the requirement for different cell-surface receptors to transmit cell-type specific intracellular signals. The structural basis for the functional specificity of src-kinases is being actively investigated. In the present report we have analysed the contribution of the area surrounding the autophosphorylation site (located at subdomain VII of the catalytic domain) in determining src-kinases activity and functional specificity. To this end we analysed the kinase activities of the lymphoid src-kinase pp56lck and a mutant of pp56lck in which this region has been exchanged for the corresponding area of the serine/threonine kinase c-Raf. Our studies indicate that the change at subdomain VII affected the ability of pp56lck to phosphorylate physiological substrates. Furthermore, when analysed in T cells, the mutant at subdomain VII failed to induce interleukin-2 production, a specific biological function of pp56lck. Thus, the area surrounding the autophosphorylation site of pp56lck plays a critical role in mediating its specific biological function.

A cAMP-activated pathway, including PKA and PI3K, regulates neuronal differentiation.

Neuronal differentiation is a complex process in which many different signalling pathways may be involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to induce neuronal differentiation and also to cooperate with NGF to induce PC12 neurite outgrowth in a Ras-dependent manner. However, the neuritogenic activities associated with cAMP are still not well understood. The purpose of this study was to investigate the potential neuritogenic activities mediated by cAMP. For this purpose, we used the human neuroblastoma cell line SH-SY5Y. These neuroblastoma cells respond to cAMP by forming neurite-like extensions. We tried to identify some essential pathways involved in the cAMP-induced neurite elongation of these cells. Our results indicated that PKA is transiently activated in this elongation model. When we blocked PKA activity, elongation did not take place. Similarly, PI3K also plays an essential role because when we blocked this kinase activity, there was no neurite elongation. Indeed, over-expression of the p110-catalytic subunit or an activating form of the p85-regulatory subunit (p65) is able to induce some degree of neurite extension. Moreover, our results showed that when elongation is initiated, PI3K is still essential for maintenance of the neuronal morphology, whereas PKA or MAPK (ERKs or p38) activation does not appear to be necessary during this process.

A cascade involving p85, Cdc42 and septin 2 regulates cytokinesis.

Mitosis, the final phase of cell division, includes the processes of nuclear division and cytosolic division (cytokinesis). Cytokinesis occurs when DNA separation terminates, and involves a number of proteins that induce furrowing at the region of cell separation, formation of new membrane, and abscission. This process is remarkably complex, and the list of proteins that regulate it is long. Our understanding is limited as to how these players are organized in space and time to ensure that the cytosol divides equally, and only after nuclear division. Class I(A) PI3K (phosphoinositide 3-kinase) is an enzyme activated by growth factor receptor stimulation, but it is re-activated in early mitosis and regulates mitosis entry. By the end of mitosis, PI3K activity is low; at this point, the class I(A) PI3K regulatory subunit p85 contributes to co-ordination of the cytoskeletal changes required for cytokinesis. The impact of these observations on current models of cytokinesis execution is discussed here.

The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP.

The study of the various protein kinases reveals that, despite their considerably diversity, they have evolved from a common origin. Eleven conserved subdomains have been described that encompass the catalytic core of these enzymes. One of these conserved regions, subdomain II, contains an invariant lysine residue present in all known protein kinase catalytic domains. Two facts have suggested that this conserved lysine of subdomain II is essential for binding ATP: (i) several investigators have demonstrated that this residue is physically proximal to the ATP molecule, and (ii) conservative substitutions at this site render the kinase inactive. However, these results are also consistent with a functional role of the conserved lysine of subdomain II in orienting or facilitating the transfer of phosphate. To study in more detail the role of subdomain II, we have generated mutants of the protein-tyrosine kinase pp56lck that have single amino acid substitutions within the area surrounding the conserved residue Lys-273 in subdomain II. When compared with wild-type pp56lck, these mutants displayed profound reductions in their phosphotransfer efficiencies and small differences in their affinities for ATP. Further, the substitution of arginine for Lys-273 resulted in a mutant protein unable to transfer the gamma-phosphate of ATP but able to bind 8-azido-ATP with an efficiency similar to that of wild-type pp56lck. These results suggest that the region including Lys-273 of subdomain II is involved in the enzymatic process of phosphate transfer, rather than in anchoring ATP.

Characterization of an active, non-myristylated, cytoplasmic form of the lymphoid protein tyrosine kinase pp56lck.

pp56lck is a member of the src family of tyrosine kinases mainly expressed in T lymphocytes. Src tyrosine kinases have been implicated in the control of cell growth and differentiation in different cell types, but the mechanism of regulation of these enzymes is poorly understood. In order to characterize the distinct species of pp56lck, we have produced high yields of enzymatically active wild type pp56lck using the eukaryotic baculovirus expression system in Spodoptera frugiperda insect cells (Sf9). We find that the various species of baculoviral pp56lck are not only differentially phosphorylated (on serine and tyrosine residues) but also heterogeneously myristylated. Surprisingly a non-myristylated, very active form of bv-pp56lck is found in the cytoplasm of Sf9 cells. Fractionation of T cells reveals that cytoplasmic pp56lck exists in T lymphocytes as well.

p56lck phosphorylation by Ca2+/calmodulin-dependent protein kinase type II.

Ca2+/calmodulin-dependent protein kinases are implicated in regulating the Ca2+ signaling involved in T cell activation and in thymocyte selection. One of the earliest events in signaling through the T cell antigen receptor is activation of the protein tyrosine kinase p56lck. Following T cell activation or signaling through the IL-2 receptor, Ca(2+)-mediated phosphorylation of p56lck occurs on serine/threonine residues. Isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinases, CaM kinase-II and CaM kinase-Gr are found in human T lymphocytes. CaM kinase-II, but not CaM kinase-Gr, phosphorylates the T cell tyrosine kinase p56lck in vitro. Tryptic phosphopeptide maps indicate that CaM kinase-II phosphorylates p56lck on multiple sites in vitro. Kinase assays of p56lck modified by CaM kinase-II indicate that CaM kinase-II modification does not appreciably affect p56lck phosphotransfer activity.

Phosphoinositide 3-kinase and Forkhead, a switch for cell division.

Cell cycle progression is a tightly controlled process. To initiate cell division, mitogens trigger a number of early signals that promote the G(0)-G(1) transition by inducing cell growth and the activation of G(1) cyclins. Activation of cyclin E/cdk2 (cyclin-dependent kinase 2) at the end of G(1) is then required to trigger DNA synthesis (S phase entry). Among the early signals induced by mitogens, activation of PI3K (phosphoinositide 3-kinase) appears essential to induce cell cycle entry, as it regulates cell growth signalling pathways, which in turn determine the rate of cell cycle progression. Another mechanisms by which PI3K and its downstream effector protein kinase B regulate cell cycle entry is by inactivation of the FOXO (Forkhead Box, subgroup O) transcription factors, which induce expression of quiescence genes such as those encoding p27(kip), p130 and cyclin G2. PI3K/FOXO then work as a complementary switch: when PI3K is active, FOXO transcription factors are inactive. The switch is turned on and off at different phases of the cell cycle, thus regulating cell cycle progression.

T cell receptor-associated alpha-phosphatidylinositol 3-kinase becomes activated by T cell receptor cross-linking and requires pp56lck.

The activation of a phosphatidylinositol (PI) 3-kinase is one of the earliest intracellular responses to T lymphocyte stimulation. We have used a human T cell line, Jurkat, and an antibody that recognizes the clonotype of its T cell receptor (TcR) to characterize the association of PI 3-kinase with the TcR and its activation in response to TcR cross-linking. We show that the TcR is constitutively associated with the alpha isoform of the PI 3-kinase p85 regulatory subunit. Stimulation of Jurkat cells through the TcR results in the rapid and transient activation of the associated PI 3-kinase. In addition, our results indicate that the tyrosine kinase pp56lck is essential for PI 3-kinase activation via the TcR.

Tyrosine kinase specific motif at subdomain VIII does not confer specificity for tyrosine.

The majority of protein kinases fall within one of the two broad classes, kinases that phosphorylate serine or threonine and kinases that phosphorylate tyrosine. The structural basis that confers residue specificity is not known. However, it has been hypothesized that a region in subdomain VIII of the catalytic domain may be involved in determining kinase specificity. This region contains a motif which is conserved among serine/threonine kinases and different from the one conserved among tyrosine kinases. We have prepared a chimera of the tyrosine kinase pp56lck in which the tyrosine kinase motif at subdomain VIII has been exchanged for the corresponding region of the serine/threonine kinase c-Raf. Our results indicate that this motif itself does not confer amino acid specificity since the chimeric kinase still displays specificity for tyrosine.

Lck unique domain influences Lck specificity and biological function.

Src-family tyrosine kinases share structural and amino acid sequence homology, particularly in the catalytic domain as well as in the SH2 and SH3 domains of the regulatory region. However, each src-family member also contains a unique domain which is specific to and characteristic of each individual tyrosine kinase. These unique or specific domains may contribute to the functional specificity of each src-family kinase. To address this possibility, we analyzed the kinase activities and substrate specificities of the lymphoid src-kinase, pp56lck, and a mutant of pp56lck lacking its specific domain. Our data show that both the wild type enzyme and the specific domain-deleted mutant displayed similar affinities for ATP and the non-physiological substrate denatured enolase. However, the specific domain-deleted mutant failed to phosphorylate a number of physiological substrates of pp56lck. In addition, the ability of pp56lck to mediate induction of the interleukin-2 promoter was strongly impaired upon deletion of its specific domain. Thus, the unique domain is not required for the intrinsic kinase activity of pp56lck, however, it influences substrate preference and contributes to the unique physiological function of this src-family tyrosine kinase.

Selective association between MHC class I-restricted T cell receptors, CDS, and activated tyrosine kinases on thymocytes undergoing positive selection.

Developing T cells undergo distinct selection processes that determine the TCR repertoire. Positive selection involves the differentiation of immature thymocytes capable of recognizing antigens complexed with self-MHC molecules to mature T cells. Besides the central role of TCR engagement by MHC in triggering selection; the interaction of CD8 and CD4 with MHC class I and class II, respectively; is thought to be important in regulating the selection process. To study potential mechanisms involved in positive selection of CD8+ cells, we have analyzed mice expressing a unique transgenic TCR. The transgenic receptor recognizes the HY male Ag in the context of the MHC class I molecule, H2-Db. We describe that CD8 and the TCR are selectively associated in thymocytes of mice expressing the restricting MHC, but not in thymocytes of mice expressing a nonrestricting MHC. pp56lck and pp59fyn, the tyrosine kinases associated with CD8 and TCR, respectively, were found to be present in this complex in an activated form. No comparable TCR-CD4 complex formation was found in thymuses undergoing positive selection to CD8+ cells. The formation of a multimolecular complex between CD8 and TCR, in which pp56lck and pp59fyn are activated, may initiate specific signaling programs involved in the maturation of CD8+ cells.

Forkhead transcription factors contribute to execution of the mitotic programme in mammals.

Cell cycle progression is a process that is tightly controlled by internal and external signals. Environmental cues, such as those provided by growth factors, activate early signals that promote cell cycle entry. Cells that have progressed past the restriction point become independent of growth factors, and cell cycle progression is then controlled endogenously. The phosphatidylinositol 3OH kinase (PI(3)K)/protein kinase B (PKB) pathway must be activated in G1 to inactivate forkhead transcription factors (FKH-TFs) and allow cell cycle entry. Here we show that subsequent attenuation of the PI(3)K/PKB pathway is required to allow transcriptional activation of FKH-TF in G2. FKH-TF activity in G2 controls mammalian cell cycle termination, as interference with FKH transcriptional activation by disrupting PI(3)K/PKB downregulation, or by expressing a transcriptionally inactive FKH mutant, induces cell accumulation in G2/M, defective cytokinesis, and delayed transition from M to G1 of the cell cycle. We demonstrate that FKH-TFs regulate expression of mitotic genes such as cyclin B and polo-like kinase (Plk). Our results support the important role of forkhead in the control of mammalian cell cycle completion, and suggest that efficient execution of the mitotic programme depends on downregulation of PI(3)K/PKB and consequent induction of FKH transcriptional activity.

Intermediate affinity interleukin-2 receptor mediates survival via a phosphatidylinositol 3-kinase-dependent pathway.

Peripheral blood T lymphocytes require two signals to enter and progress along the cell cycle from their natural quiescent state. The first activation signal is provided by the stimulation through the T cell receptor, which induces the synthesis of cyclins and the expression of the high affinity interleukin-2 receptor. The second signal, required to enter the S phase, is generated upon binding of interleukin-2 to the high affinity alphabetagamma interleukin-2 receptor. However, resting T cells already express intermediate affinity betagamma interleukin-2 receptors. As shown here, T cell stimulation through intermediate affinity receptors is capable of inducing cell rescue from the apoptosis suffered in the absence of stimulation. Characterization of the signaling pathways utilized by betagamma interleukin-2 receptors in resting T cells, indicated that pp56(lck), but not Jak1 or Jak3, is activated upon receptor triggering. Compelling evidence is presented indicating that phosphatidylinositol 3-kinase associates with the intermediate affinity interleukin-2 receptor and is activated upon interleukin-2 addition. Bcl-xL gene was also found to be induced upon betagamma interleukin-2 receptor stimulation. Finally, pharmacological inhibition of phosphatidylinositol 3-kinase blocked both interleukin-2-mediated bcl-xL induction and cell survival. We conclude that betagamma interleukin-2 receptor mediates T-cell survival via a phosphatidylinositol 3-kinase-dependent pathway, possibly involving pp56(lck) and bcl-xL as upstream and downstream effectors, respectively.

Evidence for B cell participation in the in vitro and in vivo maintenance of in vivo staphylococcal enterotoxin B-induced T cell anergy.

The mechanisms involved in the maintenance of staphylococcal enterotoxin B (SEB)-induced T cell anergy are poorly understood. Here, we demonstrate that CD4+ T cell anergy induced by SEB treatment is under partial B cell control. This effect is not mediated by anti-SEB antibodies or any in vitro B cell-produced suppresser factor. At day 13 after SEB immunization, T cells from B cell-deficient mice proliferate upon in vitro stimulation with SEB. These results suggest that SEB-induced T cell anergy is reversible and that B cells have an important function in anergy maintenance in CD4+ T cells, both in vivo and in vitro.

CD2 is involved in regulating cyclic AMP levels in T cells.

The human T lymphocyte-specific CD2 (T11) molecule, that regulates T cell activation, is capable of mediating increases of intracellular cAMP. Monoclonal antibodies directed to different epitopes of the CD2 molecule which either induce or inhibit T cell proliferation are capable of triggering an increase of cAMP comparable to that induced by reagents which activate adenylate cyclase. These results indicate that there is a relationship between the CD2 membrane molecule and the adenylate cyclase system.

Interaction between the CD45 antigen and phytohemagglutinin. Inhibitory effect on the lectin-induced T cell proliferation by anti-CD45 monoclonal antibody.

Immunoprecipitation studies from 125I-labeled T cells, previously activated with the lectin phytohemagglutinin (PHA) from Phaseolus vulgaris, showed a preferential association between the CD45 glycoprotein family containing members of 220, 205, 190 and 180 kDa, and a protein of 33 kDa. This 33-kDa protein, with an identical molecular mass as PHA, was not associated with other highly expressed molecules such as class I histocompatibility antigens (HLA-A,B,C), transferrin receptor, CDw44 and CD11a. Further immunoprecipitation analysis from peripheral blood lymphocytes incubated with 125I-labeled PHA confirmed the identification of the CD45-associated 33-kDa protein as the lectin PHA. These results prompted us to analyze the possible involvement of the CD45 molecules in the T cell activation process induced by PHA. Thus, two monoclonal antibodies specific for CD45 antigens were able to inhibit the T cell proliferation induced by the lectin. This inhibitory ability was exerted by affecting both the interleukin 2 production and the interleukin 2 receptor expression.