Altered thymic positive selection and intracellular signals in Cbl-deficient mice

Cbl is the product of the protooncogene c-cbl and is involved in T cell antigen receptor (TCR)-mediated signaling. To understand the role of Cbl for immune system development and function, we generated a Cbl-deficient mouse strain. In Cbl-deficient mice, positive selection of the thymocytes expressing major histocompatibility complex class II-restricted transgenic TCR was significantly enhanced. Two factors may have contributed to the altered thymic selection. First, Cbl deficiency markedly up-regulated the activity of ZAP-70 and mitogen-activated protein kinases. The mitogen-activated protein kinase pathway was shown previously to be involved in thymic positive selection. Second, Cbl-deficient thymocytes expressed CD3 and CD4 molecules at higher levels, which consequently may increase the avidity of TCR/major histocompatibility complex/coreceptor interaction. Thus, Cbl plays a novel role in modulating TCR-mediated multiple signaling pathways and fine-tunes the signaling threshold for thymic selection.

Dual modulation of cell survival and cell death by β2-adrenergic signaling in adult mouse cardiac myocytes

The goal of this study was to determine whether β1-adrenergic receptor (AR) and β2-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac β2-AR can activate both Gs and Gi proteins, whereas cardiac β1-AR couples only to Gs. To avoid complicated crosstalk between β-AR subtypes, we expressed β1-AR or β2-AR individually in adult β1/β2-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of β1-AR, but not β2-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, β2-AR (but not β1-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting Gi, Gβγ, or phosphoinositide 3 kinase (PI3K) with pertussis toxin, βARK-ct (a peptide inhibitor of Gβγ), or LY294002, respectively. This indicates that β2-AR activates Akt via a Gi-Gβγ-PI3K pathway. More importantly, inhibition of the Gi-Gβγ-PI3K-Akt pathway converts β2-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, β2-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the Gi-Gβγ-PI3K-Akt signaling pathway.

Cadherin Sequences That Inhibit β-Catenin Signaling: A Study in Yeast and Mammalian Cells

Drosophila Armadillo and its mammalian homologue β-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/β-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/β-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or β-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/β-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited β-catenin–mediated signaling. This suggests that the interaction between β-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

CD95/Fas induces cleavage of the GrpL/Gads adaptor and desensitization of antigen receptor signaling

The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.

Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.

Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought.

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein. Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism. Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress. However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa. Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes. Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated. These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

Inhibition of cytokines and JAK-STAT activation by distinct signaling pathways.

An important component of cytokine regulation of cell growth and differentiation is rapid transcriptional activation of genes by the JAK-STAT (signal transducer and activator of transcription) signaling pathway. Ligation of cytokine receptors results in tyrosine phosphorylation and activation of receptor-associated Jak protein tyrosine kinases and cytoplasmic STAT transcription factors, which then translocate to the nucleus. We describe the interruption of cytokine triggered JAK-STAT signals by cAMP, the calcium ionophore ionomycin, and granulocyte/macrophage colony-stimulating factor. Jak1 kinase activity, interleukin 6-induced gene activation, Stat3 tyrosine phosphorylation, and DNA-binding were inhibited, as was activation of Jak1 and Stat1 by interferon gamma. The kinetics and requirement for new RNA and protein synthesis for inhibition of interleukin 6 by ionomycin and GM-CSF differed, but both agents increased the association of Jak1 with protein tyrosine phosphatase ID (SH2-containing phosphatase 2). Our results demonstrate that crosstalk with distinct signaling pathways can inhibit JAK-STAT signal transduction, and suggest approaches for modulating cytokine activity during immune responses and inflammatory processes.

Activated Ras signals differentiation and expansion of CD4+8+ thymocytes.

We describe a novel approach to assay the ability of particular gene products to signal transitions in lymphocyte differentiation in vivo. The method involves transfection of test expression constructs into RAG-1-deficient embryonic stem cells, which are subsequently assayed by the RAG-2-deficient blastocyst complementation approach. We have used this method to demonstrate that expression of activated Ras in CD4-8- (double negative, DN) prothymocytes in vivo induces their differentiation into small CD4+8+ (double positive, DP) cortical thymocytes with accompanying expansion to normal thymocyte numbers. However, activated Ras expression in DP cells does not cause proliferation or maturation to CD4+8- or CD4-8+ (single positive) thymocytes. Therefore, signaling through Ras is sufficient for promoting differentiation of DN to DP cells, but further differentiation requires the activity of additional signaling pathways.

Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons.

Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion of PTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP[1-34] and PTH[1-34] signal via cAMP and cytosolic calcium transients. The cAMP response showed strong acute (< or = 6 h) homologous and heterologous desensitization after preincubation with PTHrP or PTH. In contrast, the acute calcium response did not desensitize after preincubation with PTHrP; in fact, preincubation dramatically recruited additional responsive neurons. Unexpectedly, C-terminal PTHrP[107-139], which does not bind or activate the cloned PTH/PTHrP receptor, signaled in neurons via cytosolic calcium but not cAMP. Although some neurons responded to both PTHrP[1-34] and PTHrP[107-139], others responded only to PTHrP[1-34]. We conclude that certain hippocampal neurons exhibit dual signaling in response to PTHrP[1-34] and that some neurons have a receptor for C-terminal PTHrP that signals only via cytosolic calcium.

Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.

Residue replacements were made at five positions (Arg-73, Asp-76, Tyr-87, Asp-106, and Asp-201) in the Halobacterium salinarium phototaxis receptor sensory rhodopsin I (SR-I) by site-specific mutagenesis. The sites were chosen for their correspondence in position to residues of functional importance in the homologous light-driven proton pump bacteriorhodopsin found in the same organism. This work identifies a residue in SR-I shown to be of vital importance to its attractant signaling function: Asp-201. The effect of the substitution with the isosteric asparagine is to convert the normally attractant signal of orange light stimulation to a repellent signal. In contrast, similar neutral substitution of the four other ionizable residues near the photoactive site allows essentially normal attractant and repellent phototaxis signaling. Wild-type two-photon repellent signaling by the receptor is intact in the Asp-201 mutant, genetically separating the wild-type attractant and repellent signal generation processes. A possible explanation and implications of the inverted signaling are discussed. Results of neutral residue substitution for Asp-76 confirm our previous evidence that proton transfer reactions involving this residue are not important to phototaxis but that Asp-76 functions as the Schiff base proton acceptor in proton translocation by transducer-free SR-I.

Prdm1 acts downstream of a sequential RA, Wnt and Fgf signaling cascade during zebrafish forelimb induction

Vertebrate limb induction is triggered in the lateral plate mesoderm (LPM) by a cascade of signaling events originating in the axial mesoderm. While it is known that Fgf, Wnt and retinoic acid (RA) signals are involved in this cascade, their precise regulatory hierarchy has not been determined in any species. tbx5 is the earliest gene expressed in the limb bud mesenchyme. Recently, another transcription factor, Prdm1, has been shown to be crucial for zebrafish forelimb development. Here, we show that Prdm1 is downstream of RA, Wnt2b and Tbx5 activity. We find that RA activity, but not Fgf signaling, is necessary for wnt2b expression. Fgf signaling is required for prdm1 expression in the fin bud, but is not necessary for the initiation of tbx5 expression. We propose a model in which RA signaling from the somitic mesoderm leads to activation of wnt2b expression in the intermediate mesoderm, which then signals to the LPM to trigger tbx5 expression. tbx5 is required for Fgf signaling in the limb bud leading to activation of prdm1 expression, which in turn is required for downstream activation of fgf10 expression.

Intraspecific and intraspecific views of colour signals in the strawberry poison frog Dendrobates Pumilio

Poison frogs in the anuran family Dendrobatidae use bright colors on their bodies to advertise toxicity. The species Dendrobates pumilio Schmidt 1858, the strawberry poison frog, shows extreme polymorphism in color and pattern in Panama. It is known that females of D. pumilio preferentially choose mates of their own color morph. Nevertheless, potential predators must clearly see and recognize all color morphs if the aposermatic signaling system is to function effectively. We examined the ability of conspecifics and a model predator to discriminate a diverse selection of D. pumilio colors from each other and from background colors. Microspectrophotometry of isolated rod and cone photoreceptors of D. pumilio revealed the presence of a trichromatic photopic visual system. A typical tetrachromatic bird system was used for the model predator. Reflectance spectra of frog and background colors were obtained, and discrimination among spectra in natural illuminants was mathematically modeled. The results revealed that both D. pumilio and the model predator discriminate most colors quite well, both from each other and from typical backgrounds, with the predator generally performing somewhat better than the conspecifics. Each color morph displayed at least one color signal that is highly visible against backgrounds to both visual systems. Our results indicate that the colors displayed by the various color morphs of D. pumilio are effective signals both to conspecifics and to a model predator.

Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

Protein quality and distribution determines anabolic signaling, cellular energetics, and body composition

Building and maintaining muscle is critical to the quality of life for adults and elderly. Physical activity and nutrition are important factors for long-term muscle health. In particular, dietary protein – including protein distribution and quality – are under-appreciated determinants of muscle health for adults. The most unequivocal evidence for the benefit of optimal dietary protein at individual meals is derived from studies of weight management. During the catabolic condition of weight loss, higher protein diets attenuate loss of lean tissue and partition weight loss to body fat when compared with commonly recommended high carbohydrate, low protein diets. Muscle protein turnover is a continuous process in which proteins are degraded, and replaced by newly synthesized proteins. Muscle growth occurs when protein synthesis exceeds protein degradation. Regulation of protein synthesis is complex, with multiple signals influencing this process. The mammalian target of rapamycin (mTORC1) pathway has been identified as a particularly important regulator of protein synthesis, via stimulation of translation initiation. Key regulatory points of translation initiation effected by mTORC1 include assembly of the eukaryotic initiation factor 4F (eIF4F) complex and phosphorylation of the 70 kilodalton ribosomal protein S6 kinase (S6K1). Assembly of the eIF4F initiation complex involves phosphorylation of the inhibitory eIF4E binding protein-1 (4E-BP1), which releases the initiation factor eIF4E and allows it to bind with eIF4G. Binding of eIF4E with eIF4G promotes preparation of the mRNA for binding to the 43S pre-initiation complex. Consumption of the amino acid leucine (Leu) is a key factor determining the anabolic response of muscle protein synthesis (MPS) and mTORC1 signaling to a meal. Research from this dissertation demonstrates that the peak activation of MPS following a complete meal is proportional to the Leu content of a meal and its ability to elevate plasma Leu. Leu has also been implicated as an inhibitor of muscle protein degradation (MPD). In particular, there is evidence suggesting that in muscle wasting conditions Leu supplementation attenuates expression of the ubiquitin-proteosome pathway, which is the primary mode of intracellular protein degradation. However, this is untested in healthy, physiological feeding models. Therefore, an experiment was performed to see if feeding isonitrogenous protein sources with different Leu contents to healthy adult rats would differentially impact ubiquitin-proteosome (protein degradation) outcomes; and if these outcomes are related to the meal responses of plasma Leu. Results showed that higher Leu diets were able to attenuate total proteasome content but had no effect on ubiquitin proteins. This research shows that dietary Leu determines postprandial muscle anabolism. In a parallel line of research, the effects of dietary Leu on changes in muscle mass overtime were investigated. Animals consuming higher Leu diets had larger gastrocnemius muscle weights; furthermore, gastrocnemius muscle weights were correlated with postprandial changes in MPS (r=0.471, P<0.01) and plasma Leu (r=0.400, P=0.01). These results show that the effect of Leu on ubiquitin-proteosome pathways is minimal for healthy adult rats consuming adequate diets. Thus, long-term changes in muscle mass observed in adult rats are likely due to the differences in MPS, rather than MPD. Factors determining the duration of Leu-stimulated MPS were further investigated. Despite continued elevations in plasma Leu and associated translation initiation factors (e.g., S6K1 and 4E-BP1), MPS returned to basal levels ~3 hours after a meal. However, administration of additional nutrients in the form of carbohydrate, Leu, or both ~2 hours after a meal was able to extend the elevation of MPS, in a time and dose dependent manner. This effect led to a novel discovery that decreases in translation elongation activity was associated with increases in activity of AMP kinase, a key cellular energy sensor. This research shows that the Leu density of dietary protein determines anabolic signaling, thereby affecting cellular energetics and body composition.