Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 μM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.

VanX, a bacterial d-alanyl-d-alanine dipeptidase: Resistance, immunity, or survival function?

The zinc-containing d-alanyl-d-alanine (d-Ala-d-Ala) dipeptidase VanX has been detected in both Gram-positive and Gram-negative bacteria, where it appears to have adapted to at least three distinct physiological roles. In pathogenic vancomycin-resistant enterococci, vanX is part of a five-gene cluster that is switched on to reprogram cell-wall biosynthesis to produce peptidoglycan chain precursors terminating in d-alanyl-d-lactate (d-Ala-d-lactate) rather than d-Ala-d-Ala. The modified peptidoglycan exhibits a 1,000-fold decrease in affinity for vancomycin, accounting for the observed phenotypic resistance. In the glycopeptide antibiotic producers Streptomyces toyocaensis and Amylocatopsis orientalis, a vanHAX operon may have coevolved with antibiotic biosynthesis genes to provide immunity by reprogramming cell-wall termini to d-Ala-d-lactate as antibiotic biosynthesis is initiated. In the Gram-negative bacterium Escherichia coli, which is never challenged by the glycopeptide antibiotics because they cannot penetrate the outer membrane permeability barrier, the vanX homologue (ddpX) is cotranscribed with a putative dipeptide transport system (ddpABCDF) in stationary phase by the transcription factor RpoS (σs). The combined action of DdpX and the permease would permit hydrolysis of d-Ala-d-Ala transported back into the cytoplasm from the periplasm as cell-wall crosslinks are refashioned. The d-Ala product could then be oxidized as an energy source for cell survival under starvation conditions.

Correlated effects of sperm competition and postmating female mortality

Adaptations in one sex may impair fitness in the opposite sex. Experiments with Drosophila melanogaster have shown that seminal fluid from the male accessory gland triggers a series of postmating responses in the female, including increased egg laying rate and lower remating propensity, but that accessory gland proteins also increase female death rate. Here, we tested the relationships among the longevity of females mated to males from 51 chromosome-extracted D. melanogaster lines, male-mating ability, and sperm-competitive ability. We found significant differences in longevity of females mated to males of different genotypes, and all mated females showed a higher death rate than control virgin females shortly after mating. Both the age-independent mortality parameter (the intercept of the female's survival function) and the slope of the mortality rate curve were significantly correlated with the proportion of progeny sired by the first male to mate relative to tester males (sperm-defense ability, P1). No significant correlation was found between the proportion of progeny sired by the second-mating male relative to tester males (sperm-offense ability, P2) and any mortality parameter. Our results support the hypothesis of a tradeoff between defensive sperm-competitive ability of males and life-history parameters of mated females.

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.

Go protein-dependent survival of primary accessory olfactory neurons

Extensive G protein-coupled receptor families in both the main and accessory olfactory systems have been implicated in axonal targeting, sensory function, and cell survival. Although sensory function seems to be mediated by G proteins, axonal guidance and cell survival may be G protein-independent processes. In the accessory olfactory system, the Go-containing neurons in the basal vomeronasal organ (VNO) project to the posterior accessory olfactory bulb (AOB), whereas more apically located VNO neurons contain Gi2 and project to the anterior AOB. Herein, we investigate the organization of the accessory olfactory system in mice with a targeted deletion in the Goα gene. The accessory olfactory system seems normal at birth; however, postnatally, the number of Go-receptor-containing VNO neurons decreases by half, and apoptotic neurons are detected. The axons of VNO neurons remain restricted to the posterior AOB. The posterior AOB is reduced in size but contains a synaptophysin-positive layer with the normal number of glomeruli. The posterior AOB has reduced mitral cell c-Fos immunoreactivity, consistent with decreased sensory activation of Go protein-coupled VNO receptor neurons. Thus, in the accessory olfactory system, receptor-coupled G proteins are required for cell survival.

The lipid phosphatase activity of PTEN is critical for its tumor supressor function

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.

PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway

To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten−/− ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G1/S transition was accompanied by down-regulation of p27KIP1, a major inhibitor for G1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten−/− cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4,5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.

Parent–progeny recognition as a function of MHC odortype identity

The several linked polymorphic genes of the MHC, which has been proposed as a prime determinant of sensed genetic individuality within species, is known to operate in mice by olfactory recognition in aspects of reproductive behavior that concern mate selection, thereby favoring outbreeding and heterozygosity, and also concern the maintenance of pregnancy. A single base-change can alter an individual MHC odortype, and the potential range of combinatorial MHC-determined odortypes is clearly vast. Following our findings that newborn mice already express their MHC odortype (which is detectable at 9 days of gestational age), we sought to determine whether MHC is involved in behavioral aspects of early development, such as rearing. In the studies presented herein, we report the ability and proclivity of mothers to recognize and preferentially retrieve syngeneic (genetically identical) pups from other pups differing only for MHC. Reciprocally, we report the ability of pups to recognize their familial environment, regardless of whether they had been nursed by their biological mothers or by foster mothers. Early learning experiences of the MHC environment are apparently a key element in survival, assuring maternal protection and promoting outbreeding.

Survival motor neuron protein modulates neuron-specific apoptosis

Spinal muscular atrophy (SMA) is attributed to mutations in the SMN1 gene, leading to loss of spinal cord motor neurons. The neurotropic Sindbis virus vector system was used to investigate a role for the survival motor neuron (SMN) protein in regulating neuronal apoptosis. Here we show that SMN protects primary neurons and differentiated neuron-like stem cells, but not cultured cell lines from virus-induced apoptotic death. SMN also protects neurons in vivo and increases survival of virus-infected mice. SMN mutants (SMNΔ7 and SMN-Y272C) found in patients with SMA not only lack antiapoptotic activity but also are potently proapoptotic, causing increased neuronal apoptosis and animal mortality. Full-length SMN is proteolytically processed in brains undergoing apoptosis or after ischemic injury. Mutation of an Asp-252 of SMN abolished cleavage of SMN and increased the antiapoptotic function of full-length SMN in neurons. Taken together, deletions or mutations of the C terminus of SMN that result from proteolysis, splicing (SMNΔ7), or germ-line mutations (e.g., Y272C), produce a proapoptotic form of SMN that may contribute to neuronal death in SMA and perhaps other neurodegenerative disorders.

Regulation of CD40 function by its isoforms generated through alternative splicing

CD40 is a member of the tumor necrosis factor receptor superfamily. The interaction between CD40 and CD40 ligand (CD154) activates NF-κB, Jun N-terminal kinase, and Janus kinase/signal transducers and activators of transcription pathways and promotes B cell growth, differentiation, and survival as well as IL-12 production in macrophages and dendritic cells. We demonstrate here the existence of multiple isoforms of CD40 mRNA generated by alternative splicing and show that their expression is regulated differentially in activated macrophages and dendritic cells. Pre-CD40 RNA is spliced preferentially out to signal-transducible CD40 mRNA in the early stage of activation; half of the CD40 mRNA is replaced by the signal-nontransducible CD40 mRNAs in the later stages (24 h). Using IL-12 p40 gene expression as a reporter for CD40 signaling, we show that three of the alternative isoforms can disable signaling through CD40. The major alternative isoform lacks the membrane-associated endodomain and seems to reduce the amount of the signal-transducible form available on the cell surface. It would seem, therefore, that CD40 expression is controlled by posttranscriptional and posttranslational regulation through alternative splicing. Modulation of isoform expression may provide a mechanism by which cells regulate their susceptibility to CD40L signaling.

Profilin I is essential for cell survival and cell division in early mouse development

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1ko) allele in mice. Homozygous pfn1ko/ko mice are not viable. Pfn1ko/ko embryos died as early as the two-cell stage, and no pfn1ko/ko blastocysts were detectable. Adult pfn1ko/wt mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1ko/wt embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

Cardiac βARK1 inhibition prolongs survival and augments β blocker therapy in a mouse model of severe heart failure

Chronic human heart failure is characterized by abnormalities in β-adrenergic receptor (βAR) signaling, including increased levels of βAR kinase 1 (βARK1), which seems critical to the pathogenesis of the disease. To determine whether inhibition of βARK1 is sufficient to rescue a model of severe heart failure, we mated transgenic mice overexpressing a peptide inhibitor of βARK1 (βARKct) with transgenic mice overexpressing the sarcoplasmic reticulum Ca2+-binding protein, calsequestrin (CSQ). CSQ mice have a severe cardiomyopathy and markedly shortened survival (9 ± 1 weeks). In contrast, CSQ/βARKct mice exhibited a significant increase in mean survival age (15 ± 1 weeks; P < 0.0001) and showed less cardiac dilation, and cardiac function was significantly improved (CSQ vs. CSQ/βARKct, left ventricular end diastolic dimension 5.60 ± 0.17 mm vs. 4.19 ± 0.09 mm, P < 0.005; % fractional shortening, 15 ± 2 vs. 36 ± 2, P < 0.005). The enhancement of the survival rate in CSQ/βARKct mice was substantially potentiated by chronic treatment with the βAR antagonist metoprolol (CSQ/βARKct nontreated vs. CSQ/βARKct metoprolol treated, 15 ± 1 weeks vs. 25 ± 2 weeks, P < 0.0001). Thus, overexpression of the βARKct resulted in a marked prolongation in survival and improved cardiac function in a mouse model of severe cardiomyopathy that can be potentiated with β-blocker therapy. These data demonstrate a significant synergy between an established heart-failure treatment and the strategy of βARK1 inhibition.

Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism

The Ser/Thr kinase Raf-1 is a protooncogene product that is a central component in many signaling pathways involved in normal cell growth and oncogenic transformation. Upon activation, Raf-1 phosphorylates mitogen-activated protein kinase kinase (MEK), which in turn activates mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs), leading to the propagation of signals. Depending on specific stimuli and cellular environment, the Raf-1–MEK–ERK cascade regulates diverse cellular processes such as proliferation, differentiation, and apoptosis. Here, we describe a MEK–ERK-independent prosurvival function of Raf-1. We found that Raf-1 interacts with the proapoptotic, stress-activated protein kinase ASK1 (apoptosis signal-regulating kinase 1) in vitro and in vivo. Deletion analysis localized the Raf-1 binding site to the N-terminal regulatory fragment of ASK1. This interaction allows Raf-1 to act independently of the MEK–ERK pathway to inhibit apoptosis. Furthermore, catalytically inactive forms of Raf-1 can mimic the wild-type effect, raising the possibility of a kinase-independent function of Raf-1. Thus, Raf-1 may promote cell survival through its protein–protein interactions in addition to its established MEK kinase function.

Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investigate the mechanism by which CD4 cross-linking induces cell death. We have found that CD4 cross-linking results in a small but rapid increase in levels of cell surface Fas, a member of the tumor necrosis factor receptor family implicated in apoptotic death and maintenance of immune homeostasis. Importantly, CD4 cross-linking triggered the ability of Fas to function as a death molecule. Subsequent to CD4 cross-linking, CD4+ splenocytes cultured overnight became sensitive to Fas-mediated death. Death was Fas-dependent, as demonstrated by cell survival in the absence of plate-bound anti-Fas antibody, and by the lack of CD4-induced death in cells from Fas-defective lymphoproliferative (lpr) mice. We demonstrate here that CD4 regulates the ability of Fas to induce cell death in Cd4+ T cells.

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.