Unstimulated State Research Articles

Epigenetic Control of Inducible Gene Expression in the Immune System

It has been well documented that active genes, and their promoters and enhancers have a different chromatin or epigenomic environment compared with unexpressed genes. In addition, the epigenome may influence not only which genes are expressed, but also which genes can be induced in response to activation or differentiation signals. Immune cells respond to activation signals by rapidly inducing the expression of specific gene sets, and therefore this is a good system in which to examine the role of the epigenome in gene activation and cell differentiation. Several studies have now found that many immediate-early inducible genes exist in a similar epigenomic environment to active genes even in the unstimulated state. Some studies suggest that subsets of these genes may even have RNA polymerase II at their promoters and induction may be controlled downstream of its recruitment. Other inducible genes, however, undergo changes to histone modifications, levels or variant composition upon activation. In this article, we discuss how the epigenome of immune cells regulates inducible gene expression and discuss the differences between the immediate responses to activation signals and the longer term changes observed during differentiation.

Lipid Rafts and Caveolin-1 Coordinate Interleukin-1β (IL-1β)-dependent Activation of NFκB by Controlling Endocytosis of Nox2 and IL-1β Receptor 1 from the Plasma Membrane

Recent evidence suggests that signaling by the proinflammatory cytokine interleukin-1beta (IL-1beta) is dependent on reactive oxygen species derived from NADPH oxidase. Redox signaling in response to IL-1beta is known to require endocytosis of its cognate receptor (IL-1R1) following ligand binding and the formation of redox-active signaling endosomes that contain Nox2 (also called redoxosomes). The consequent generation of reactive oxygen species by redoxosomes is responsible for the downstream recruitment of IL-1R1 effectors (IRAK, TRAF6, and IkappaB kinase kinases) and ultimately for activation of the transcription factor NFkappaB. Despite this knowledge of the signaling events that occur downstream of redoxosome formation, an understanding of the mechanisms that coordinate the genesis of redoxosomes following IL-1beta stimulation has been lacking. Here, we demonstrate that lipid rafts play an important role in this process. We show that Nox2 and IL-1R1 localize to plasma membrane lipid rafts in the unstimulated state and that IL-1beta signals caveolin-1-dependent endocytosis of both proteins into the redoxosome. We also show that inhibiting lipid raft-mediated endocytosis prevents NFkappaB activation. Finally, we demonstrate that Vav1, a Rac1 guanine exchange factor and activator of Nox2, is recruited to lipid rafts following IL-1beta stimulation and that it is required for NFkappaB activation. Our results fill in an important mechanistic gap in the understanding of early IL-1R1 and Nox2 signaling events that control NFkappaB activation, a redox-dependent process important in inflammation.

The Partitioning and Domain-Specific Multi-Site Phosphorylation "Zip Codes" of Endothelial Nitric Oxide Synthase in Uterine Artery Endothelial Cells.

Pregnancy-associated rises in uterine blood flow occurs via changes in endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production at the level of uterine arteries. In arterial endothelial cells, the eNOS protein has been associated with lipid-ordered microdomains called caveolae and is tonically inhibited by the major caveolar scaffolding protein caveolin-1 (cav-1). However, the spatial and temporal dynamics of eNOS, including domain-specific post-translational modification via multi-site phosphorylation that are critical for enzyme activation and NO production, in response to a physiologic calcium mobilizing agonist-like ATP, especially during pregnancy are unknown. The aim of this study was to determine the spatial and temporal dynamics of eNOS in response to the calcium mobilizing physiologic agonist ATP in endothelial cells from the uterine arteries of pregnant ewes when the rises in UBF and NO are greatest. Primary uterine arteries of late gestation ewes were dissected and the endothelial cells were isolated and validated. Temporal and spatial dynamics of eNOS, and domain-specific multi-site phosphorylation were studied by density centrifugation, confocal microscopy, immunoisolation, native gel electrophoresis and Western blotting. Under unstimulated conditions, eNOS was predominantly located in specialized caveolar microdomains. Treatment with ATP resulted in time-dependent re-partitioning of eNOS between the caveolar and non-caveolar domains. The abundance of cav-1 bound eNOS significantly decreased in response to ATP (one way ANOVA, P = 0.039); compared to the unstimulated state, the magnitude of cav-1 bound eNOS was significantly lower at 2.5 (P = 0.002), 5 (P = 0.02) and 10 (P = 0.02) minutes after ATP treatment. eNOS exhibited a domain-specific multi-site phosphorylation "zip code." In unstimulated cells, P-Thr497eNOS but not P-Ser635eNOS or P-Ser1179eNOS was detected in the caveolar domain. In contrast, P-Ser114eNOS was detected only in the non-caveolar pool. Upon ATP stimulation, P-Ser1179eNOS was mainly detectable only in the caveolar domain whereas P-Ser635eNOS was detectable in both the caveolar and the non-caveolar pools. Moreover with ATP stimulation, P-Thr497eNOS was barely detectable in the caveolar domain and P-Ser114eNOS was not detectable in any of the fractions. These observations were confirmed by non-denaturing gel electrophoresis where ATP receptor activation increased cav-1 free-dimerized eNOS by 54% (F1,5 = 15.8; P = 0.011). Further, ATP resulted in a significant increase in the magnitude of P14cav-1. Confocal microscopy utilizing double immunofluorescence staining showed the temporal and spatial partition of post-translationally modified eNOS and total eNOS between the caveolar and cytoplasmic domains to be consistent with immunoblots. Conclusion: This is the first study to systematically investigate the temporal and spatial partition of eNOS in uterine artery endothelial cells. We show the novel observation that multi-site phosphorylation state of eNOS acts as a "zip code" for the intracellular location of the enzyme, and gives direct clues to the state of the enzyme activity. These data suggest that the regulatory mechanisms pertaining temporal and spatial dynamics of eNOS may delineate a greater understanding of the etiologies of gestational diseases. NIH HL49210, HD38843, HL87144, HL86939, HL70562 and HL74947. (poster)

Adenylyl cyclase type 6 regulates Akt activity via PHLPP2

Adenylyl cyclase (AC) is the enzyme that catalyzes ATP to generate cyclic AMP (cAMP). Cardiac‐directed expression of adenylyl cyclase type 6 (AC6) has pronounced favorable effects on heart function in normal and failing hearts. The mechanisms explaining the favorable effects of AC6 are not entirely known, but may reflect effects on cellular events that are not replicated by cAMP production. Because of the central role of Akt in intracellular signaling, and its importance to cardiac myocyte survival and function, we tested the hypothesis that increased AC6 expression influences Akt signaling. Studies were performed in cultured neonatal cardiac myocytes; AC6 gene transfer was performed using recombinant adenovirus encoding AC6 (Ad.AC6). In the basal (unstimulated) state, AC6 gene transfer increased Akt phosphorylation and activity 5‐fold. However, when these cardiac myocytes were stimulated with a ß‐adrenergic receptor agonist (isoproterenol) or with an adenylyl cyclase activator (forskolin), AC6‐associated Akt phosphorylation rapidly was reduced (<2 min). This reduction in Akt phosphorylation required an Akt‐specific phosphatase, PH domain leucine‐rich phosphatase (PHLPP2), but was cAMP‐independent and reversible. The Mg2+ binding domain in the C1 region of AC6 is required for agonist‐induced activation of PHLPP2. This novel bidirectional regulation of Akt activity may contribute to the unexpected favorable effects of AC6 on the failing heart.

Activation of c-Src and Fyn Kinases by Protein-tyrosine Phosphatase RPTPα Is Substrate-specific and Compatible with Lipid Raft Localization

Src family tyrosine kinases (SFKs) function in multiple signaling pathways, raising the question of how appropriate regulation and substrate choice are achieved. SFK activity is modulated by several protein-tyrosine phosphatases, among which RPTPalpha and SHP2 are the best established. We studied how RPTPalpha affects substrate specificity and regulation of c-Src and Fyn in response to epidermal growth factor and platelet-derived growth factor. We find that RPTPalpha, in a growth factor-specific manner, directs the specificity of these kinases toward a specific subset of SFK substrates, particularly the focal adhesion protein Paxillin and the lipid raft scaffolding protein Cbp/PAG. A significant fraction of RPTPalpha is present in lipid rafts, where its targets Fyn and Cbp/PAG reside, and growth factor-mediated SFK activation within this compartment is strictly dependent on RPTPalpha. Forced concentration of RPTPalpha into lipid rafts is compatible with activation of Fyn. Finally, RPTPalpha-induced phosphorylation of Paxillin and Cbp/PAG induces recruitment of the SFK inhibitory kinase Csk, indicative of negative feedback loops limiting SFK activation by RPTPalpha. Our findings indicate that individual SFK-controlling PTPs play important and specific roles in dictating SFK substrate specificity and regulatory mechanism.

Plasticity of Drosophila Stat DNA binding shows an evolutionary basis for Stat transcription factor preferences

In vertebrates, seven signal transducer and activator of transcription (STAT) proteins bind to palindromic sites separated by spacers of two or three nucleotides (STAT1), four nucleotides (STAT6) or three nucleotides (STAT2 to STAT5a/b). This diversity of binding sites provides specificity to counter semiredundancy and was thought to be a recent evolutionary acquisition. Here, we examine the natural DNA-binding sites of the single Drosophila Stat and show that this is not the case. Rather, Drosophila Stat92E is able to bind to and activate target gene expression through both 3n and 4n spaced sites. Our experiments indicate that Stat92E has a higher binding affinity for 3n sites than for 4n sites and suggest that the levels of target gene expression can be modulated by insertion and/or deletion of single bases. Our results indicate that the ancestral STAT protein had the capacity to bind to 3n and 4n sites and that specific STAT binding preferences evolved with the radiation of the vertebrate STAT family.

Spontaneous expression of IL-4 mRNA in lymphocytes from children with atopic dermatitis.

Normal lymphocytes do not generally produce or secrete lymphokines in the resting or unstimulated state and only express or release cytokines following activation. Recently, the spontaneous production of intracellular interferon-gamma (IFN-gamma) and spontaneous secretion of IL-6 has been documented in patients with atopic dermatitis. These findings indicated that lymphocytes had been previously activated in vivo. Such in vivo activation may also be associated with spontaneous production of IL-4. As measurement of IL-4 secretion by immunoassay is complicated by poor sensitivity, and only provides information on the net amount of cytokine present after secretion, adsorption, consumption and degradation have occurred, IL-4 mRNA expression in peripheral blood lymphocytes from children with atopic dermatitis and controls was examined by polymerase chain reaction (PCR)-assisted mRNA amplification. Spontaneous expression of IL-4 mRNA was detected in four of eight patients with severe atopic dermatitis. Following stimulation in vitro, seven of eight atopic patients demonstrated detectable IL-4 mRNA. In comparison, no spontaneous expression of IL-4 mRNA was found in controls, and only six of 10 controls expressed IL-4 mRNA in stimulated cultures. The spontaneous expression of IL-4 mRNA in unstimulated cultures from children with atopic dermatitis supports the possibility that previous in vivo activation has occurred, and suggests that IL-4 production is increased in vivo in atopic dermatitis. This in vivo activation together with the constitutive expression of IL-4 mRNA are likely to contribute to the spontaneous in vitro production of IgE in atopic patients.

Alveolar macrophage phagocytosis is impaired in children with poorly controlled asthma

Lower respiratory tract infection is a differentiating feature of children with poorly controlled asthma. Given the role of alveolar macrophages (AMs) in innate immunity, we hypothesized that AM phagocytosis might be impaired in poorly controlled asthma. Bronchoalveolar lavage fluid AMs were isolated from 28 asthmatic children (moderate asthma, n = 12; severe asthma, n = 16), 10 nonasthmatic children with chronic cough treated with inhaled corticosteroids, and 10 healthy adult control subjects. AMs were stimulated with LPS and exposed to fluorescein isothiocyanate-conjugated Staphylococcus aureus for 2 hours. Phagocytosis was quantified by using a phagocytic index (PI) calculated from the percentage of phagocytic cells multiplied by the relative fluorescence (RFU) units of S. aureus per cell. Apoptosis was determined from the percentage of cells positive for poly (adenosine diphosphate-ribose) polymerase. Phagocytosis as measured by using the unstimulated PI was decreased in subjects with poorly controlled asthma (healthy control subjects, 9330 +/- 3992 RFU; chronic cough, 9042 +/- 5976 RFU; moderate asthma, 4361 +/- 2536 RFU; severe asthma, 3153 +/- 1886 RFU; P < .001) and remained unchanged with LPS stimulation. Children with severe asthma also had increased AM apoptosis, both the unstimulated and LPS-simulated states (P < .001), which correlated with the PI. AM function is compromised in children with poorly controlled asthma and is characterized by decreased phagocytosis and increased apoptosis.

Conferral of Enhanced Natural Killer Cell Function by KIR3DS1 in Early Human Immunodeficiency Virus Type 1 Infection

A flurry of recent reports on the role of activating and inhibitory forms of the killer cell immunoglobulin-like receptors (KIR) in natural killer (NK) cell activity against human immunodeficiency virus type 1 (HIV-1) have yielded widely divergent results. The role of the activating NK receptor encoded by the KIR3DS1 allele and its putative ligands, members of the HLA class I Bw4Ile80 cluster, in early HIV-1 disease is controversial. We selected 60 treatment-naïve adults for study from the OPTIONS cohort of individuals with early HIV-1 infection in San Francisco. We performed NK cell functional assays measuring gamma interferon (IFN-gamma) and CD107a expression by NK cells in the unstimulated state and after stimulation by the major histocompatibility complex class I-deficient 721.221 B-lymphoblastoid cell line. In addition, we measured CD38 expression (a T-cell activation marker) on T and NK cells. Persons who have at least one copy of the KIR3DS1 gene had higher IFN-gamma and CD107a expression in the unstimulated state compared to those who do not possess this gene. After stimulation, both groups experienced a large induction of IFN-gamma and CD107a, with KIR3DS1 carriers achieving a greater amount of IFN-gamma expression. Differences in effector activity correlating with KIR3DS1 were not attributable to joint carriage of HLA Bw4Ile80 and KIR3DS1. We detected a partial but not complete dependence of KIR3DS1 on the members of B*58 supertype (B*57 and B*58) leading to higher NK cell function. Possessing KIR3DS1 was associated with lower expression of CD38 on both CD8(+) T and NK cells and with a loss or weakening of the known strong associations between CD8(+) T-cell expression of CD38 mean fluorescence intensity and the HIV-1 viral load. We observed that possessing KIR3DS1 was associated with higher NK cell effector functions in early HIV-1 disease, despite the absence of HLA Bw4Ile80, a putative ligand of KIR3DS1. Carriage of KIR3DS1 was associated with diminished CD8(+) T-cell activation, as determined by expression of CD38, and a disruption of the traditional relationship between viral load and activation in HIV-1 disease, which may lead to better clinical outcomes for these individuals.

Extracellular point mutations in FGFR2 result in elevated ERK1/2 activation and perturbation of neuronal differentiation

Two independent gain-of-function point mutations (S252W and P253R) in the extracellular region of the FGFR2 (fibroblast growth factor receptor 2) increase the binding affinity for the growth factor. The effect of this enhanced growth factor binding by these mutants is expected to be an increase in activation of regular signalling pathways from FGFR2 as a result of more receptors being engaged by ligand at any given time. Using PC12 (pheochromocytoma) cells as a model cell system we investigated the effect of these mutations on protein phosphorylation including the receptor, the activation of downstream signalling pathways and cell differentiation. Our results show that the effects of both of these extracellular mutations have unexpected intracellular phenotypes and cellular responses. Receptor phosphorylation was altered in both the ligand-stimulated and unstimulated states. The mutants also resulted in differential phosphorylation of a number of intracellular proteins. Both mutations resulted in enhanced ERK1/2 (extracellular-signalregulated kinase1/2) activation. Although ERK1/2 activation is believed to transduce signals resulting in cell differentiation, this response was abrogated in the cells expressing the mutant receptors. The results of the present study demonstrate that single extracellular point mutations in the FGFR2 have a profound effect on intracellular signalling and ultimately on cell fate.

A Moving Story

When activated by their transmembrane receptors, heterotrimeric guanine nucleotide-binding proteins (G proteins) dissociate into α and βγ subunits to act on plasma membrane-associated targets. Thus, G proteins are typically thought to reside in association with the cytoplasmic surface of the plasma membrane. Two articles from the same group described research using various fluorescence imaging methods in live cells--including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and photoactivation of a fluorescent protein--to directly examine G protein localization. Chisari et al . focused on G proteins in the basal (unstimulated) state; they found that fluorescently tagged α o , β 1 , and various γ subunits shuttled between the plasma membrane and the internal membranes of CHO cells, with a t 1/2 of less than a minute. G protein movement was not blocked by pharmacological inhibitors of vesicular trafficking and was still rapid at 10 o C, which, together with the rapidity of the movement, suggested that it depended on diffusion rather than vesicular trafficking. Treatment with 2-bromopalmitate, however, which inhibits palmitoylation, inhibited shuttling. Fluorescence resonance energy transfer (FRET) analysis indicated that G proteins were resident as heterotrimers in internal membranes as well as in the plasma membrane. Saini et al ., investigated the movement of βγ when β 1 was complexed with different fluorescently labeled γ subunits after receptor-mediated activation in CHO cells stably expressing M2 muscarinic receptors. Treatment with the muscarinic agonist carbachol led to the movement of βγ containing 6 of 12 γ subunit types from the plasma membrane to internal membranes, whereas treatment with antagonist or agonist withdrawal led to translocation back to the plasma membrane. Intriguingly, βγ subunits were targeted to distinct compartments, depending on specific γ subunit identity. γ 1 , γ 5 , γ 9 , and γ 11 translocated to one compartment, identified as the Golgi, whereas γ 13 translocated to another, identified as the endoplasmic reticulum. The rates of translocation differed as well, falling into either a "rapid" (t 1/2 ~ 6 to 13 s) or a "slow" (t 1/2 ~ 39 to 85 s) group. The ability of specific subunits to translocate, and the speed with which they did so, was closely related to their structure, and mutation of C-terminal residues enabled nontranslocating subunits to do so. Translocation occurred in various cell types after activation by various receptors. Like the shuttling of heterotrimers in the basal state, translocation was inhibited by 2-bromopalmitate and was likely diffusion mediated. Thus, the authors conclude that both likely involve the same process and speculate that the ability of only certain γ subunits to translocate and their targeting to distinct compartments add additional layers of complexity to regulation of G protein signaling. M. Chisari, D. K. Saini, V. Kalyanaraman, N. Gautam, Shuttling of G protein subunits between the plasma membrane and intracellular membranes. J. Biol. Chem. 282 , 24092-24098 (2007). [Abstract] [Full Text] D. K. Saini, V. Kalyanaraman, M. Chisari, N. Gautam, A family of G protein βγ subunits translocate reversibly from the plasma membrane to endomembranes on receptor activation. J. Biol. Chem. 282 , 24099-24108 (2007). [Abstract] [Full Text]

Optimal deep brain stimulation of the subthalamic nucleus—a computational study

Deep brain stimulation (DBS) of the subthalamic nucleus, typically with periodic, high frequency pulse trains, has proven to be an effective treatment for the motor symptoms of Parkinson's disease (PD). Here, we use a biophysically-based model of spiking cells in the basal ganglia (Terman et al., Journal of Neuroscience, 22, 2963-2976, 2002; Rubin and Terman, Journal of Computational Neuroscience, 16, 211-235, 2004) to provide computational evidence that alternative temporal patterns of DBS inputs might be equally effective as the standard high-frequency waveforms, but require lower amplitudes. Within this model, DBS performance is assessed in two ways. First, we determine the extent to which DBS causes Gpi (globus pallidus pars interna) synaptic outputs, which are burstlike and synchronized in the unstimulated Parkinsonian state, to cease their pathological modulation of simulated thalamocortical cells. Second, we evaluate how DBS affects the GPi cells' auto- and cross-correlograms. In both cases, a nonlinear closed-loop learning algorithm identifies effective DBS inputs that are optimized to have minimal strength. The network dynamics that result differ from the regular, entrained firing which some previous studies have associated with conventional high-frequency DBS. This type of optimized solution is also found with heterogeneity in both the intrinsic network dynamics and the strength of DBS inputs received at various cells. Such alternative DBS inputs could potentially be identified, guided by the model-free learning algorithm, in experimental or eventual clinical settings.

Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes

This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.

Protein Phosphatase 6 Down-regulates TAK1 Kinase Activation in the IL-1 Signaling Pathway

TAK1 (transforming growth factor beta-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.

Stable Association between Gαq and Phospholipase Cβ1 in Living Cells

Signal transduction through G alpha(q) involves stimulation of phospholipase C beta (PLC beta) that results in increased intracellular Ca2+ and activation of protein kinase C. We have measured complex formation between G alpha(q) and PLC beta1 in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC beta1 will bind to G alpha(q)(guanosine 5'-3-O-(thio)triphosphate) and also to G alpha(q)(GDP), and the latter association has a different protein-protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G alpha(q) is localized almost entirely to the plasma membrane, whereas PLC beta1 has a significant cytosolic population. By using fluorescence resonance energy transfer, we found that these proteins are pre-associated in the unstimulated state in PC12 and HEK293 cells. By determining the cellular levels of the two proteins in transfected versus nontransfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G alpha(q)-PLC beta1 complexes are observed in both single cell measurements and measurements of a large (i.e. 10(6)) cell suspension. The high level (approximately 40% maximum) of FRET is surprising considering that G alpha(q) is more highly expressed than PLC beta1 and that not all PLC beta1 is plasma membrane-localized. Our measurements suggest a model in which G proteins and effectors can exist in stable complexes prior to activation and that activation is achieved through changes in intermolecular interactions rather than diffusion and association. These pre-formed complexes in turn give rise to rapid, localized signals.

Ligand-regulated Peptide Aptamers that Inhibit the 5′-AMP-activated Protein Kinase

In an effort to extend the peptide aptamer approach, we have developed a scaffold protein that allows small molecule ligand control over the presentation of a peptide aptamer. This scaffold, a fusion of three protein domains, FKBP12, FRB, and GST, presents a peptide linker region for target protein binding only in the absence of the small molecule Rapamycin or other non-immunosuppressive Rapamycin derivatives. Here we describe the characterization of ligand-regulated peptide aptamers that interact with and inhibit the 5′-AMP-activated protein kinase (AMPK). AMPK, a central regulator of cellular energy homeostasis, responds to high cellular AMP/ATP ratios by promoting energy producing pathways and inhibiting energy consuming biosynthetic pathways. We have characterized 15 LiRPs of similar, poly-basic sequence and have determined that they interact with the substrate peptide binding region of both AMPK α1 and α2. These proteins, some of which serve as poor substrates of AMPK, inhibit the kinase as pseudosubstrates in a Rapamycin-regulated fashion in vitro, an effect that is largely competitive with substrate peptide and mediated by an increase in the kinase's apparent K m for substrate peptide. This pseudosubstrate inhibition of AMPK by LiRP proteins reduced the AMP stimulation of AMPK in vitro and caused the inhibited state of the kinase to kinetically resemble the basal, unstimulated state of AMPK, providing potential insight into the molecular mechanisms of AMP stimulation of AMPK.

Agonist-stimulated β-Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover

Stimulation of beta-adrenergic receptors (betaARs) leads to sequential recruitment of beta-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for betaAR endocytosis is not known. In this study, we have used beta(1)- and beta(2) ARs, two ubiquitously expressed members of the betaAR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of beta(2)ARs, beta(1)ARs, and mutants of beta(1)ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit beta-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for beta(2)AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of beta(2)AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated beta(2)AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of beta(2)AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal betaAR internalization.

ATM Activation by Ionizing Radiation Requires BRCA1-associated BAAT1

ATM (ataxia telangiectasia mutated) is required for the early response to DNA-damaging agents such as ionizing radiation (IR) that induce DNA double-strand breaks. Cells deficient in ATM are extremely sensitive to IR. It has been shown that IR induces immediate phosphorylation of ATM at Ser(1981), leading to catalytic activation of the protein. We recently isolated a novel BRCA1-associated protein, BAAT1 (BRCA1-associated protein required for ATM activation-1), by yeast two-hybrid screening and found that BAAT1 also binds to ATM, localizes to double-strand breaks, and is required for Ser(1981) phosphorylation of ATM. Small interfering RNA-mediated stable or transient reduction of BAAT1 resulted in decreased phosphorylation of both ATM at Ser(1981) and CHK2 at Thr(68). Treatment of BAAT1-depleted cells with okadaic acid greatly restored phosphorylation of ATM at Ser(1981), suggesting that BAAT1 is involved in the regulation of ATM phosphatase. Protein phosphatase 2A-mediated dephosphorylation of ATM was partially blocked by purified BAAT1 in vitro. Significantly, acute loss of BAAT1 resulted in increased p53, leading to apoptosis. These results demonstrate that DNA damage-induced ATM activation requires a coordinated assembly of BRCA1, BAAT1, and ATM.

Biphasic Effects of Statins on Angiogenesis-Mediated Processes.

Background: Statins inhibit HMG-CoA reductase, resulting in the reduction of cholesterol synthesis as well as isoprenoids that modulate various cell functions.Methods: The present study was undertaken to determine the effects of different statins (lovastatin, fluvastatin, simvastatin, and pravastatin) on endothelial cell functions and angiogenesis. The effect of statins on endothelial cell migration toward matrix proteins and stimulated with tissue factor (TF)/factor VIIa was examined using modified Boyden chamber technique. Additionally, the effect of statins on endothelial differentiation and tube formation was determined in unstimulated and basic fibroblast growth factor (FGF-2) -stimulated endothelial cell tube formation. Furthermore, the effect of statins on the modulation of new blood vessel generation in the chick chorioallantoic membrane (CAM) model was determined under unstimulated and stimulated angiogenesis by FGF-2 or insulin.Results: Endothelial cell migration toward vitronectin and stimulated by TF/VIIa was shown to be inhibited by the various statins in a concentration (0.001–10 μM) -dependent manner. Endothelial cell differentiation and tube formation were enhanced by statins at low concentrations (0.01–0.001 μM) but inhibited at relatively high concentrations (0.1–10 μM) either in unstimulated or FGF-2-stimulated conditions, with different IC50s depending on the statin. In FGF-2- or insulin-stimulated angiogenesis in the CAM model, lovastatin demonstrated significant inhibition (40-75%) at 1–5 μg. In contrast, in unstimulated state lovastatin enhanced angiogenesis in the CAM model by 20–40% at 1–5 μg. Statins have proangiogenesis effects at low concentration in unstimulated or stimulated states but angiostatic effects at high concentrations in stimulated state. Similar biphasic effects on angiogenesis-mediated processes were determined with pravastatin, fluvastatin, or simvastatin, but with different potency. The antiangiogenesis efficacy was significantly (P <0.01) greater for the different statins in inhibiting insulin versus FGF-2-induced angiogenesis.Conclusion: Statins have a biphasic dose-dependent effect on angiogenesis-mediated processes that depend on the microenvironment and status of the proangiogenesis stimuli. At clinically relevant doses, statins may modulate angiogenesis depending on the proangiogenesis/antiangiogenesis hemostasis.Table 1. Modulation of Angiogenesis by Statins in the Chick Chorioallantoic Membrane ModelTreatmentNo. of Branch Points ± SEM% Inhibition ± SEMPBS (control)76 ± 10-----PBS + lovastatin (5 ug)120 ± 12-----FGF-2189 ± 13-----FGF-2 + lovastatin (5 ug)108 ± 865 ± 8FGF = fibroblast growth factor; PBS = phosphate-buffered saline.Table 2. Statins and Angiogenesis Modulation in the Chick Chorioallantoic Membrane ModelTreatmentBranch Points ± SEM% Inhibition ± SEMPBS (control)72 ± 8-----Insulin (1 ug)163 ± 12-----PBS + lovastatin (5 ug)119 ± 8-----PBS + pravastatin (5 ug)109 ± 11-----Insulin + lovastatin (5 ug)117 ± 950 ± 10Insulin + pravastatin (5 ug)113 ± 854 ± 9PBS = phosphate-buffered saline.

Cell surface sialic acids do not affect primary CD22 interactions with CD45 and surface IgM nor the rate of constitutive CD22 endocytosis.

CD22/Siglec-2 is a B cell-specific molecule modulating surface IgM (sIgM) signaling via cytosolic tyrosine-based motifs. CD22 recognizes alpha2-6-linked sialic acids (Sias) via an amino-terminal Ig-like domain. This Sia-binding site is typically masked by unknown sialylated ligands on the same cell surface, an interaction required for optimal signaling function. We studied the effect of cell surface Sias on specific interactions of CD22 with other molecules and on its turnover via endocytosis. A novel approach for simultaneous biotinylation and cross-linking showed that CD22 associates with CD45 and sIgM at much higher levels than reported in prior studies, possibly involving cell surface multimers of CD22. Sia removal or mutation of a CD22 arginine residue required for Sia recognition did not affect these associations even in human:mouse heterologous systems, indicating that they are primarily determined by evolutionarily conserved protein-protein interactions. Thus masking of the Sia-binding site of CD22 involves many cell surface sialoglycoproteins, without requiring specific ligand(s) and/or is mediated by secondary interactions with Sias on CD45 and sIgM. Abrogating Sia interactions also does not affect constitutive CD22 endocytosis. Sia removal does enhance the much faster rate of anti-CD22 antibody-triggered endocytosis, as well as killing by an anti-CD22 immunotoxin. In contrast to the unstimulated state, sIgM cross-linking inhibits both antibody-induced endocytosis and immunotoxin killing. Thus the signal- modulating activity of CD22 Sia recognition cannot be explained by mediation of primary interactions with specific molecules, nor by effects on constitutive endocytosis. The effects on antibody-mediated endocytosis could be of relevance to immunotoxin treatment of lymphomas.