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Biomedical subjects

Tony Hunter

Publications and source records attributed to Tony Hunter.

At least 19 recordsLinked to original sources

Museum specimens reveal the genomic consequences of long-term population decline in an insect pollinator.

Global insect pollinator populations are under threat, with reported declines attributed to increasing habitat loss, pesticide use, and disease. Tracking how genetic diversity has changed over time could reveal the rate and extent of these declines, and the adaptive capacity of affected species-providing an important complement to habitat-based conservation efforts. However, few studies have been able to reconstruct suitable historical baselines to link genomic changes with population change. Here, we use whole genome data from 101 museum specimens of the declining moss carder bumblebee (Bombus muscorum) collected across Britain and Ireland between 1894 and 2019 to reveal a dramatic drop in genetic diversity over the last century. We find a substantial (∼24.6%) reduction in genome-wide heterozygosity across Britain during this period. In England and Wales, where habitat fragmentation is most pronounced, we observe a 2.86-fold increase in runs of homozygosity, commensurate with population fragmentation and isolation. Our results reveal the extent to which human-induced environmental change can lead to severe decadal-scale genomic erosion in a functionally important insect. Identified using DNA from historic museum collections, our approach has widespread applicability for insect conservation and understanding the evolutionary consequences of environmental change.

Animals↗

MEKK1 mediates the ubiquitination and degradation of c-Jun in response to osmotic stress.

c-Jun, a major transcription factor in the activating protein 1 family of regulatory proteins, is activated by many physiologic and pathological stimuli. We show here that c-Jun was downregulated in response to osmotic stress via ubiquitination-dependent degradation by the PHD/RING finger domain of MEKK1, which exhibited E3 ubiquitin ligase activity toward c-Jun in vitro and in vivo. The reduced c-Jun protein level resulting from exogenous expression of wild-type MEKK1 and the opposite effect induced by expression of a MEKK1 PHD/RING finger domain mutant were consistent with a higher level of c-Jun protein in MEKK1(-/-) cells than in corresponding wild-type cells. The deficiency of MEKK1 blocked posttranslational downregulation of c-Jun in response to osmotic stress. Furthermore, apoptosis induced by osmotic stress was suppressed by overexpression of c-Jun, indicating that the downregulation of c-Jun promotes apoptosis.

Animals↗

Proteasome inhibitor drugs on the rise.

In May 2003, the U.S. Food and Drug Administration granted the proteasome inhibitor bortezomib (Velcade) fast-track status for the treatment of multiple myeloma. This landmark represented the first approval of a drug targeting the ubiquitin-proteasome system (UPS) for any indication. More recently, at the AACR Special Conference "Ubiquitin and Cancer: From Molecular Targets and Mechanisms to the Clinic" (Orlando, FL, January 18-22, 2006), it became evident that drug discovery in the UPS is experiencing another round of great excitement. The reason--new clinical applications found for bortezomib, along with the promised success of new types of proteasome inhibitors reaching the clinic.

Antineoplastic Agents↗

SMK-1, an essential regulator of DAF-16-mediated longevity.

Insulin/IGF-1 signaling (IIS) regulates aging in worms, flies, and mice through a well-characterized, highly conserved core set of components. IIS also regulates early developmental decisions, the reproductive status of the animal, innate immunity, and stress-resistance functions. In C. elegans, the sole insulin/IGF-1 receptor, DAF-2, negatively regulates the FOXO transcription factor, DAF-16. We report here on a new component of the IIS longevity pathway, SMK-1, which specifically influences DAF-16-dependent regulation of the aging process in C. elegans by regulating the transcriptional specificity of DAF-16 activity. Localization analysis of DAF-16 places SMK-1 downstream of DAF-16's phosphorylation-dependent relocation to the nucleus. Physiological and transcription analyses indicate that smk-1 is required for the innate immune, UV, and oxidative stress but not the thermal stress functions of DAF-16. SMK-1 therefore plays a role in longevity by modulating DAF-16 transcriptional specificity without affecting other processes regulated by IIS.

Aging↗

Turning the replication checkpoint on and off.

The replication checkpoint monitors the progress of DNA replication forks during S phase, and delays the firing of later replication origins when active replication forks are stalled due to collisions with damaged or abnormally structured DNA. Key components of the replication checkpoint pathway are the apical protein kinase, ATR, and its downstream target kinase, Chk1. Defects in either ATR or Chk1 function result in loss of DNA replication fidelity and cell viability, even in the absence of extrinsic genotoxic stress. Moreover, several clinically important antitumor agents, such as the camptothecins (CPTs), exert their antitumor effects by interfering with DNA replication, and hence the therapeutic response to these drugs is intimately related to signaling through the replication checkpoint. A recent report from this laboratory adds a new facet to the regulatory mechanisms that control the function and duration of checkpoint signaling through the ATR-Chk1 pathway. The results indicate that replication stress induced by a variety of agents, including CPT and deep hypoxia, triggers the ubiquitin-dependent degradation of the checkpoint kinase Chk1 in both normal and transformed human cells. This review provides an overview of the study's major findings, together with their implications for both replication checkpoint function and tumor responsiveness to CPT and related anticancer drugs.

Checkpoint Kinase 1↗

Essential role of tuberous sclerosis genes TSC1 and TSC2 in NF-kappaB activation and cell survival.

The TSC1-TSC2 complex has recently been implicated in cell survival responses. We observed that NF-kappaB signaling is attenuated in TSC1- and TSC2-deficient MEFs concomitant with reduced survival following DNA damage or TNFalpha stimulation. Reconstitution of TSC2 expression in TSC2(-/-) MEFs rescued survival in an NF-kappaB activity-dependent manner. Furthermore, in TSC2(-/-) MEFs, the rapamycin-mediated inhibition of deregulated mTOR activity restored NF-kappaB activation and survival. This rapamycin-mediated effect was reversed by inhibition of NF-kappaB transcriptional activation or by inhibition of ERK1/2 MAP kinase or PI-3K pathways, which lie on signaling cascades that lead to NF-kappaB activation. These results provide evidence for a crosstalk between the TSC/Rheb/mTOR pathway and the NF-kappaB induction pathways and indicate that NF-kappaB functions as an important survival factor that regulates TSC2-dependent cell survival.

Animals↗

Critical roles of the p160 transcriptional coactivators p/CIP and SRC-1 in energy balance.

Several transcriptional coactivators have been implicated in modulating the transcriptional activities of nuclear hormone receptors in vitro. Potential roles of these cofactors in important physiological processes such as energy homeostasis remain unknown. We report here that a developmental arrest in interscapular brown fat and defective adaptive thermogenesis occur in mice lacking both the p160 family transcriptional coactivators SRC-1 and p/CIP due to a failure in induction of selective PPARgamma target genes involved in adipogenesis and mitochondrial uncoupling. In the absence of p/CIP and SRC-1, mice eat more food on both regular chow and a high-fat diet because of decreased blood leptin levels. However, the p/CIP(-/-)/SRC-1(-/-) mice are lean and resistant to high-fat-diet-induced obesity. They exhibit increased basal metabolic rates and heightened levels of physical activity. Therefore, p/CIP and SRC-1 play critical roles in energy balance by controlling both energy intake and energy expenditure.

Adipogenesis↗

Turnover of the active fraction of IRS1 involves raptor-mTOR- and S6K1-dependent serine phosphorylation in cell culture models of tuberous sclerosis.

The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.

Adaptor Proteins, Signal Transducing↗

Protein phosphatase 2A antagonizes ATM and ATR in a Cdk2- and Cdc7-independent DNA damage checkpoint.

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts.

Animals↗

CaMKII structure--an elegant design.

Crystal structures of protein kinases continue to reveal new mechanisms for the regulation of catalytic activity of these enzymes. In this issue of Cell, Rosenberg et al. (2005) report the structure of the catalytic and regulatory domains of CaMKII, a protein kinase important in the cellular response to changes in intracellular calcium ion concentration. This study provides new mechanistic insights into the workings of this finely tuned molecular machine.

Animals↗

Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways.

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCF(beta-TrCP). The F-box protein beta-TrCP (beta-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to beta-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved beta-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in beta-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to beta-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of beta-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second beta-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.

Amino Acid Sequence↗

Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

Amino Acid Sequence↗

Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains.

Contact-dependent signaling between membrane-linked ligands and receptors such as the ephrins and Eph receptor tyrosine kinases controls a wide range of developmental and pathological processes. Paradoxically, many cell types coexpress both ligands and receptors, raising the question of how specific signaling readouts are achieved under these conditions. Here, we studied the signaling activities exerted by coexpressed EphA receptors and GPI-linked ephrin-A ligands in spinal motor neuron growth cones. We demonstrate that coexpressed Eph and ephrin proteins segregate laterally into distinct membrane domains from which they signal opposing effects on the growth cone: EphAs direct growth cone collapse/repulsion and ephrin-As signal motor axon growth/attraction. This subcellular arrangement of Eph-ephrin proteins enables axons to discriminate between cis- versus trans-configurations of ligand/receptor proteins, thereby allowing the utilization of both Ephs and ephrins as functional guidance receptors within the same neuronal growth cone.

Animals↗

Mitotic phosphorylation rescues Abl from F-actin-mediated inhibition.

We have previously shown that F-actin exerts a negative effect on Abl tyrosine kinase activity. This inhibition results from a direct association of F-actin with the C terminus of Abl and accounts, in part, for the loss of Abl activity in detached fibroblasts. We report here that Abl from mitotic cells or cells treated with the protein phosphatase inhibitor okadaic acid remains active when detached from the extracellular matrix. Aspartic acid substitution of Thr(566), which is phosphorylated in mitotic or okadaic acid-treated cells, is sufficient to abolish F-actin-mediated inhibition and to maintain Abl activity despite cell detachment. A recent crystal structure of the Abl N-terminal region has revealed autoinhibitory interactions among the Src homology 3 (SH3), SH2, and kinase domains. We found that deletion of the SH2 domain also abolished the negative effect of F-actin on kinase activity. Immediately following the kinase domain in Abl is a proline-rich linker (PRL) that binds to several SH3 adaptor proteins. Interestingly, binding of the Crk N-terminal SH3 domain to the PRL also disrupted F-actin-mediated inhibition of Abl kinase. These results suggest that F-actin may reinforce the autoinhibitory interactions to regulate Abl kinase and that inhibition can be relieved through phosphorylation and/or protein interactions with the Abl PRL region.

Actins↗

Tuberous sclerosis and insulin resistance. Unlikely bedfellows reveal a TORrid affair.

The TSC1-TSC2 tumor suppressor complex serves as an interface between insulin and nutrient signaling pathways and the cell growth machinery. Recent work has indicated that the TSC1-TSC2 complex plays a role in the pathobiology of a number of tumor predisposition syndromes, including tuberous sclerosis (TSC1/2), Peutz-Jeghers syndrome (LKB1), and Cowden's syndrome (PTEN), in which the TSC/Rheb/mTOR axis is inappropriately active secondary to loss of tumor suppressor function. Recent work has demonstrated that TSC deficiency imposes a negative autoregulatory loop that suppresses insulin signaling at the post-receptor level, effectively resulting in cell autonomous insulin resistance. Exploitation of this insulin signaling deficiency may hold promise among tailored clinical therapies designed to manage tuberous sclerosis.

Animals↗

Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1.

The leucine-rich-repeat receptor serine/threonine kinase, BRI1, is a cell-surface receptor for brassinosteroids (BRs), the steroid hormones of plants, yet its activation mechanism is unknown. Here, we report a unique autoregulatory mechanism of BRI1 activation. Removal of BRI1's C terminus leads to a hypersensitive receptor, indicated by suppression of dwarfism of BR-deficient and BR-perception mutants and by enhanced BR signaling as a result of elevated phosphorylation of BRI1. Several sites in the C-terminal region can be phosphorylated in vitro, and transgenic Arabidopsis expressing BRI1 mutated at these sites demonstrates an essential role of phosphorylation in BRI1 activation. BRI1 is a ligand-independent homo-oligomer, as evidenced by the transphosphorylation of BRI1 kinase in vitro, the dominant-negative effect of a kinase-inactive BRI1 in transgenic Arabidopsis, and coimmunoprecipitation experiments. Our results support a BRI1-activation model that involves inhibition of kinase activity by its C-terminal domain, which is relieved upon ligand binding to the extracellular domain.

Amino Acid Sequence↗

Kinomics: methods for deciphering the kinome.

Phosphorylation by protein kinases is the most widespread and well-studied signaling mechanism in eukaryotic cells. Phosphorylation can regulate almost every property of a protein and is involved in all fundamental cellular processes. Cataloging and understanding protein phosphorylation is no easy task: many kinases may be expressed in a cell, and one-third of all intracellular proteins may be phosphorylated, representing as many as 20,000 distinct phosphoprotein states. Defining the kinase complement of the human genome, the kinome, has provided an excellent starting point for understanding the scale of the problem. The kinome consists of 518 kinases, and every active protein kinase phosphorylates a distinct set of substrates in a regulated manner. Deciphering the complex network of phosphorylation-based signaling is necessary for a thorough and therapeutically applicable understanding of the functioning of a cell in physiological and pathological states. We review contemporary techniques for identifying physiological substrates of the protein kinases and studying phosphorylation in living cells.

Adenosine Triphosphate↗

Reconstruction of cellular signalling networks and analysis of their properties.

The study of cellular signalling over the past 20 years and the advent of high-throughput technologies are enabling the reconstruction of large-scale signalling networks. After careful reconstruction of signalling networks, their properties must be described within an integrative framework that accounts for the complexity of the cellular signalling network and that is amenable to quantitative modelling.

Animals↗