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Biomedical subjects

Michael E Greenberg

Publications and source records attributed to Michael E Greenberg.

16 recordsLinked to original sources
PubMed2003

BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis.

Glycolysis and apoptosis are considered major but independent pathways that are critical for cell survival. The activity of BAD, a pro-apoptotic BCL-2 family member, is regulated by phosphorylation in response to growth/survival factors. Here we undertook a proteomic analysis to assess whether BAD might also participate in mitochondrial physiology. In liver mitochondria, BAD resides in a functional holoenzyme complex together with protein kinase A and protein phosphatase 1 (PP1) catalytic units, Wiskott-Aldrich family member WAVE-1 as an A kinase anchoring protein, and glucokinase (hexokinase IV). BAD is required to assemble the complex in that Bad-deficient hepatocytes lack this complex, resulting in diminished mitochondria-based glucokinase activity and blunted mitochondrial respiration in response to glucose. Glucose deprivation results in dephosphorylation of BAD, and BAD-dependent cell death. Moreover, the phosphorylation status of BAD helps regulate glucokinase activity. Mice deficient for BAD or bearing a non-phosphorylatable BAD(3SA) mutant display abnormal glucose homeostasis including profound defects in glucose tolerance. This combination of proteomics, genetics and physiology indicates an unanticipated role for BAD in integrating pathways of glucose metabolism and apoptosis.

Animals↗
PubMed2003

The Drosophila forkhead transcription factor FOXO mediates the reduction in cell number associated with reduced insulin signaling.

BACKGROUND: Forkhead transcription factors belonging to the FOXO subfamily are negatively regulated by protein kinase B (PKB) in response to signaling by insulin and insulin-like growth factor in Caenorhabditis elegans and mammals. In Drosophila, the insulin-signaling pathway regulates the size of cells, organs, and the entire body in response to nutrient availability, by controlling both cell size and cell number. In this study, we present a genetic characterization of dFOXO, the only Drosophila FOXO ortholog. RESULTS: Ectopic expression of dFOXO and human FOXO3a induced organ-size reduction and cell death in a manner dependent on phosphoinositide (PI) 3-kinase and nutrient levels. Surprisingly, flies homozygous for dFOXO null alleles are viable and of normal size. They are, however, more sensitive to oxidative stress. Furthermore, dFOXO function is required for growth inhibition associated with reduced insulin signaling. Loss of dFOXO suppresses the reduction in cell number but not the cell-size reduction elicited by mutations in the insulin-signaling pathway. By microarray analysis and subsequent genetic validation, we have identified d4E-BP, which encodes a translation inhibitor, as a relevant dFOXO target gene. CONCLUSION: Our results show that dFOXO is a crucial mediator of insulin signaling in Drosophila, mediating the reduction in cell number in insulin-signaling mutants. We propose that in response to cellular stresses, such as nutrient deprivation or increased levels of reactive oxygen species, dFOXO is activated and inhibits growth through the action of target genes such as d4E-BP.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2003

Bad-deficient mice develop diffuse large B cell lymphoma.

The proapoptotic activity of the "BH3-only" molecule BAD can be differentially regulated by survival factor signaling. Bad-deficient mice lacking both BAD long and BAD short proteins proved viable, and most cell types appeared to develop normally. BAD did not exclusively account for cell death after withdrawal of survival factors, but it was an intermediate for epidermal growth factor- or insulin-like growth factor I-countered apoptosis, consistent with a "sensitizing" BH3-only molecule. Lymphocytes developed normally with no premalignant hyperplasia, but they displayed subtle abnormalities in proliferation and IgG production. Despite the minimal phenotype, Bad-deficient mice progressed, with aging, to diffuse large B cell lymphoma of germinal center origin. Exposure of Bad-null mice to sublethal gamma-irradiation resulted in an increased incidence of pre-T cell and pro-/pre-B cell lymphoblastic leukemia/lymphoma. Thus, proapoptotic BAD suppresses tumorigenesis in the lymphocyte lineage.

Animals↗
PubMed2003

Basic helix-loop-helix factors in cortical development.

Transcription factors with bHLH motifs modulate critical events in the development of the mammalian neocortex. Multipotent cortical progenitors are maintained in a proliferative state by bHLH factors from the Id and Hes families. The transition from proliferation to neurogenesis involves a coordinate increase in the activity of proneural bHLH factors (Mash1, Neurogenin1, and Neurogenin2) and a decrease in the activity of Hes and Id factors. As development proceeds, inhibition of proneural bHLH factors in cortical progenitors promotes the formation of astrocytes. Finally, the formation of oligodendrocytes is triggered by an increase in the activity of bHLH factors Olig1 and Olig2 that may be coupled with a decrease in Id activity. Thus, bHLH factors have key roles in corticogenesis, affecting the timing of differentiation and the specification of cell fate.

Animals↗
PubMed2003

Upstream stimulatory factors are mediators of Ca2+-responsive transcription in neurons.

To identify molecular mechanisms that control activity-dependent gene expression in the CNS, we have characterized the factors that mediate activity-dependent transcription of BDNF promoter III. We report the identification of a Ca(2+)-responsive E-box element, CaRE2, within BDNF promoter III that binds upstream stimulatory factors 1 and 2 (USF1/2) and show that USFs are required for the activation of CaRE2-dependent transcription from BDNF promoter III. We find that the transcriptional activity of the USFs is regulated by Ca(2+)-activated signaling pathways in neurons and that the USFs bind to the promoters of a number of neuronal activity-regulated genes in vivo. These results suggest a new function for the USFs in the regulation of activity-dependent transcription in neurons.

Animals↗
PubMed2003

The many forks in FOXO's road.

The FOXO family of transcription factors constitute an evolutionarily conserved subgroup within the larger family known as winged helix or Forkhead transcriptional regulators. Building upon work in the nematode, researchers have uncovered a role for these proteins in a diverse set of cellular responses that include glucose metabolism, stress response, cell cycle regulation, and apoptosis. At the organismal level, FOXO transcription factors are believed to function in various pathological processes ranging from cancer and diabetes to organismal aging. A number of studies have also shed light on the signaling pathways that regulate FOXO activity in response to external stimuli and have identified multiple FOXO target genes that mediate this varied set of biological responses.

Aging↗
PubMed2002

Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction in mammalian cells.

The ability to make sensitive measurements of protein-protein interaction kinetics in single neurons is critical for understanding the molecular and cellular basis of neuronal function. We have developed a reporter technology based on the differential induction of Escherichia coli TEM-1 beta-lactamase (Bla) enzymatic activity that can function as a sensor of the interaction state of two target proteins within single neurons in vivo. To modulate Bla enzymatic activity, we first split the enzyme into two separate, complementary protein fragments that we identified by using a functional screening approach based on circular permutation of the Bla enzyme. The split enzyme was then brought together by the phosphorylation-dependent association of the kinase inducible domain of the cAMP response element binding protein (CREB) and the KIX domain of the CREB binding protein. Using an intracellular substrate whose fluorescence spectrum changes after hydrolysis by Bla, we performed time-lapse ratiometric imaging measurements of Bla enzymatic induction after association of the CREB and CREB binding protein interaction domains. This approach permits direct imaging of protein-protein interactions in single cells with high signal discrimination.

Cell Line↗
PubMed2002

Analysis of phosphorylation-dependent protein-protein interactions using a bacterial two-hybrid system.

Phosphorylation-dependent protein-protein interactions provide the foundation for a multitude of intracellular signal transduction pathways. One of the goals of signal transduction research is to more precisely understand the nature of these phosphorylation-dependent interactions. Here, we describe a bacterial two-hybrid assay that allows for the rapid, efficient analysis of phosphorylation-dependent protein-protein interactions. In this system, the interacting protein domains are provided as fusion proteins in Escherichia coli. cells that contain a eukaryotic kinase. Specific phosphorylation of one of the fused protein domains results in a protein-protein interaction that can be detected as a change in the expression of a reporter gene. We also describe how this system can be modified to permit the use of cDNA libraries to identify either novel binding partners for a phosphorylated substrate or novel kinases that can induce a specific protein-protein interaction.

Bacteriophage lambda↗
PubMed2002

DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein.

The signaling pathway from phosphoinositide 3-kinase to the protein kinase Akt controls organismal life-span in invertebrates and cell survival and proliferation in mammals by inhibiting the activity of members of the FOXO family of transcription factors. We show that mammalian FOXO3a also functions at the G2 to M checkpoint in the cell cycle and triggers the repair of damaged DNA. By gene array analysis, FOXO3a was found to modulate the expression of several genes that regulate the cellular response to stress at the G2-M checkpoint. The growth arrest and DNA damage response gene Gadd45a appeared to be a direct target of FOXO3a that mediates part of FOXO3a's effects on DNA repair. These findings indicate that in mammals FOXO3a regulates the resistance of cells to stress by inducing DNA repair and thereby may also affect organismal life-span.

Animals↗
PubMed2002

CREB transcriptional activity in neurons is regulated by multiple, calcium-specific phosphorylation events.

The transcription factor CREB mediates diverse responses in the nervous system. It is not known how CREB induces specific patterns of gene expression in response to different extracellular stimuli. We find that Ca(2+) influx into neurons induces CREB phosphorylation at Ser133 and two additional sites, Ser142 and Ser143. While CREB Ser133 phosphorylation is induced by many stimuli, phosphorylation at Ser142 and Ser143 is selectively activated by Ca(2+) influx. The triple phosphorylation of CREB is required for effective Ca(2+) stimulation of CREB-dependent transcription, but the phosphorylation of Ser142 and Ser143, in addition to Ser133, disrupts the interaction of CREB with its cofactor CBP. These results suggest that Ca(2+) influx triggers a specific program of gene expression in neurons by selectively regulating CREB phosphorylation.

Amino Acid Sequence↗
PubMed2002

14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport.

14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal alpha-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.

14-3-3 Proteins↗
PubMed2002

A calcium-responsive transcription factor, CaRF, that regulates neuronal activity-dependent expression of BDNF.

Transcription of the brain-derived neurotrophic factor (BDNF) gene is regulated in a calcium- and neuron-selective manner; however, the mechanisms that underlie this selectivity are not known. We have characterized a new calcium-response element, CaRE1, that is required for activity-dependent transcription of BDNF exon III and have cloned a transcription factor, CaRF, that activates transcription from BDNF promoter III in a CaRE1-dependent manner. The transcriptional activity of CaRF is regulated in a calcium- and neuron-selective manner, suggesting that CaRF may confer selectivity upon the activity-dependent induction of BDNF exon III expression.

1-Methyl-3-isobutylxanthine↗
PubMed2002

The IGF-1/Akt pathway is neuroprotective in Huntington's disease and involves Huntingtin phosphorylation by Akt.

In the search for neuroprotective factors in Huntington's disease, we found that insulin growth factor 1 via activation of the serine/threonine kinase Akt/PKB is able to inhibit neuronal death specifically induced by mutant huntingtin containing an expanded polyglutamine stretch. The IGF-1/Akt pathway has a dual effect on huntingtin-induced toxicity, since activation of this pathway also results in a decrease in the formation of intranuclear inclusions of mutant huntingtin. We demonstrate that huntingtin is a substrate of Akt and that phosphorylation of huntingtin by Akt is crucial to mediate the neuroprotective effects of IGF-1. Finally, we show that Akt is altered in Huntington's disease patients. Taken together, these results support a potential role of the Akt pathway in Huntington's disease.

Cell Death↗
PubMed2002

Survival factor-mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis.

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, we generated mice with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. We show that BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo.

Animals↗
PubMed2001

Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors.

Protein-protein interactions and calcium entry through the N-methyl-d-aspartate (NMDA)-type glutamate receptor regulate synaptic development and plasticity in the central nervous system. The EphB receptor tyrosine kinases are localized at excitatory synapses where they cluster and associate with NMDA receptors. We identified a mechanism whereby EphBs modulate NMDA receptor function. EphrinB2 activation of EphB in primary cortical neurons potentiates NMDA receptor-dependent influx of calcium. Treatment of cells with ephrinB2 led to NMDA receptor tyrosine phosphorylation through activation of the Src family of tyrosine kinases. These ephrinB2-dependent events result in enhanced NMDA receptor-dependent gene expression. Our findings indicate that ephrinB2 stimulation of EphB modulates the functional consequences of NMDA receptor activation and suggest a mechanism whereby activity-independent and activity-dependent signals converge to regulate the development and remodeling of synaptic connections.

Animals↗