Actin binding to WH2 domains regulates nuclear import of the multifunctional actin regulator JMY

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 23 (2012): 853-863, doi:10.1091/mbc.E11-12-0992.Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. In response to DNA damage, JMY accumulates in the nucleus and promotes p53-dependent apoptosis. JMY's actin-regulatory activity relies on a cluster of three actin-binding Wiskott–Aldrich syndrome protein homology 2 (WH2) domains that nucleate filaments directly and also promote nucleation activity of the Arp2/3 complex. In addition to these activities, we find that the WH2 cluster overlaps an atypical, bipartite nuclear localization sequence (NLS) and controls JMY's subcellular localization. Actin monomers bound to the WH2 domains block binding of importins to the NLS and prevent nuclear import of JMY. Mutations that impair actin binding, or cellular perturbations that induce actin filament assembly and decrease the concentration of monomeric actin in the cytoplasm, cause JMY to accumulate in the nucleus. DNA damage induces both cytoplasmic actin polymerization and nuclear import of JMY, and we find that damage-induced nuclear localization of JMY requires both the WH2/NLS region and importin β. On the basis of our results, we propose that actin assembly regulates nuclear import of JMY in response to DNA damage.This work was supported by grants from the National Institutes of Health, an American Heart Association Predoctoral Fellowship (J.B.Z.), the Robert Day Allen Fellowship Fund (J.B.Z.), and a National Science Foundation Predoctoral Fellowship (B.B.)

Hypoxia-driven cell motility reflects the interplay between JMY and HIF-1α.

Junction-mediating and regulatory protein (JMY) is a novel p53 cofactor that regulates p53 activity during stress. JMY interacts with p300/CBP, which are ubiquitous transcriptional co-activators that interact with a variety of sequence-specific transcription factors, including hypoxia-inducible factor-1α (HIF-1α). In addition, JMY is an actin-nucleating protein, which, through its WH2 domains, stimulates cell motility. In this study, we show that JMY is upregulated during hypoxia in a HIF-1α-dependent manner. The JMY gene contains HIF-responsive elements in its promoter region and HIF-1α is recruited to its promoter during hypoxia. HIF-1α drives transcription of JMY, which accounts for its induction under hypoxia. Moreover, the enhanced cell motility and invasion that occurs during hypoxia requires JMY, as depleting JMY under hypoxic conditions causes decreased cell motility. Our results establish the interplay between JMY and HIF-1α as a new mechanism that controls cell motility under hypoxic stress

RhoD regulates cytoskeletal dynamics via the actin nucleation-promoting factor WASp homologue associated with actin Golgi membranes and microtubules

The Rho GTPases have mainly been studied in association with their roles in the regulation of actin filament organization. These studies have shown that the Rho GTPases are essential for basic cellular processes, such as cell migration, contraction, and division. In this paper, we report that RhoD has a role in the organization of actin dynamics that is distinct from the roles of the better-studied Rho members Cdc42, RhoA, and Rac1. We found that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM), as well as the related filamin A–binding protein FILIP1. Of these two RhoD-binding proteins, WHAMM was found to bind to the Arp2/3 complex, while FILIP1 bound filamin A. WHAMM was found to act downstream of RhoD in regulating cytoskeletal dynamics. In addition, cells treated with small interfering RNAs for RhoD and WHAMM showed increased cell attachment and decreased cell migration. These major effects on cytoskeletal dynamics indicate that RhoD and its effectors control vital cytoskeleton-driven cellular processes. In agreement with this notion, our data suggest that RhoD coordinates Arp2/3-dependent and FLNa-dependent mechanisms to control the actin filament system, cell adhesion, and cell migration

Tumor Suppressor RASSF1A Promoter: p53 Binding and Methylation

Oncogenes and tumor suppressors work in concert to regulate cell growth or death, which is a pair of antagonist factors for regulation of tumorigenesis. Here we show promoter characteristic of tumor suppressor RASSF1A, which revealed a p53 binding site in the distal and a GC-rich region in the proximal promoter region of RASSF1A, in despite of TATA box-less. The GC-rich region, which is ∼300 bp upstream from the RASSF1A ATG, showed the strongest promoter activity in an assay of RASSF1A-driving GFP expression. Methylation analysis of the CpG island showed that 78.57% of the GC sties were methylated in testis tumor samples compared with methylation-less in normal testis. Hypermethylation of the GC-rich region is associated with RASSF1A silencing in human testis tumors. In addition, electrophoretic mobility shift assay indicated that p53 protein bound to the RASSF1A promoter. Further chromatin immunoprecipitation confirmed p53 binding to the RASSF1A. Moreover, p53 binding to the promoter down-regulated RASSF1A expression. These results suggest that p53 protein specifically binds to the RASSF1A promoter and inhibits its expression. Our results provide new insight into the mechanism of action of tumor suppressors and may be a starting point for development of new approaches to cancer treatment

Partitioning the Proteome: Phase Separation for Targeted Analysis of Membrane Proteins in Human Post-Mortem Brain

Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain

A Novel Pathway of TEF Regulation Mediated by MicroRNA-125b Contributes to the Control of Actin Distribution and Cell Shape in Fibroblasts

BACKGROUND: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF. PRINCIPAL FINDINGS: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53. CONCLUSIONS/SIGNIFICANCE: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts

Arp2/3 Complex Regulates Asymmetric Division and Cytokinesis in Mouse Oocytes

Mammalian oocyte meiotic maturation involves oocyte polarization and a unique asymmetric division, but until now, the underlying mechanisms have been poorly understood. Arp2/3 complex has been shown to regulate actin nucleation and is widely involved in a diverse range of processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Whether Arp2/3 complex participates in oocyte polarization and asymmetric division is unknown. The present study investigated the expression and functions of Arp2/3 complex during mouse oocyte meiotic maturation. Immunofluorescent staining showed that the Arp2/3 complex was restricted to the cortex, with a thickened cap above the meiotic apparatus, and that this localization pattern was depended on actin. Disruption of Arp2/3 complex by a newly-found specific inhibitor CK666, as well as by Arpc2 and Arpc3 RNAi, resulted in a range of effects. These included the failure of asymmetric division, spindle migration, and the formation and completion of oocyte cytokinesis. The formation of the actin cap and cortical granule-free domain (CGFD) was also disrupted, which further confirmed the disruption of spindle migration. Our data suggest that the Arp2/3 complex probably regulates oocyte polarization through its effect on spindle migration, asymmetric division and cytokinesis during mouse oocyte meiotic maturation

Evolution of the eukaryotic ARP2/3 activators of the WASP family: WASP, WAVE, WASH, and WHAMM, and the proposed new family members WAWH and WAML

<p>Abstract</p> <p>Background</p> <p>WASP family proteins stimulate the actin-nucleating activity of the ARP2/3 complex. They include members of the well-known WASP and WAVE/Scar proteins, and the recently identified WASH and WHAMM proteins. WASP family proteins contain family specific N-terminal domains followed by proline-rich regions and C-terminal VCA domains that harbour the ARP2/3-activating regions.</p> <p>Results</p> <p>To reveal the evolution of ARP2/3 activation by WASP family proteins we performed a "holistic" analysis by manually assembling and annotating all homologs in most of the eukaryotic genomes available. We have identified two new families: the WAML proteins (WASP and MIM like), which combine the membrane-deforming and actin bundling functions of the IMD domains with the ARP2/3-activating VCA regions, and the WAWH protein (WASP without WH1 domain) that have been identified in amoebae, Apusozoa, and the anole lizard. Surprisingly, with one exception we did not identify any alternative splice forms for WASP family proteins, which is in strong contrast to other actin-binding proteins like Ena/VASP, MIM, or NHS proteins that share domains with WASP proteins.</p> <p>Conclusions</p> <p>Our analysis showed that the last common ancestor of the eukaryotes must have contained a homolog of WASP, WAVE, and WASH. Specific families have subsequently been lost in many taxa like the WASPs in plants, algae, Stramenopiles, and Euglenozoa, and the WASH proteins in fungi. The WHAMM proteins are metazoa specific and have most probably been invented by the Eumetazoa. The diversity of WASP family proteins has strongly been increased by many species- and taxon-specific gene duplications and multimerisations. All data is freely accessible via <url>http://www.cymobase.org</url>.</p

Neurons and neuronal activity control gene expression in astrocytes to regulate their development and metabolism

The influence that neurons exert on astrocytic function is poorly understood. To investigate this, we first developed a system combining cortical neurons and astrocytes from closely related species, followed by RNA-seq and in silico species separation. This approach uncovers a wide programme of neuron-induced astrocytic gene expression, involving Notch signalling, which drives and maintains astrocytic maturity and neurotransmitter uptake function, is conserved in human development, and is disrupted by neurodegeneration. Separately, hundreds of astrocytic genes are acutely regulated by synaptic activity via mechanisms involving cAMP/PKA-dependent CREB activation. This includes the coordinated activity-dependent upregulation of major astrocytic components of the astrocyte-neuron lactate shuttle, leading to a CREB-dependent increase in astrocytic glucose metabolism and elevated lactate export. Moreover, the groups of astrocytic genes induced by neurons or neuronal activity both show age-dependent decline in humans. Thus, neurons and neuronal activity regulate the astrocytic transcriptome with the potential to shape astrocyte-neuron metabolic cooperation

Kif13b Regulates PNS and CNS Myelination Through the Dlg1 Scaffold

Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS) and central nervous system (CNS) myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK)-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1), a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K)/v-AKT murine thymoma viral oncogene homolog (AKT) pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS