Barbara Birkaya

The signaling mucins Msb2 and Hkr1 differentially regulate the filamentation mitogen-activated protein kinase pathway and contribute to a multimodal response.

A central question in the area of signal transduction is why pathways utilize common components. In the budding yeast Saccharomyces cerevisiae, the HOG and filamentous growth (FG) MAPK pathways require overlapping components but are thought to be induced by different stimuli and specify distinct outputs. To better understand the regulation of the FG pathway, we examined FG in one of yeasts native environments, the grape-producing plant Vitis vinifera. In this setting, different aspects of FG were induced in a temporal manner coupled to the nutrient cycle, which uncovered a multimodal feature of FG pathway signaling. FG pathway activity was modulated by the HOG pathway, which led to the finding that the signaling mucins Msb2p and Hkr1p, which operate at the head of the HOG pathway, differentially regulate the FG pathway. The two mucins exhibited different expression and secretion patterns, and their overproduction induced nonoverlapping sets of target genes. Moreover, Msb2p had a function in cell polarization through the adaptor protein Sho1p that Hkr1p did not. Differential MAPK activation by signaling mucins brings to light a new point of discrimination between MAPK pathways.

Shedding of the Mucin-Like Flocculin Flo11p Reveals a New Aspect of Fungal Adhesion Regulation

Cell adhesion is a key feature in the regulation of many biological processes. In the budding yeast Saccharomyces cerevisiae, Flo11p is the major adhesion molecule that controls filamentous growth [1-3] and the expansion of interconnected cells in mats or biofilms [4]. We show here that Flo11p is shed from cells. Flo11p shedding attenuated adherence and contributed to the overall balance in adherence properties that was optimal for filamentous growth and mat formation. Shed Flo11p comprised an essential component of a fluid layer surrounding yeast mats that may be functionally analogous to the mucus secretions of higher eukaryotes. Genome-wide secretion profiling of Flo11p identified new regulatory proteins, including the furin protease Kex2p, which was required for cleavage and maturation of the Flo11p protein. Secreted mucin-like proteins may play unexpected roles in the adherence properties and virulence of microbial pathogens.

Multiple signals converge on a differentiation MAPK pathway.

An important emerging question in the area of signal transduction is how information from different pathways becomes integrated into a highly coordinated response. In budding yeast, multiple pathways regulate filamentous growth, a complex differentiation response that occurs under specific environmental conditions. To identify new aspects of filamentous growth regulation, we used a novel screening approach (called secretion profiling) that measures release of the extracellular domain of Msb2p, the signaling mucin which functions at the head of the filamentous growth (FG) MAPK pathway. Secretion profiling of complementary genomic collections showed that many of the pathways that regulate filamentous growth (RAS, RIM101, OPI1, and RTG) were also required for FG pathway activation. This regulation sensitized the FG pathway to multiple stimuli and synchronized it to the global signaling network. Several of the regulators were required for MSB2 expression, which identifies the MSB2 promoter as a target “hub” where multiple signals converge. Accessibility to the MSB2 promoter was further regulated by the histone deacetylase (HDAC) Rpd3p(L), which positively regulated FG pathway activity and filamentous growth. Our findings provide the first glimpse of a global regulatory hierarchy among the pathways that control filamentous growth. Systems-level integration of signaling circuitry is likely to coordinate other regulatory networks that control complex behaviors.

Schizophrenia: A neurodevelopmental disorder — Integrative genomic hypothesis and therapeutic implications from a transgenic mouse model

Schizophrenia is a neurodevelopmental disorder featuring complex aberrations in the structure, wiring, and chemistry of multiple neuronal systems. The abnormal developmental trajectory of the brain appears to be established during gestation, long before clinical symptoms of the disease appear in early adult life. Many genes are associated with schizophrenia, however, altered expression of no one gene has been shown to be present in a majority of schizophrenia patients. How does altered expression of such a variety of genes lead to the complex set of abnormalities observed in the schizophrenic brain? We hypothesize that the protein products of these genes converge on common neurodevelopmental pathways that affect the development of multiple neural circuits and neurotransmitter systems. One such neurodevelopmental pathway is Integrative Nuclear FGFR1 Signaling (INFS). INFS integrates diverse neurogenic signals that direct the postmitotic development of embryonic stem cells, neural progenitors and immature neurons, by direct gene reprogramming. Additionally, FGFR1 and its partner proteins link multiple upstream pathways in which schizophrenia-linked genes are known to function and interact directly with those genes. A th-fgfr1(tk-) transgenic mouse with impaired FGF receptor signaling establishes a number of important characteristics that mimic human schizophrenia - a neurodevelopmental origin, anatomical abnormalities at birth, a delayed onset of behavioral symptoms, deficits across multiple domains of the disorder and symptom improvement with typical and atypical antipsychotics, 5-HT antagonists, and nicotinic receptor agonists. Our research suggests that altered FGF receptor signaling plays a central role in the developmental abnormalities underlying schizophrenia and that nicotinic agonists are an effective class of compounds for the treatment of schizophrenia.

Role of the Cell Wall Integrity and Filamentous Growth Mitogen-Activated Protein Kinase Pathways in Cell Wall Remodeling during Filamentous Growth

ABSTRACT Many fungal species including pathogens exhibit filamentous growth (FG) as a means of foraging for nutrients. Genetic screens were performed to identify genes required for FG in the budding yeast Saccharomyces cerevisiae. Genes encoding proteins with established functions in transcriptional activation (MCM1, MATα2, PHD1, MSN2, SIR4, and HMS2), cell wall integrity (MPT5, WSC2, and MID2), and cell polarity (BUD5) were identified as potential regulators of FG. The transcription factors MCM1 and MATα2 induced invasive growth by promoting diploid-specific bipolar budding in haploid cells. Components of the cell wall integrity pathway including the cell surface proteins Slg1p/Wsc1p, Wsc2p, Mid2p, and the mitogen-activated protein kinase (MAPK) Slt2p/Mpk1p contributed to multiple aspects of the FG response including cell elongation, cell-cell adherence, and agar invasion. Mid2p and Wsc2p stimulated the FG MAPK pathway through the signaling mucin Msb2p and components of the MAPK cascade. The FG pathway contributed to cell wall integrity in parallel with the cell wall integrity pathway and in opposition with the high osmolarity glycerol response pathway. Mass spectrometry approaches identified components of the filamentous cell wall including the mucin-like proteins Msb2p, Flo11p, and subtelomeric (silenced) mucin Flo10p. Secretion of Msb2p, which occurs as part of the maturation of the protein, was inhibited by the ß-1,3-glucan layer of the cell wall, which highlights a new regulatory aspect to cell wall remodeling in this organism. Disruption of ß-1,3-glucan linkages induced mucin shedding and resulted in defects in cell-cell adhesion and invasion of cells into the agar matrix.

A novel nuclear FGF Receptor‐1 partnership with retinoid and Nur receptors during developmental gene programming of embryonic stem cells

FGF Receptor‐1 (FGFR1), a membrane‐targeted protein, is also involved in independent direct nuclear signaling. We show that nuclear accumulation of FGFR1 is a common response to retinoic acid (RA) in pluripotent embryonic stem cells (ESC) and neural progenitors and is both necessary and sufficient for neuronal‐like differentiation and accompanying neuritic outgrowth. Dominant negative nuclear FGFR1, which lacks the tyrosine kinase domain, prevents RA‐induced differentiation while full‐length nuclear FGFR1 elicits differentiation in the absence of RA. Immunoprecipitation and GST assays demonstrate that FGFR1 interacts with RXR, RAR and their Nur77 and Nurr1 partners. Conditions that promote these interactions decrease the mobility of nuclear FGFR1 and RXR in live cells. RXR and FGFR1 co‐associate with 5′‐Fluorouridine‐labeled transcription sites and with RA Responsive Elements (RARE). RA activation of neuronal (tyrosine hydroxylase) and neurogenic (fgf‐2 and fgfr1) genes is accompanied by increased FGFR1, Nur, and histone H3.3 binding to their regulatory sequences. Reporter‐gene assays show synergistic activations of RARE, NBRE, and NurRE by FGFR1, RAR/RXR, and Nurs. As shown for mESC differentiation, FGFR1 mediates gene activation by RA and augments transcription in the absence of RA. Cooperation of FGFR1 with RXR/RAR and Nurs at targeted genomic sequences offers a new mechanism in developmental gene regulation. J. Cell. Biochem. 113: 2920–2936, 2012.

NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)

Nerve growth factor (NGF) is the founding member of the polypeptide neurotrophin family responsible for neuronal differentiation. To determine whether the effects of NGF rely upon novel Integrative Nuclear FGF Receptor-1 (FGFR1) Signaling (INFS) we utilized the PC12 clonal cell line, a long-standing benchmark model of sympathetic neuronal differentiation. We demonstrate that NGF increases expression of the fgfr1 gene and promotes trafficking of FGFR1 protein from cytoplasm to nucleus by inhibiting FGFR1 nuclear export. Nuclear-targeted dominant negative FGFR1 antagonizes NGF-induced neurite outgrowth, doublecortin (dcx) expression and activation of the tyrosine hydroxylase (th) gene promoter, while active constitutive nuclear FGFR1 mimics the effects of NGF. NGF increases the expression of dcx, th, βIII tubulin, nurr1 and nur77, fgfr1and fibroblast growth factor-2 (fgf-2) genes, while enhancing binding of FGFR1and Nur77/Nurr1 to those genes. NGF activates transcription from isolated NurRE and NBRE motifs. Nuclear FGFR1 transduces NGF activation of the Nur dimer and raises basal activity of the Nur monomer. Cooperation of nuclear FGFR1 with Nur77/Nurr1 in NGF signaling expands the integrative functions of INFS to include NGF, the first discovered pluripotent neurotrophic factor.

α7 Nicotinic receptor agonist reactivates neurogenesis in adult brain

Reactivation of neurogenesis by endogenous Neural Stem/Progenitor Cells (NS/PC) in the adult brain or spinal cord holds the key for treatment of CNS injuries as well as neurodegenerative disorders, which are major healthcare issues for the worlds aging population. Recent studies show that targeting the α7 nicotinic acetylcholine receptors (α7nAChR) with a specific TC-7020 agonist inhibits proliferation and stimulates neuronal differentiation of NS/PC in subventricular zone (SVZ) in the adult mouse brain. TC-7020-induced neuronogenesis is observed in different brain regions, including: (1) βIII Tubulin-expressing cortical neurons, (2) calretinin expressing hippocampal neurons and (3) cells in substantia nigra (SN) expressing predopaminergic Nurr1+phenotype. Reactivation of developmental integrative nuclear FGFR1 signaling (INFS), via gene transfection reinstates neurogenesis in the adult brain by promoting neuronal differentiation of brain NS/PC. TC-7020 neuronogenic effect is associated with a robust accumulation of endogenous FGFR1 in the nuclei of differentiating cells. Furthermore, direct in vitro stimulation of neural stem/progenitor cells with α7nAChR agonist activates INFS and neuronal-like differentiation and activation of neuronal genes. The α7nAChR upregulation of early neuronal βIII-Tubulin gene involves neurogenic FGFR1-Nur signaling and direct FGFR1 interaction with the gene promoter. The reactivation of developmental INFS and neurogenesis in adult brain by the α7nAChR agonist may offer new strategy to treat brain injuries, neurodegenerative and neurodevelopmental diseases.

Activation of Developmental Nuclear Fibroblast Growth Factor Receptor 1 Signaling and Neurogenesis in Adult Brain by α7 Nicotinic Receptor Agonist

Reactivation of endogenous neurogenesis in the adult brain or spinal cord holds the key for treatment of central nervous system injuries and neurodegenerative disorders, which are major health care issues for the worlds aging population. We have previously shown that activation of developmental integrative nuclear fibroblast growth factor receptor 1 (FGFR1) signaling (INFS), via gene transfection, reactivates neurogenesis in the adult brain by promoting neuronal differentiation of brain neural stem/progenitor cells (NS/PCs). In the present study, we report that targeting the α7 nicotinic acetylcholine receptors (α7nAChRs) with a specific TC‐7020 agonist led to a robust accumulation of endogenous FGFR1 in the cell nucleus. Nuclear FGFR1 accumulation was accompanied by an inhibition of proliferation of NS/PCs in the subventricular zone (SVZ) and by the generation of new neurons. Neuronal differentiation was observed in different regions of the adult mouse brain, including (a) βIII‐Tubulin‐expressing cortical neurons, (b) calretinin‐expressing hippocampal neurons, and (c) cells in substantia nigra expressing the predopaminergic Nurr1+ phenotype. Furthermore, we showed that in vitro stimulation of neural stem/progenitor cells with α7nAChR agonist directly activated INFS and neuronal‐like differentiation. TC‐7020 stimulation of the βIII‐Tubulin gene was accompanied by increased binding of FGFR1, CREB binding protein, and RNA polymerase II to a Nur77 targeted promoter region. TC‐7020 augmented Nur77‐dependent activation of nerve growth factor inducible‐B protein responsive element, indicating that α7nAChR upregulation of βIII‐Tubulin involves neurogenic FGFR1‐Nur signaling. The reactivation of INFS and neurogenesis in adult brain by the α7nAChR agonist may offer a new strategy to treat brain injuries, neurodegenerative diseases, and neurodevelopmental diseases.

Global Developmental Gene Programing Involves a Nuclear Form of Fibroblast Growth Factor Receptor-1 (FGFR1)

Genetic studies have placed the Fgfr1 gene at the top of major ontogenic pathways that enable gastrulation, tissue development and organogenesis. Using genome-wide sequencing and loss and gain of function experiments the present investigation reveals a mechanism that underlies global and direct gene regulation by the nuclear form of FGFR1, ensuring that pluripotent Embryonic Stem Cells differentiate into Neuronal Cells in response to Retinoic Acid. Nuclear FGFR1, both alone and with its partner nuclear receptors RXR and Nur77, targets thousands of active genes and controls the expression of pluripotency, homeobox, neuronal and mesodermal genes. Nuclear FGFR1 targets genes in developmental pathways represented by Wnt/β-catenin, CREB, BMP, the cell cycle and cancer-related TP53 pathway, neuroectodermal and mesodermal programing networks, axonal growth and synaptic plasticity pathways. Nuclear FGFR1 targets the consensus sequences of transcription factors known to engage CREB-binding protein, a common coregulator of transcription and established binding partner of nuclear FGFR1. This investigation reveals the role of nuclear FGFR1 as a global genomic programmer of cell, neural and muscle development.