scholarly journals Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

2012 ◽  
Vol 11 (10) ◽  
pp. 1239-1248 ◽  
Author(s):  
Susan Won ◽  
Alexander V. Michkov ◽  
Svetlana Krystofova ◽  
Amruta V. Garud ◽  
Katherine A. Borkovich
Keyword(s):  
Neurospora Crassa ◽  
G Proteins ◽  
Negative Control ◽  
Physical Interaction ◽  
Heterotrimeric G Proteins ◽  
Content Type ◽  
Binding Partners ◽  
Genetic Epistasis ◽  
Physical Interactions ◽  
Submerged Conidiation

ABSTRACTHeterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungusNeurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gβγ dimer is currently lacking. In this study, the threeN. crassaGα subunits are analyzed for genetic epistasis withgnb-1and for physical interaction with the Gβγ dimer. We created double mutants lacking one Gα gene andgnb-1and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δgnb-1background. Genetic analysis revealed thatgna-3is epistatic tognb-1with regard to negative control of submerged conidiation.gnb-1is epistatic togna-2andgna-3for aerial hyphal height, whilegnb-1appears to act upstream ofgna-1andgna-2during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1mutants, and thegna-3Q208Lallele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from thegng-1-FLAG strain demonstrated that the three Gα proteins interact with the Gβγ dimer. The finding that the Gβγ dimer interacts with all three Gα proteins is supported by epistasis betweengnb-1andgna-1,gna-2, andgna-3for at least one function.

scholarly journals Heterotrimeric G-Proteins Are Indispensable for FLT3-ITD autophosphorylation and Oncogenic Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2712-2712
Author(s):  
Maike Rehage ◽  
Susanne Wingert ◽  
Nadine Haetscher ◽  
Sabrina Bothur ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
G Protein ◽  
G Proteins ◽  
B Cell Lymphoma ◽  
Physical Interaction ◽  
Heterotrimeric G Proteins ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Oncogenic Function

Abstract Heterotrimeric G-proteins transmit signals of G-protein coupled receptors and regulate many basic cellular functions. However, their role in normal and malignant hematopoietic stem cells remains obscure. Activating mutations in the heterotrimeric G-protein Gaq were found in various cancers and its expression is enhanced in diffuse large B-cell lymphoma and T-ALL. Our previous data suggested the involvement of heterotrimeric G-proteins in Flt3-ITD-mediated leukemic transformation. FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is a frequent oncoprotein in acute myeloid leukemia causing constitutive active STAT5 signaling. Here, we investigated a novel role of Gaq in Flt3-ITD-induced leukemic transformation. We could show that Gaq is indispensable for aberrant FLT3-ITD activation and oncogenic function as Gaq activity is necessary to maintain the autophosphorylation of FLT3-ITD. Gaq abrogation resulted in diminished cell proliferation and colony formation as well as delayed leukemogenesis in vivo of Flt3-ITD leukemic cells. Importantly, the growth inhibition could be rescued by addition of IL3 and did not occur in the presence of FLT3 ligand-activated FLT3 wildtype receptor, demonstrating the specificity of Gaq requirement for FLT3-ITD oncogenic signaling. Interestingly, co-immunoprecipitations revealed a direct physical interaction between FLT3-ITD and Gaq which did not require phosphorylation of the receptor tyrosine kinase. Hence, FLT3-ITD hyperphosphorylation seems to be rather a consequence of the interaction than a prerequisite. Flt3-ITD-induced transformation of murine hematopoietic stem/progenitor cells (HSPCs) strictly depended on the presence of Gaq, and the ablation of Gaq/11 in transplanted Flt3-ITD-transduced HSPCs from conditional Gaq/11 double knock-out mice delayed leukemic burden. These findings of an unexpected, yet critical, role of Gaq place the molecule as an important target for antileukemic strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals The G alpha i homologue gna-1 controls multiple differentiation pathways in Neurospora crassa.

1996 ◽  
Vol 7 (8) ◽  
pp. 1283-1297 ◽  
Author(s):  
F D Ivey ◽  
P N Hodge ◽  
G E Turner ◽  
K A Borkovich
Keyword(s):  
Neurospora Crassa ◽  
G Proteins ◽  
Solid Medium ◽  
Sexual Cycle ◽  
Heterotrimeric G Proteins ◽  
Wild Type ◽  
Multiple Phenotypes ◽  
Multiple Differentiation ◽  
Differentiation Pathways ◽  
Type Strains

Heterotrimeric G proteins are components of principal signaling pathways in eukaryotes. In higher organisms, alpha subunits of G proteins have been divided into four families, Gi, Gs, Gq, and G12. We previously identified a G alpha i homologue gna-1 in the filamentous fungus Neurospora crassa. Now we report that deletion of gna-1 leads to multiple phenotypes during the vegetative and sexual cycles in N. crassa. On solid medium, delta gna-1 strains have a slower rate of hyphal apical extension than wild type, a rate that is more pronounced under hyperosmotic conditions or in the presence of a cellophane overlay. delta gna-1 mutants accumulate less mass than wild-type strains, and their mass accumulation is not affected in the same way by exposure to light. delta gna-1 strains are defective in macroconidiation, possessing aerial hyphae that are shorter, contain abnormal swellings, and differentiate adherent macroconidia. During the sexual cycle, delta gna-1 strains are fertile as males. However, the mutants are female-sterile, producing small, aberrant female reproductive structures. After fertilization, delta gna-1 female structures do not enlarge and develop normally, and no sexual spores are produced. Thus, mutation of gna-1 results in sex-specific loss of fertility.

scholarly journals Contribution of Physical Interactions to Signaling Specificity between a Diguanylate Cyclase and Its Effector

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Kurt M. Dahlstrom ◽  
Krista M. Giglio ◽  
Alan J. Collins ◽  
Holger Sondermann ◽  
George A. O’Toole
Keyword(s):  
Protein Interactions ◽  
Second Messenger ◽  
Target Protein ◽  
Physical Interaction ◽  
Content Type ◽  
Signaling Specificity ◽  
Cellular Processes ◽  
Diguanylate Cyclases ◽  
Cellular Behaviors ◽  
Physical Interactions

ABSTRACT Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such “local signaling” is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network. IMPORTANCE An important question in microbiology is how bacteria make decisions using a signaling network made up of proteins that make, break, and bind the second messenger c-di-GMP, which is responsible for controlling many cellular behaviors. Previous work has shown that a given DGC enzyme will signal for specific cellular outputs, despite making the same diffusible molecule as its sibling DGCs in the unpartitioned space of the bacterial cell. Understanding how one DGC differentiates its output from the dozens of other such enzymes in the cell is synonymous with understanding a large component of the bacterial decision-making machinery. We present evidence for a helix on a DGC used to physically associate with its target protein, which is necessary to achieve maximal signaling.

scholarly journals Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis

2012 ◽  
Vol 194 (18) ◽  
pp. 4941-4950 ◽  
Author(s):  
Melissa de Francesco ◽  
Jake Z. Jacobs ◽  
Filipa Nunes ◽  
Mónica Serrano ◽  
Peter T. McKenney ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interactions ◽  
Mutational Analysis ◽  
Physical Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Protein Protein Interactions ◽  
Content Type ◽  
Morphogenetic Protein ◽  
Physical Interactions

ABSTRACTEndospore formation byBacillus subtilisis a complex and dynamic process. One of the major challenges of sporulation is the assembly of a protective, multilayered, proteinaceous spore coat, composed of at least 70 different proteins. Spore coat formation can be divided into two distinct stages. The first is the recruitment of proteins to the spore surface, dependent on the morphogenetic protein SpoIVA. The second step, known as encasement, involves the migration of the coat proteins around the circumference of the spore in successive waves, a process dependent on the morphogenetic protein SpoVID and the transcriptional regulation of individual coat genes. We provide genetic and biochemical evidence supporting the hypothesis that SpoVID promotes encasement of the spore by establishing direct protein-protein interactions with other coat morphogenetic proteins. It was previously demonstrated that SpoVID directly interacts with SpoIVA and the inner coat morphogenetic protein, SafA. Here, we show by yeast two-hybrid and pulldown assays that SpoVID also interacts directly with the outer coat morphogenetic protein, CotE. Furthermore, by mutational analysis, we identified a specific residue in the N-terminal domain of SpoVID that is essential for the interaction with CotE but dispensable for the interaction with SafA. We propose an updated model of coat assembly and spore encasement that incorporates several physical interactions between the principal coat morphogenetic proteins.

scholarly journals G-Alpha Subunit Abundance and Activity Differentially Regulate β-Catenin Signaling

2018 ◽  
Vol 39 (5) ◽  
Author(s):  
Arshiya Banu ◽  
Karen J. Liu ◽  
Alistair J. Lax ◽  
Agamemnon E. Grigoriadis
Keyword(s):  
G Proteins ◽  
Pasteurella Multocida ◽  
Alpha Subunit ◽  
Dependent Manner ◽  
Heterotrimeric G Proteins ◽  
Wild Type ◽  
Content Type ◽  
Alpha Subunits ◽  
Licl Treatment ◽  
Signal Transduction Proteins

ABSTRACT Heterotrimeric G proteins are signal transduction proteins involved in regulating numerous signaling events. In particular, previous studies have demonstrated a role for G-proteins in regulating β-catenin signaling. However, the link between G-proteins and β-catenin signaling is controversial and appears to depend on G-protein specificity. We describe a detailed analysis of a link between specific G-alpha subunits and β-catenin using G-alpha subunit genetic knockout and knockdown approaches. The Pasteurella multocida toxin was utilized as a unique tool to activate G-proteins, with LiCl treatment serving as a β-catenin signaling agonist. The results show that Pasteurella multocida toxin (PMT) significantly enhanced LiCl-induced active β-catenin levels in HEK293T cells and mouse embryo fibroblasts. Evaluation of the effect of specific G-alpha proteins on the regulation of β-catenin showed that Gq/11 and G12/13 knockout cells had significantly higher levels of active and total β-catenin than wild-type cells. The stimulation of active β-catenin by PMT and LiCl was lost upon both constitutive and transient knockdown of G12 and G13 but not Gq. Based on our results, we conclude that endogenous G-alpha proteins are negative regulators of active β-catenin; however, PMT-activated G-alpha subunits positively regulate LiCl-induced β-catenin expression in a G12/13-dependent manner. Hence, G-alpha subunit regulation of β-catenin is context dependent.

scholarly journals Noncanonical G-Protein-Dependent Modulation of Osteoclast Differentiation and Bone Resorption Mediated by Pasteurella multocida Toxin

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Julia Strack ◽  
Hannah Heni ◽  
Ralf Gilsbach ◽  
Lutz Hein ◽  
Klaus Aktories ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Bone Resorption ◽  
G Protein ◽  
G Proteins ◽  
Pasteurella Multocida ◽  
Osteoclast Differentiation ◽  
Mitogen Activated Protein Kinase ◽  
Atrophic Rhinitis ◽  
Heterotrimeric G Proteins ◽  
Content Type

ABSTRACT Pasteurella multocida toxin (PMT) induces atrophic rhinitis in animals, which is characterized by a degradation of nasal turbinate bones, indicating an effect of the toxin on bone cells such as osteoblasts and osteoclasts. The underlying molecular mechanism of PMT was defined as a persistent activation of heterotrimeric G proteins by deamidation of a specific glutamine residue. Here, we show that PMT acts directly on osteoclast precursor cells such as bone marrow-derived CD14+ monocytes and RAW246.7 cells to induce osteoclastogenesis as measured by expression of osteoclast-specific markers such as tartrate-resistant acid phosphatase and bone resorption activity. Treatment performed solely with PMT stimulates osteoclast differentiation, showing a receptor activator of nuclear factor-κB ligand (RANKL)-independent action of the toxin. The underlying signal transduction pathway was defined as activation of the heterotrimeric G proteins Gαq/11 leading to the transactivation of Ras and the mitogen-activated protein kinase pathway. Gαq/11 transactivates Ras via its effector phospholipase Cβ-protein kinase C (PKC) involving proline-rich tyrosine kinase 2 (Pyk2). PMT-induced activation of the mitogen-activated protein kinase pathway results in stimulation of the osteoclastogenic transcription factors AP-1, NF-κB, and NFATc1. In addition, Ca2+-dependent calcineurin activation of NFAT is crucial for PMT-induced osteoclastogenesis. The data not only elucidate a rationale for PMT-dependent bone loss during atrophic rhinitis but also highlight a noncanonical, G-protein-dependent pathway toward bone resorption that is distinct from the RANKL-RANK pathway but mimics it. We define heterotrimeric G proteins as as-yet-underestimated entities/players in the maturation of osteoclasts which might be of pharmacological relevance. IMPORTANCE Pasteurella multocida toxin (PMT) induces degradation of nasal turbinate bones, leading to the syndrome of atrophic rhinitis. Recently, the molecular mechanism and substrate specificity of PMT were identified. The toxin activates heterotrimeric G proteins by a covalent modification. However, the mechanism by which PMT induces bone degradation is poorly understood. Our report demonstrates a direct effect of PMT on osteoclast precursor cells, leading to maturation of bone-degrading osteoclasts. Interestingly, PMT stimulates osteoclastogenesis independently of the cytokine RANKL, which is a key factor in induction of osteoclast differentiation. This implicates a noncanonical osteoclastogenic signaling pathway induced by PMT. The elucidated Gαq/11-dependent osteoclastogenic signal transduction pathway ends in osteoclastogenic NFAT signaling. The noncanonical, heterotrimeric G protein-dependent osteoclast differentiation process may be of pharmacological relevance, as members of this pathway are highly druggable. In particular, modulation of G protein-coupled receptor activity in osteoclast progenitors by small molecules might be of specific interest.

scholarly journals Olfactory desensitization requires membrane targeting of receptor kinase mediated by beta gamma-subunits of heterotrimeric G proteins.

1994 ◽  
Vol 269 (1) ◽  
pp. 37-40
Author(s):  
I. Boekhoff ◽  
J. Inglese ◽  
S. Schleicher ◽  
W.J. Koch ◽  
R.J. Lefkowitz ◽  
...  
Keyword(s):  
G Proteins ◽  
Membrane Targeting ◽  
Heterotrimeric G Proteins ◽  
Receptor Kinase ◽  
Gamma Subunits

scholarly journals Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 222
Author(s):  
Agnieszka Polit ◽  
Paweł Mystek ◽  
Ewa Błasiak
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Lipid Membranes ◽  
Signaling Proteins ◽  
Heterotrimeric G Proteins ◽  
Membrane Attachment ◽  
The Individual ◽  
Multicellular Organisms ◽  
G Protein Coupled

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

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