scholarly journals Depletion of Ric-8B leads to reduced mTORC2 activity

2019 ◽  
Author(s):  
Maíra H. Nagai ◽  
Luciana M. Gutiyama ◽  
Victor P. S. Xavier ◽  
Cleiton F. Machado ◽  
Alice H. Reis ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Neural Tube ◽  
Adult Brain ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Hypomorphic Mutation ◽  
Downstream Target ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

AbstractmTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway.Phosphorylation of Akt at Ser 473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. In contrast, phosphorylation of S6, a downstream target of mTORC1, is unaltered. Knockdown of the endogenous Ric-8B gene in HEK293T cells leads to reduced phosphorylation levels of Akt at Ser 473, but not of S6, further supporting the selective involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2 activity.Author SummaryGene inactivation in mice can be used to identify genes that are involved in important biological processes and that may contribute to disease. By using this approach, we found that the Ric-8B gene is essential for embryogenesis and for the normal development of the nervous system. Ric-8B mutant mouse embryos are smaller than their wild type littermates and show neural tube defects at the cranial region. This approach also allowed us to identify the biological pathways that are involved in the observed phenotypes, the G protein and mTORC2 signaling pathways. mTORC2 plays particular important roles also in the adult brain, and has been implicated in neurological disorders. Ric-8B is highly conserved in mammals, including humans. Our mutant mice provide a model to study the complex molecular and cellular processes underlying the interplay between Ric-8B and mTORC2 in neuronal function.

scholarly journals The Intermolecular Interaction of Ephexin4 Leads to Autoinhibition by Impeding Binding of RhoG

Cells ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 211 ◽  
Author(s):  
Kwanhyeong Kim ◽  
Juyeon Lee ◽  
Hyunji Moon ◽  
Sang-Ah Lee ◽  
Deokhwan Kim ◽  
...  
Keyword(s):  
Intermolecular Interaction ◽  
Apoptotic Cells ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Migration ◽  
Conserved Residue

Ephexin4 is a guanine nucleotide-exchange factor (GEF) for RhoG and is involved in various RhoG-related cellular processes such as phagocytosis of apoptotic cells and migration of cancer cells. Ephexin4 forms an oligomer via an intermolecular interaction, and its GEF activity is increased in the presence of Elmo, an Ephexin4-interacting protein. However, it is uncertain if and how Ephexin4 is autoinhibited. Here, using an Ephexin4 mutant that abrogated the intermolecular interaction, we report that this interaction impeded binding of RhoG to Ephexin4 and thus inhibited RhoG activation. Mutation of the glutamate residue at position 295, which is a highly conserved residue located in the region of Ephexin4 required for the intermolecular interaction, to alanine (Ephexin4E295A) disrupted the intermolecular interaction and increased binding of RhoG, resulting in augmented RhoG activation. In addition, phagocytosis of apoptotic cells and formation of membrane ruffles were increased more by expression of Ephexin4E295A than by expression of wild-type Ephexin4. Taken together, our data suggest that Ephexin4 is autoinhibited through its intermolecular interaction, which impedes binding of RhoG.

scholarly journals Induction of cell retraction by the combined actions of Abl–CrkII and Rho–ROCK1 signaling

2008 ◽  
Vol 183 (4) ◽  
pp. 711-723 ◽  
Author(s):  
XiaoDong Huang ◽  
Diana Wu ◽  
Hua Jin ◽  
Dwayne Stupack ◽  
Jean Y.J. Wang
Keyword(s):  
Nucleotide Exchange ◽  
Downstream Target ◽  
Abl Tyrosine Kinase ◽  
Guanine Nucleotide Exchange ◽  
Cell Matrix ◽  
Nucleotide Exchange Factor ◽  
Wide Range ◽  
Important Regulator ◽  
Guanosine Triphosphatase ◽  
Cell Retraction

Dynamic modulation of cell adhesion is integral to a wide range of biological processes. The small guanosine triphosphatase (GTPase) Rap1 is an important regulator of cell–cell and cell–matrix adhesions. We show here that induced expression of activated Abl tyrosine kinase reduces Rap1-GTP levels through phosphorylation of Tyr221 of CrkII, which disrupts interaction of CrkII with C3G, a guanine nucleotide exchange factor for Rap1. Abl-dependent down-regulation of Rap1-GTP causes cell rounding and detachment only when the Rho–ROCK1 pathway is also activated, for example, by lysophosphatidic acid (LPA). During ephrin-A1–induced retraction of PC3 prostate cancer cells, we show that endogenous Abl is activated and disrupts the CrkII–C3G complex to reduce Rap1-GTP. Interestingly, ephrin-A1–induced PC3 cell retraction also requires LPA, which stimulates Rho to a much higher level than that is activated by ephrin-A1. Our results establish Rap1 as another downstream target of the Abl–CrkII signaling module and show that Abl–CrkII collaborates with Rho–ROCK1 to stimulate cell retraction.

scholarly journals Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1

2003 ◽  
Vol 160 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Metello Innocenti ◽  
Emanuela Frittoli ◽  
Isabella Ponzanelli ◽  
John R. Falck ◽  
Saskia M. Brachmann ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Physical Interaction ◽  
Nucleotide Exchange ◽  
Downstream Target ◽  
Signaling Complex ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphoinositide 3 Kinase

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

scholarly journals The RAC1 activator Tiam1 regulates centriole duplication through controlling PLK4 levels

2020 ◽  
Author(s):  
Andrew P. Porter ◽  
Gavin R. M. White ◽  
Erinn-Lee Ogg ◽  
Helen J. Whalley ◽  
Angeliki Malliri
Keyword(s):  
Cell Cycle ◽  
Guanine Nucleotide Exchange Factor ◽  
S Phase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Centriole Duplication ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
F Box Protein

SummaryCentriole duplication is tightly controlled to maintain correct centriole number through the cell cycle. A key component of this control is the regulated degradation of PLK4, the master regulator of centriole duplication. Here we show that the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 localises to centrosomes during S-phase, where it is required for maintenance of normal centriole number. Depletion of Tiam1 leads to an increase in centrosomal PLK4, centriole overduplication and ultimately to lagging chromosomes at anaphase and aneuploidy. The effects of Tiam1 depletion can be rescued by re-expression of wild-type Tiam1 and catalytically inactive (GEF*) Tiam1, but not by Tiam1 mutants unable to bind to the F-box protein βTRCP, implying that Tiam1 regulates PLK4 levels through promoting βTRCP-mediated degradation.

scholarly journals Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3

2014 ◽  
Vol 25 (10) ◽  
pp. 1608-1619 ◽  
Author(s):  
Gus Lawrence ◽  
Christopher C. Brown ◽  
Blake A. Flood ◽  
Surya Karunakaran ◽  
Margarita Cabrera ◽  
...  
Keyword(s):  
Fusion Reaction ◽  
Casein Kinase ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphatidic Acid Phosphatase ◽  
Phosphatidylinositol 3

Maturation of organelles in the endolysosomal pathway requires exchange of the early endosomal GTPase Rab5/Vps21 for the late endosomal Rab7/Ypt7. The Rab exchange depends on the guanine nucleotide exchange factor activity of the Mon1-Ccz1 heterodimer for Ypt7. Here we investigate vacuole binding and recycling of Mon1-Ccz1. We find that Mon1-Ccz1 is absent on vacuoles lacking the phosphatidic acid phosphatase Pah1, which also lack Ypt7, the phosphatidylinositol 3-kinase Vps34, and the lipid phosphatidylinositol 3-phosphate (PI3P). Interaction of Mon1-Ccz1 with wild-type vacuoles requires PI3P, as shown in competition experiments. We also find that Mon1 is released from vacuoles during the fusion reaction and its release requires its phosphorylation by the type 1 casein kinase Yck3. In contrast, Mon1 is retained on vacuoles lacking Yck3 or when Mon1 phosphorylation sites are mutated. Phosphorylation and release of Mon1 is restored with addition of recombinant Yck3. Together the results show that Mon1 is recruited to endosomes and vacuoles by PI3P and, likely after activating Ypt7, is phosphorylated and released from vacuoles for recycling.

scholarly journals The Rac activator Tiam1 controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex

2005 ◽  
Vol 170 (7) ◽  
pp. 1029-1037 ◽  
Author(s):  
Alexander E.E. Mertens ◽  
Tomasz P. Rygiel ◽  
Cristina Olivo ◽  
Rob van der Kammen ◽  
John G. Collard
Keyword(s):  
Cell Polarity ◽  
Epidermal Keratinocytes ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Invasion And Metastasis ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
T Lymphoma ◽  
Maturation Defect ◽  
Membrane Sealing

The GTPases Rac and Cdc42 play a pivotal role in the establishment of cell polarity by stimulating biogenesis of tight junctions (TJs). In this study, we show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis) controls the cell polarity of epidermal keratinocytes. Similar to wild-type (WT) keratinocytes, Tiam1-deficient cells establish primordial E-cadherin–based adhesions, but subsequent junction maturation and membrane sealing are severely impaired. Tiam1 and V12Rac1 can rescue the TJ maturation defect in Tiam1-deficient cells, indicating that this defect is the result of impaired Tiam1–Rac signaling. Tiam1 interacts with Par3 and aPKCζ, which are two components of the conserved Par3–Par6–aPKC polarity complex, and triggers biogenesis of the TJ through the activation of Rac and aPKCζ, which is independent of Cdc42. Rac is activated upon the formation of primordial adhesions (PAs) in WT but not in Tiam1-deficient cells. Our data indicate that Tiam1-mediated activation of Rac in PAs controls TJ biogenesis and polarity in epithelial cells by association with and activation of the Par3–Par6–aPKC polarity complex.

scholarly journals A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling

2021 ◽  
Vol 17 (8) ◽  
pp. e1009902
Author(s):  
Anthony Davidson ◽  
Joe Tyler ◽  
Peter Hume ◽  
Vikash Singh ◽  
Vassilis Koronakis
Keyword(s):  
Kinase Domain ◽  
Nucleotide Exchange ◽  
Independent Function ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Negative Effect ◽  
P21 Activated Kinase ◽  
Actin Remodelling ◽  
Proline Rich Region

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.

scholarly journals A NUMB–EFA6B–ARF6 recycling route controls apically restricted cell protrusions and mesenchymal motility

2018 ◽  
Vol 217 (9) ◽  
pp. 3161-3182 ◽  
Author(s):  
Martina Zobel ◽  
Andrea Disanza ◽  
Francesca Senic-Matuglia ◽  
Michel Franco ◽  
Ivan Nicola Colaluca ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Negative Regulator ◽  
Migration And Invasion ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Endocytic Protein ◽  
Membrane Protrusions

The endocytic protein NUMB has been implicated in the control of various polarized cellular processes, including the acquisition of mesenchymal migratory traits through molecular mechanisms that have only been partially defined. Here, we report that NUMB is a negative regulator of a specialized set of understudied, apically restricted, actin-based protrusions, the circular dorsal ruffles (CDRs), induced by either PDGF or HGF stimulation. Through its PTB domain, NUMB binds directly to an N-terminal NPLF motif of the ARF6 guanine nucleotide exchange factor, EFA6B, and promotes its exchange activity in vitro. In cells, a NUMB–EFA6B–ARF6 axis regulates the recycling of the actin regulatory cargo RAC1 and is critical for the formation of CDRs that mark the acquisition of a mesenchymal mode of motility. Consistently, loss of NUMB promotes HGF-induced cell migration and invasion. Thus, NUMB negatively controls membrane protrusions and the acquisition of mesenchymal migratory traits by modulating EFA6B–ARF6 activity.

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