Dock7: A GEF for Rho-family GTPases and a novel myosin VI-binding partner in neuronal PC12 cells

2012 ◽  
Vol 90 (4) ◽  
pp. 565-574 ◽  
Author(s):  
Łukasz Majewski ◽  
Magdalena Sobczak ◽  
Serhiy Havrylov ◽  
Jolanta Jóźwiak ◽  
Maria Jolanta Rędowicz
Keyword(s):  
Actin Cytoskeleton ◽  
Pc12 Cells ◽  
Hippocampal Neurons ◽  
Small Gtpases ◽  
Myosin Vi ◽  
Tail Domain ◽  
Nucleotide Exchange ◽  
Binding Partner ◽  
Cytoskeleton Organization ◽  
Neuronal Pc12 Cells

Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI–Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization.

scholarly journals The RhoGEF Trio: A Protein with a Wide Range of Functions in the Vascular Endothelium

2021 ◽  
Vol 22 (18) ◽  
pp. 10168
Author(s):  
Lanette Kempers ◽  
Amber J. M. Driessen ◽  
Jos van Rijssel ◽  
Martijn A. Nolte ◽  
Jaap D. van Buul
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Function ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Endothelial Cell Function ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Wide Range ◽  
Range Of Functions

Many cellular processes are controlled by small GTPases, which can be activated by guanine nucleotide exchange factors (GEFs). The RhoGEF Trio contains two GEF domains that differentially activate the small GTPases such as Rac1/RhoG and RhoA. These small RhoGTPases are mainly involved in the remodeling of the actin cytoskeleton. In the endothelium, they regulate junctional stabilization and play a crucial role in angiogenesis and endothelial barrier integrity. Multiple extracellular signals originating from different vascular processes can influence the activity of Trio and thereby the regulation of the forementioned small GTPases and actin cytoskeleton. This review elucidates how various signals regulate Trio in a distinct manner, resulting in different functional outcomes that are crucial for endothelial cell function in response to inflammation.

scholarly journals Identification of a Novel Sequence in PDZ-RhoGEF That Mediates Interaction with the Actin Cytoskeleton

2004 ◽  
Vol 15 (4) ◽  
pp. 1760-1775 ◽  
Author(s):  
Jayashree Banerjee ◽  
Philip B. Wedegaertner
Keyword(s):  
Actin Cytoskeleton ◽  
Plasma Membranes ◽  
Small Gtpases ◽  
Protein Signaling ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Necessary And Sufficient ◽  
Cytoskeleton Rearrangements

Small GTPases of the Rho family are crucial regulators of actin cytoskeleton rearrangements. Rho is activated by members of the Rho guanine-nucleotide exchange factor (GEF) family; however, mechanisms that regulate RhoGEFs are not well understood. This report demonstrates that PDZ-RhoGEF, a member of a subfamily of RhoGEFs that contain regulator of G protein signaling domains, is partially localized at or near the plasma membranes in 293T, COS-7, and Neuro2a cells, and this localization is coincident with cortical actin. Disruption of the cortical actin cytoskeleton in cells by using latrunculin B prevents the peri-plasma membrane localization of PDZ-RhoGEF. Coimmunoprecipitation and F-actin cosedimentation assays demonstrate that PDZ-RhoGEF binds to actin. Extensive deletion mutagenesis revealed the presence of a novel 25-amino acid sequence in PDZ-RhoGEF, located at amino acids 561–585, that is necessary and sufficient for localization to the actin cytoskeleton and interaction with actin. Last, PDZ-RhoGEF mutants that fail to interact with the actin cytoskeleton display enhanced Rho-dependent signaling compared with wild-type PDZ-RhoGEF. These results identify interaction with the actin cytoskeleton as a novel function for PDZ-RhoGEF, thus implicating actin interaction in organizing PDZ-RhoGEF signaling.

scholarly journals Interaction of myosin VI and its binding partner DOCK7 plays an important role in NGF-stimulated protrusion formation in PC12 cells

2016 ◽  
Vol 1863 (7) ◽  
pp. 1589-1600 ◽  
Author(s):  
Magdalena Sobczak ◽  
Vira Chumak ◽  
Paweł Pomorski ◽  
Emilia Wojtera ◽  
Łukasz Majewski ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Myosin Vi ◽  
Binding Partner

scholarly journals Very-KIND, a KIND domain–containing RasGEF, controls dendrite growth by linking Ras small GTPases and MAP2

2007 ◽  
Vol 179 (3) ◽  
pp. 539-552 ◽  
Author(s):  
Jinhong Huang ◽  
Asako Furuya ◽  
Teiichi Furuichi
Keyword(s):  
Hippocampal Neurons ◽  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Small Gtpases ◽  
Dendrite Growth ◽  
Nucleotide Exchange ◽  
Cerebellar Granule ◽  
Nucleotide Exchange Factor ◽  
Dendritic Shaft ◽  
Developing Mouse Brain

The regulation of cytoskeletal components in the dendritic shaft core is critical for dendrite elongation and branching. Here, we report that a brain-specific Ras guanine nucleotide exchange factor (RasGEF) carrying two kinase non-catalytic C-lobe domains (KINDs), very-KIND (v-KIND), regulates microtubule-associated protein 2 (MAP2). v-KIND is expressed in developing mouse brain, predominantly in the cerebellar granule cells. v-KIND not only activates Ras small GTPases via the C-terminal RasGEF domain, but also specifically binds to MAP2 via the second KIND domain (KIND2), leading to threonine phosphorylation of MAP2. v-KIND overexpression suppresses dendritic extension and branching of hippocampal neurons and cerebellar granule cells, whereas knockdown of endogenous v-KIND expression promotes dendrite growth. These findings suggest that v-KIND mediates a signaling pathway that links Ras and MAP2 to control dendrite growth.

scholarly journals Dual Role of Cdc42 in Spindle Orientation Control of Adherent Cells

2009 ◽  
Vol 29 (10) ◽  
pp. 2816-2827 ◽  
Author(s):  
Masaru Mitsushima ◽  
Fumiko Toyoshima ◽  
Eisuke Nishida
Keyword(s):  
Actin Cytoskeleton ◽  
Small Gtpases ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Adherent Cells ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Independent Manner ◽  
Oriented Parallel

ABSTRACT The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and PtdIns(3,4,5)P3. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. βPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-βPix-actin pathway for this mechanism.

scholarly journals Rabin8 regulates neurite outgrowth in both GEF activity–dependent and –independent manners

2016 ◽  
Vol 27 (13) ◽  
pp. 2107-2118 ◽  
Author(s):  
Yuta Homma ◽  
Mitsunori Fukuda
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Membrane Trafficking ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Recycling Endosomes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Activity Dependent

Many aspects of membrane-trafficking events are regulated by Rab-family small GTPases. Neurite outgrowth requires massive addition of proteins and lipids to the tips of growing neurites by membrane trafficking, and although several Rabs, including Rab8, Rab10, and Rab11, have been implicated in this process, their regulatory mechanisms during neurite outgrowth are poorly understood. Here, we show that Rabin8, a Rab8-guanine nucleotide exchange factor (GEF), regulates nerve growth factor (NGF)–induced neurite outgrowth of PC12 cells. Knockdown of Rabin8 results in inhibition of neurite outgrowth, whereas overexpression promotes it. We also find that Rab10 is a novel substrate of Rabin8 and that both Rab8 and Rab10 function during neurite outgrowth downstream of Rabin8. Surprisingly, however, a GEF activity–deficient isoform of Rabin8 also promotes neurite outgrowth, indicating the existence of a GEF activity–independent role of Rabin8. The Arf6/Rab8-positive recycling endosomes (Arf6/Rab8-REs) and Rab10/Rab11-positive REs (Rab10/Rab11-REs) in NGF-stimulated PC12 cells are differently distributed. Rabin8 localizes on both RE populations and appears to activate Rab8 and Rab10 there. These localizations and functions of Rabin8 are Rab11 dependent. Thus Rabin8 regulates neurite outgrowth both by coordinating with Rab8, Rab10, and Rab11 and by a GEF activity–independent mechanism.

scholarly journals Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

2005 ◽  
Vol 169 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Daniela A. Sahlender ◽  
Rhys C. Roberts ◽  
Susan D. Arden ◽  
Giulietta Spudich ◽  
Marcus J. Taylor ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rna Interference ◽  
Vesicular Stomatitis Virus ◽  
Protein Interaction ◽  
Golgi Complex ◽  
Myosin Vi ◽  
Binding Partner ◽  
Binding Partners ◽  
Vesicular Stomatitis ◽  
Golgi Organization

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

scholarly journals Sesamin Modulates Tyrosine Hydroxylase, Superoxide Dismutase, Catalase, Inducible No Synthase and Interleukin-6 Expression in Dopaminergic Cells Under Mpp+-Induced Oxidative Stress

2008 ◽  
Vol 1 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Vicky Lahaie-Collins ◽  
Julie Bournival ◽  
Marilyn Plouffe ◽  
Julie Carange ◽  
Maria-Grazia Martinoli
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Tyrosine Hydroxylase ◽  
Protein Expression ◽  
Pc12 Cells ◽  
Interleukin 6 ◽  
Estrogen Receptor Alpha ◽  
Strong Increase ◽  
Mrna Levels ◽  
Neuronal Pc12 Cells

Oxidative stress is regarded as a mediator of nerve cell death in several neurodegenerative disorders, such as Parkinson's disease. Sesamin, a lignan mainly found in sesame oil, is currently under study for its anti-oxidative and possible neuroprotective properties. We used 1-methyl-4-phenyl-pyridine (MPP+) ion, the active metabolite of the potent parkinsonism-causing toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, to produce oxidative stress and neurodegeneration in neuronal PC12 cells, which express dopamine, as well as neurofilaments. Our results show that picomolar doses of sesamin protected neuronal PC12 cells from MPP+-induced cellular death, as revealed by colorimetric measurements and production of reactive oxygen species. We also demonstrated that sesamin acted by rescuing tyrosine hydroxylase levels from MPP+-induced depletion. Sesamin, however, did not modulate dopamine transporter levels, and estrogen receptor-alpha and -beta protein expression. By examining several parameters of cell distress, we found that sesamin also elicited a strong increase in superoxide dismutase activity as well as protein expression and decreased catalase activity and the MPP+stimulated inducible nitric oxide synthase protein expression, in neuronal PC12 cells. Finally, sesamin possessed significant anti-inflammatory properties, as disclosed by its potential to reduce MPP+-induced interleukin-6 mRNA levels in microglia. From these studies, we determined the importance of the lignan sesamin as a neuroprotective molecule and its possible role in complementary and/or preventive therapies of neurodegenerative diseases.

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