scholarly journals Interaction between ROCK II and Nucleophosmin/B23 in the Regulation of Centrosome Duplication

2006 ◽  
Vol 26 (23) ◽  
pp. 9016-9034 ◽  
Author(s):  
Zhiyong Ma ◽  
Masayuki Kanai ◽  
Kenji Kawamura ◽  
Kozo Kaibuchi ◽  
Keqiang Ye ◽  
...  
Keyword(s):  
Cyclin E ◽  
Active Form ◽  
Small Gtpase ◽  
Centrosome Duplication ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Physical Separation ◽  
Initial Event ◽  
Activity Dependent

ABSTRACT Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr199 by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr199 remains at centrosomes. It has been shown that Thr199 phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr199. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr199-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

Extracellular Release of ILEI/FAM3C and Amyloid-β Is Associated with the Activation of Distinct Synapse Subpopulations

2021 ◽  
pp. 1-16
Author(s):  
Masaki Nakano ◽  
Yachiyo Mitsuishi ◽  
Lei Liu ◽  
Naoki Watanabe ◽  
Emi Hibino ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Epithelial Mesenchymal Transition ◽  
Surrogate Marker ◽  
Amyloid Β ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Extracellular Release ◽  
Activity Dependent ◽  
Aβ Production

Background: Brain amyloid-β (Aβ) peptide is released into the interstitial fluid (ISF) in a neuronal activity-dependent manner, and Aβ deposition in Alzheimer’s disease (AD) is linked to baseline neuronal activity. Although the intrinsic mechanism for Aβ generation remains to be elucidated, interleukin-like epithelial-mesenchymal transition inducer (ILEI) is a candidate for an endogenous Aβ suppressor. Objective: This study aimed to access the mechanism underlying ILEI secretion and its effect on Aβ production in the brain. Methods: ILEI and Aβ levels in the cerebral cortex were monitored using a newly developed ILEI-specific ELISA and in vivo microdialysis in mutant human Aβ precursor protein-knockin mice. ILEI levels in autopsied brains and cerebrospinal fluid (CSF) were measured using ELISA. Results: Extracellular release of ILEI and Aβ was dependent on neuronal activation and specifically on tetanus toxin-sensitive exocytosis of synaptic vesicles. However, simultaneous monitoring of extracellular ILEI and Aβ revealed that a spontaneous fluctuation of ILEI levels appeared to inversely mirror that of Aβ levels. Selective activation and inhibition of synaptic receptors differentially altered these levels. The evoked activation of AMPA-type receptors resulted in opposing changes to ILEI and Aβ levels. Brain ILEI levels were selectively decreased in AD. CSF ILEI concentration correlated with that of Aβ and were reduced in AD and mild cognitive impairment. Conclusion: ILEI and Aβ are released from distinct subpopulations of synaptic terminals in an activity-dependent manner, and ILEI negatively regulates Aβ production in specific synapse types. CSF ILEI might represent a surrogate marker for the accumulation of brain Aβ.

scholarly journals Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G1 Cyclin-Dependent Kinase Complexes In Vivo

2001 ◽  
Vol 21 (14) ◽  
pp. 4773-4784 ◽  
Author(s):  
Sergei A. Ezhevsky ◽  
Alan Ho ◽  
Michelle Becker-Hapak ◽  
Penny K. Davis ◽  
Steven F. Dowdy
Keyword(s):  
Cyclin E ◽  
Active Form ◽  
Dominant Negative ◽  
Tumor Suppressor Protein ◽  
Cyclin D ◽  
Cyclin Dependent Kinase ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G0 quiescent cells and becomes hypophosphorylated (∼2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (∼10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT–dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.

scholarly journals ADP-ribosylation factor–like 4A interacts with Robo1 to promote cell migration by regulating Cdc42 activation

2019 ◽  
Vol 30 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Tsai-Shin Chiang ◽  
Ming-Chieh Lin ◽  
Meng-Chen Tsai ◽  
Chieh-Hsin Chen ◽  
Li-Ting Jang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Molecular Mechanisms ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Adp Ribosylation ◽  
Promote Cell Migration ◽  
Cytoskeleton Remodeling ◽  
Adp Ribosylation Factor

Cell migration is a highly regulated event that is initiated by cell membrane protrusion and actin reorganization. Robo1, a single-pass transmembrane receptor, is crucial for neuronal guidance and cell migration. ADP-ribosylation factor (Arf)–like 4A (Arl4A), an Arf small GTPase, functions in cell morphology, cell migration, and actin cytoskeleton remodeling; however, the molecular mechanisms of Arl4A in cell migration are unclear. Here, we report that the binding of Arl4A to Robo1 modulates cell migration by promoting Cdc42 activation. We found that Arl4A interacts with Robo1 in a GTP-dependent manner and that the Robo1 amino acid residues 1394–1398 are required for this interaction. The Arl4A-Robo1 interaction is essential for Arl4A-induced cell migration and Cdc42 activation but not for the plasma membrane localization of Robo1. In addition, we show that the binding of Arl4A to Robo1 decreases the association of Robo1 with the Cdc42 GTPase-activating protein srGAP1. Furthermore, Slit2/Robo1 binding down-regulates the Arl4A-Robo1 interaction in vivo, thus attenuating Cdc42-mediated cell migration. Therefore, our study reveals a novel mechanism by which Arl4A participates in Slit2/Robo1 signaling to modulate cell motility by regulating Cdc42 activity.

scholarly journals A mechanism of Rap1-induced stabilization of endothelial cell–cell junctions

2011 ◽  
Vol 22 (14) ◽  
pp. 2509-2519 ◽  
Author(s):  
Jian J. Liu ◽  
Rebecca A. Stockton ◽  
Alexandre R. Gingras ◽  
Ararat J. Ablooglu ◽  
Jaewon Han ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Small Gtpases ◽  
Cell Junctions ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Cardiovascular Development ◽  
Cavernous Malformations ◽  
Cell Cell

Activation of Rap1 small GTPases stabilizes cell–cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1–Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ∼40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell–cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell–cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.

A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5052-5062 ◽  
Author(s):  
Gaël Ménasché ◽  
Mickaël M. Ménager ◽  
Juliette M. Lefebvre ◽  
Einat Deutsch ◽  
Rafika Athman ◽  
...  
Keyword(s):  
Target Cell ◽  
Affinity Purification ◽  
Cytotoxic T Cells ◽  
Active Form ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Granule Secretion ◽  
Immunologic Synapse ◽  
Cytotoxic Granule

Abstract Cytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules—indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.

scholarly journals Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

2015 ◽  
Vol 26 (16) ◽  
pp. 2939-2954 ◽  
Author(s):  
Sónia Barbosa ◽  
Suzanne Carreira ◽  
Daniel Bailey ◽  
Fernando Abaitua ◽  
Peter O'Hare
Keyword(s):  
Target Gene ◽  
Active Form ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Critical Determinant ◽  
Phosphatase Treatment ◽  
Steady State Levels ◽  
Treatment Use

CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1aE3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV.

scholarly journals Modulation of Synaptic Plasticity by Glutamatergic Gliotransmission: A Modeling Study

2016 ◽  
Vol 2016 ◽  
pp. 1-30 ◽  
Author(s):  
Maurizio De Pittà ◽  
Nicolas Brunel
Keyword(s):  
Synaptic Plasticity ◽  
Glutamate Release ◽  
Spike Timing ◽  
Biophysical Model ◽  
Dependent Manner ◽  
Inward Currents ◽  
Functional Relevance ◽  
And Function ◽  
Activity Dependent

Glutamatergic gliotransmission, that is, the release of glutamate from perisynaptic astrocyte processes in an activity-dependent manner, has emerged as a potentially crucial signaling pathway for regulation of synaptic plasticity, yet its modes of expression and function in vivo remain unclear. Here, we focus on two experimentally well-identified gliotransmitter pathways, (i) modulations of synaptic release and (ii) postsynaptic slow inward currents mediated by glutamate released from astrocytes, and investigate their possible functional relevance on synaptic plasticity in a biophysical model of an astrocyte-regulated synapse. Our model predicts that both pathways could profoundly affect both short- and long-term plasticity. In particular, activity-dependent glutamate release from astrocytes could dramatically change spike-timing-dependent plasticity, turning potentiation into depression (and vice versa) for the same induction protocol.

scholarly journals An XA21-Associated Kinase (OsSERK2) regulates immunity mediated by the XA21 and XA3 immune receptors

2013 ◽  
Author(s):  
Xuewei Chen ◽  
Shimin Zuo ◽  
Benjamin Schwessinger ◽  
Mawsheng Chern ◽  
Patrick Canlas ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
Immune Receptor ◽  
Immune Receptors ◽  
Yeast Two Hybrid System ◽  
Intracellular Domains ◽  
Activity Dependent

The rice XA21 immune receptor kinase and the structurally related XA3 receptor, confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast-two-hybrid system in a kinase activity dependent manner. OsSERK2 undergoes bidirectional trans-phosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. Taken together, these results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function.

scholarly journals Title: In vivo phosphatidylserine variations steer Rho GTPase signaling in a cell-context dependent manner

2018 ◽  
Author(s):  
Matthieu Pierre Platre ◽  
Vincent Bayle ◽  
Laia Armengot ◽  
Joseph Bareille ◽  
Maria Mar Marques-Bueno ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Plant Root ◽  
Super Resolution ◽  
Small Gtpase ◽  
Auxin Signaling ◽  
Dependent Manner ◽  
Downstream Signaling

AbstractRho GTPases are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. Here, we show that the phospholipid phosphatidylserine acts as a developmentally-controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live super-resolution single molecule imaging revealed that RHO-OF-PLANT6 (ROP6) is stabilized by phosphatidylserine into plasma membrane (PM) nanodomains, which is required for auxin signaling. Furthermore, we uncovered that the PM phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work reveals that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.One Sentence SummaryPhosphatidylserine acts as a developmentally-controlled lipid rheostat that regulates cellular auxin sensitivity and plant development.

scholarly journals Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke

2021 ◽  
Vol 118 (32) ◽  
pp. e2018850118
Author(s):  
Hiroo Takahashi ◽  
Ryo Asahina ◽  
Masayuki Fujioka ◽  
Takeshi K. Matsui ◽  
Shigeki Kato ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cortical Neurons ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Small Gtpase ◽  
Neuroprotective Effects ◽  
Necessary And Sufficient ◽  
Activity Dependent

Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

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