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 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.

Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B

2001 ◽  
Vol 356 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Mireille CORMONT ◽  
Nadine GAUTIER ◽  
Karine ILC ◽  
Yannick Le MARCHAND-BRUSTEL
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Active Form ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Phosphatidylinositol 3 Kinase ◽  
Isolated Adipocytes ◽  
Phosphatidylinositol 3

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 ΔCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 ΔCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 ΔCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 ΔCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.

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 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.

scholarly journals COPI Recruitment Is Modulated by a Rab1b-dependent Mechanism

2003 ◽  
Vol 14 (5) ◽  
pp. 2116-2127 ◽  
Author(s):  
Cecilia Alvarez ◽  
Rafael Garcia-Mata ◽  
Elizabeth Brandon ◽  
Elizabeth Sztul
Keyword(s):  
Active Form ◽  
Brefeldin A ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Inactive Form ◽  
Exact Function ◽  
Guanine Nucleotide Binding

The small GTPase Rab1b is essential for endoplasmic reticulum (ER) to Golgi transport, but its exact function remains unclear. We have examined the effects of wild-type and three mutant forms of Rab1b in vivo. We show that the inactive form of Rab1b (the N121I mutant with impaired guanine nucleotide binding) blocks forward transport of cargo and induces Golgi disruption. The phenotype is analogous to that induced by brefeldin A (BFA): it causes resident Golgi proteins to relocate to the ER and induces redistribution of ER-Golgi intermediate compartment proteins to punctate structures. The COPII exit machinery seems to be functional in cells expressing the N121I mutant, but COPI is compromised, as shown by the release of β-COP into the cytosol. Our results suggest that Rab1b function influences COPI recruitment. In support of this, we show that the disruptive effects of N121I can be reversed by expressing known mediators of COPI recruitment, the GTPase ARF1 and its guanine nucleotide exchange factor GBF1. Further evidence is provided by the finding that cells expressing the active form of Rab1b (the Q67L mutant with impaired GTPase activity) are resistant to BFA. Our data suggest a novel role for Rab1b in ARF1- and GBF1-mediated COPI recruitment pathway.

scholarly journals Ras Binding Triggers Ubiquitination of the Ras Exchange Factor Ras-GRF2

2001 ◽  
Vol 21 (6) ◽  
pp. 2107-2117 ◽  
Author(s):  
Carmen L. de Hoog ◽  
Jackie A. Koehler ◽  
Marni D. Goldstein ◽  
Paul Taylor ◽  
Daniel Figeys ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpase ◽  
Dominant Negative ◽  
26S Proteasome ◽  
Guanine Nucleotide ◽  
Sequence Motifs ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange

ABSTRACT Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.

scholarly journals The T-Cell Receptor Regulates Akt (Protein Kinase B) via a Pathway Involving Rac1 and Phosphatidylinositide 3-Kinase

2000 ◽  
Vol 20 (15) ◽  
pp. 5469-5478 ◽  
Author(s):  
Elisabeth M. Genot ◽  
Cecile Arrieumerlou ◽  
Gregory Ku ◽  
Boudewijn M. T. Burgering ◽  
Arthur Weiss ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase B ◽  
Active Form ◽  
Cell Receptor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
Dominant Negative Mutant

ABSTRACT The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.

The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference

2012 ◽  
Vol 443 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Julia Knöckel ◽  
Ingrid B. Müller ◽  
Sabine Butzloff ◽  
Bärbel Bergmann ◽  
Rolf D. Walter ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Vitamin B6 ◽  
De Novo ◽  
Active Form ◽  
Cellular Level ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Transcription Analysis

The malaria parasite Plasmodium falciparum is able to synthesize de novo PLP (pyridoxal 5′-phosphate), the active form of vitamin B6. In the present study, we have shown that the de novo synthesized PLP is used by the parasite to detoxify 1O2 (singlet molecular oxygen), a highly destructive reactive oxygen species arising from haemoglobin digestion. The formation of 1O2 and the response of the parasite were monitored by live-cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite. Pull-down experiments of transgenic parasites overexpressing the vitamin B6-biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 μM in wild-type parasites to 36.6 μM in the PfPdx1/PfPdx2-overexpressing cells and thus to a higher tolerance towards 1O2. In contrast, by applying the dominant-negative effect on the cellular level using inactive mutants of PfPdx1 and PfPdx2, P. falciparum becomes susceptible to 1O2. Our results demonstrate clearly the crucial role of vitamin B6 biosynthesis in the detoxification of 1O2 in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes, this second important task of the vitamin B6de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.

scholarly journals Cortical actin properties controlled by Drosophila Fos aid macrophage infiltration against surrounding tissue resistance

2020 ◽  
Author(s):  
Vera Belyaeva ◽  
Stephanie Wachner ◽  
Igor Gridchyn ◽  
Markus Linder ◽  
Shamsi Emtenani ◽  
...  
Keyword(s):  
Immune Cells ◽  
Immune Cell ◽  
Active Form ◽  
Dominant Negative ◽  
Surrounding Tissue ◽  
Sequencing Analysis ◽  
Mrna Levels ◽  
Cortical Actin ◽  
Forward Movement

ABSTRACTThe infiltration of immune cells into tissues underlies the establishment of tissue resident macrophages, and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio which are themselves required for invasion. Cortical F-actin levels are critical as expressing a dominant active form of Diaphanous, a actin polymerizing Formin, can rescue the Dfos Dominant Negative macrophage invasion defect. In vivo imaging shows that Dfos is required to enhance the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the mechanical properties of the macrophage nucleus from affecting tissue entry. We thus identify tuning the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

Rac1 GTPase Targeting Inhibits p53-Deficiency Mediated Lymphomagenesis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1764-1764
Author(s):  
Emily E Bosco ◽  
James F Johnson ◽  
Wenjun Ni ◽  
Yi Zheng
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Tumor Formation ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Lymphoma Cell ◽  
Dominant Negative Mutant ◽  
Rac1 Activity ◽  
T Lymphoma

Abstract Mutation of the p53 tumor suppressor is linked to therapeutic resistance in several tumor types. p53 mutation is generally associated with disease progression and poor prognosis in patients with lymphoid malignancies and can occur in approximately 50% of Burkitt’s lymphomas [1, 2]. Thus, new therapies are needed to specifically target p53-deficient lymphomas with increased efficacy. Rac1 small GTPase signaling is linked to tumorigenesis and Rac1 activity is increased by p53 loss of function. Previous studies in primary cell culture have shown that suppression of Rac1 activity can preferentially inhibit p53-deficiency induced hyperproliferation and cause increase apoptosis [3], indicating that targeting Rac1 may have therapeutic potential for p53-deficient malignancies. In the current study, the specific impact of inhibition of Rac1 on p53-deficient B and T lymphoma cell proliferation, apoptosis, tumor formation, and spontaneous lymphoma development in in vivo mouse lymphoma models was interrogated. p53-deficiency resulted in increased Rac1 activity in both human B and murine T lymphoma lines. Suppression of Rac activity using a dominant negative mutant or the small molecule inhibitor NSC23766 was able to abrogate p53-deficient lymphoma cell proliferation through a G1 checkpoint as determined by cell cycle analysis. Immunoblot analysis revealed that the anti-proliferative effect of Rac1 targeting in lymphoma cells may involve PAK, LIMK, and Akt signaling pathways rather than the MAP kinase pathway. Further, Rac1 targeting by shRNA resulted in an increase in expression of cleaved caspase 3 and cytochrome c by immunoblot concurrent with an elevation in the annexinV/7-AAD positive apoptotic cell population. These data indicate that loss of Rac1 activity can increase apoptosis via a p53-independent mechanism in p53 mutated lymphoma lines. These effects of inhibition of active Rac1 were extended in vivo where Rac1 targeting was able to impair p53-deficient lymphoma cell growth in xenograft models and postpone lymphomagenesis onset in murine transplant models. Taken together, our studies demonstrate that Rac1 activity is inversely regulated by functional p53 and that Rac1 contributes to p53-deficiency induced hyperproliferation by modulating both cell cycle and apoptosis in B and T lymphomas. Because the Rac signaling axis constitutes a critical determinant of cell survival and tumorigenesis associated with p53 defects, it may represent an important target for therapy in the treatment of p53-deficient lymphomas.

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