scholarly journals Raf-1 regulates Rho signaling and cell migration

2005 ◽  
Vol 168 (6) ◽  
pp. 955-964 ◽  
Author(s):  
Karin Ehrenreiter ◽  
Daniela Piazzolla ◽  
Vanishree Velamoor ◽  
Izabela Sobczak ◽  
J. Victor Small ◽  
...  
Keyword(s):  
Wild Type ◽  
Cortical Actin ◽  
Downstream Signaling ◽  
Normal Wound Healing ◽  
Gene Ablation ◽  
Ras Activation ◽  
Upstream Kinase ◽  
And Migration

Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1–deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-α to the plasma membrane. Raf-1 physically associates with Rok-α in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Impaired T-cell priming in vivo resulting from dysfunction of WASp-deficient dendritic cells

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4278-4284 ◽  
Author(s):  
Gerben Bouma ◽  
Siobhan Burns ◽  
Adrian J. Thrasher
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Immune Cell ◽  
Wild Type ◽  
Cytoskeletal Dynamics ◽  
T Cell Priming ◽  
And Migration ◽  
Priming Activity

The Wiskott-Aldrich syndrome (WAS) is characterized by defective cytoskeletal dynamics affecting multiple immune cell lineages, and leading to immunodeficiency and autoimmunity. The contribution of dendritic cell (DC) dysfunction to the immune dysregulation has not been defined, although both immature and mature WAS knockout (KO) DCs exhibit significant abnormalities of chemotaxis and migration. To exclude environmental confounders as a result of WAS protein (WASp) deficiency, we studied migration and priming activity of WAS KO DCs in vivo after adoptive transfer into wild-type recipient mice. Homing to draining lymph nodes was reduced and WAS KO DCs failed to localize efficiently in T-cell areas. Priming of both CD4+ and CD8+ T lymphocytes by WAS KO DCs preloaded with antigen was significantly decreased. At low doses of antigen, activation of preprimed wild-type CD4+ T lymphocytes by WAS KO DCs in vitro was also abrogated, suggesting that there is a threshold-dependent impairment even if successful DC–T cell colocalization is achieved. Our data indicate that intrinsic DC dysfunction due to WASp deficiency directly impairs the T-cell priming response in vivo, most likely as a result of inefficient migration, but also possibly influenced by suboptimal DC-mediated cognate interaction.

scholarly journals New insights into cadherin function in epidermal sheet formation and maintenance of tissue integrity

2008 ◽  
Vol 105 (40) ◽  
pp. 15405-15410 ◽  
Author(s):  
Christopher L. Tinkle ◽  
H. Amalia Pasolli ◽  
Nicole Stokes ◽  
Elaine Fuchs
Keyword(s):  
Adherens Junction ◽  
Actin Dynamics ◽  
Loss Of Function ◽  
Cortical Actin ◽  
Tissue Integrity ◽  
Gene Ablation ◽  
Rnai Technology ◽  
Physiological Relevance

Co-expression and gene linkage have hampered elucidating the physiological relevance of cadherins in mammalian tissues. Here, we combine conditional gene ablation and transgenic RNA interference to uncover new roles for E- and P-cadherins in epidermal sheet formation in vitro and maintenance of epidermal integrity in vivo. By devising skin-specific RNAi technology, we demonstrate that cadherin inhibition in vivo impairs junction formation and intercellular adhesion and increases apoptosis. These defects compromise epidermal barrier function and tissue integrity. In vitro, with only E-cadherin missing, epidermal sheet formation is delayed, but when both cadherins are suppressed, defects extend to adherens junctions, desmosomes, tight junctions and cortical actin dynamics. Using different rescue strategies, we show that cadherin level rather than subtype is critical. Finally, by comparing conditional loss-of-function studies of epidermal catenins and cadherins, we dissect cadherin-dependent and independent roles of adherens junction components in tissue physiology.

scholarly journals Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy

2010 ◽  
Vol 298 (2) ◽  
pp. H688-H698 ◽  
Author(s):  
Hui Li ◽  
Weiwei Li ◽  
Arun K. Gupta ◽  
Peter J. Mohler ◽  
Mark E. Anderson ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Muscle Hypertrophy ◽  
Central Feature ◽  
Ang Ii ◽  
Downstream Signaling ◽  
And Migration ◽  
Medial Hypertrophy

Despite our understanding that medial smooth muscle hypertrophy is a central feature of vascular remodeling, the molecular pathways underlying this pathology are still not well understood. Work over the past decade has illustrated a potential role for the multifunctional calmodulin-dependent kinase CaMKII in smooth muscle cell contraction, growth, and migration. Here we demonstrate that CaMKII is enriched in vascular smooth muscle (VSM) and that CaMKII inhibition blocks ANG II-dependent VSM cell hypertrophy in vitro and in vivo. Specifically, systemic CaMKII inhibition with KN-93 prevented ANG II-mediated hypertension and medial hypertrophy in vivo. Adenoviral transduction with the CaMKII peptide inhibitor CaMKIIN abrogated ANG II-induced VSM hypertrophy in vitro, which was augmented by overexpression of CaMKII-δ2. Finally, we identify the downstream signaling components critical for ANG II- and CaMKII-mediated VSM hypertrophy. Specifically, we demonstrate that CaMKII induces VSM hypertrophy by regulating histone deacetylase 4 (HDAC4) activity, thereby stimulating activity of the hypertrophic transcription factor MEF2. MEF2 transcription is activated by ANG II in vivo and abrogated by the CaMKII inhibitor KN-93. Together, our studies identify a complete pathway for ANG II-triggered arterial VSM hypertrophy and identify new potential therapeutic targets for chronic human hypertension.

K-Ras is essential for normal fetal liver erythropoiesis

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3538-3541 ◽  
Author(s):  
Waleed F. Khalaf ◽  
Hilary White ◽  
Mary Jo Wenning ◽  
Attilio Orazi ◽  
Reuben Kapur ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Erythroid Cell ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Kit Ligand ◽  
Ras Activation ◽  
Ras Isoforms ◽  
Fetal Erythropoiesis

AbstractIn vitro studies suggest that Ras activation is necessary for erythroid cell development. However, genetic inactivation of the Ras isoforms H-Ras, N-Ras, and K-Ras in mice reportedly did not affect adult or fetal erythropoiesis, though K-Ras-/- embryos were anemic. Given these discrepancies, we performed a more detailed analysis of fetal erythropoiesis in K-Ras-/- embryos. Day-13.5 K-Ras-/- embryos were pale with a marked reduction of mature erythrocytes in their fetal livers. The frequency and number of both early (erythroid burst-forming unit [BFU-E]) and late erythroid progenitors (erythroid colony-forming unit [CFU-E]) were reduced in K-Ras-/- fetal livers compared with wild-type controls and displayed a delay in terminal erythroid cell maturation. Further, K-Ras-/- hematopoietic progenitors had reduced proliferation in response to erythropoietin and Kit ligand compared with control cells. Thus, these studies identify K-Ras as a unique Ras isoform that is essential for regulating fetal erythropoiesis in vivo.

scholarly journals Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl)

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1669-1677 ◽  
Author(s):  
Klaus Podar ◽  
Marc S. Raab ◽  
Jing Zhang ◽  
Douglas McMillin ◽  
Iris Breitkreutz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Xenograft Model ◽  
Multidrug Resistant ◽  
The Novel ◽  
Pkc Inhibitor ◽  
Downstream Signaling ◽  
Synergistic Cytotoxicity ◽  
And Migration

Abstract In multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester–induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCμ. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.

scholarly journals Transmembrane glycoprotein gp150 is a substrate for receptor tyrosine phosphatase DPTP10D in Drosophila cells.

1997 ◽  
Vol 17 (12) ◽  
pp. 6859-6867 ◽  
Author(s):  
S J Fashena ◽  
K Zinn
Keyword(s):  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Cytoplasmic Domain ◽  
Receptor Protein ◽  
S2 Cells ◽  
Wild Type ◽  
Downstream Signaling ◽  
Transmembrane Glycoprotein

We have begun to explore the downstream signaling pathways of receptor protein tyrosine phosphatases (RPTPs) that control axon guidance decisions in the Drosophila central nervous system. We have focused our studies on the adhesion molecule-like gp150 protein, which binds directly to and is an in vitro substrate for the RPTP DPTP10D. Here we show that gp150 and DPTP10D form stable complexes in Drosophila Schneider 2 (S2) cells and in wild-type larval tissue. We also demonstrate that the DPTP10D cytoplasmic domain is sufficient to confer binding to gp150. gp150 has a short cytoplasmic domain containing four tyrosines, all found within sequences similar to immunoreceptor family tyrosine-based activation motifs (ITAMs). We demonstrate that gp150 is tyrosine phosphorylated in wild-type larvae. In S2 cells, gp150 becomes tyrosine phosphorylated following incubation with PTP inhibitors or upon coexpression of the Dsrc tyrosine kinase. Phosphorylated Dsrc and an unknown 40-kDa phosphoprotein form stable complexes with gp150, thereby implicating them in a putative gp150 signaling pathway. When coexpressed with gp150, either full-length DPTP10D or its cytoplasmic domain mediates gp150 dephosphorylation whereas a catalytically inactive DPTP10D cytoplasmic domain does not. The neural RPTP DPTP99A can also induce gp150 dephosphorylation but does not coimmunoprecipitate with gp150. Taken together, the results suggest that gp150 transduces signals via phosphorylation of its ITAM-like elements. Phosphotyrosines on gp150 might function as binding sites for downstream signaling molecules, thereby initiating a signaling cascade that could be modulated in vivo by RPTPs such as DPTP10D.

scholarly journals αvβ3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration

2010 ◽  
Vol 189 (2) ◽  
pp. 369-383 ◽  
Author(s):  
Daniel C. Worth ◽  
Kairbaan Hodivala-Dilke ◽  
Stephen D. Robinson ◽  
Samantha J. King ◽  
Penny E. Morton ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Focal Adhesions ◽  
Αvβ3 Integrin ◽  
Β1 Integrin ◽  
Downstream Signaling ◽  
Β3 Integrin ◽  
And Migration ◽  
2D And 3D

Integrins are fundamental to the control of protrusion and motility in adherent cells. However, the mechanisms by which specific members of this receptor family cooperate in signaling to cytoskeletal and adhesion dynamics are poorly understood. Here, we show that the loss of β3 integrin in fibroblasts results in enhanced focal adhesion turnover and migration speed but impaired directional motility on both 2D and 3D matrices. These motility defects are coupled with an increased rate of actin-based protrusion. Analysis of downstream signaling events reveals that loss of β3 integrin results in a loss of protein kinase A–dependent phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP). Dephosphorylated VASP in β3-null cells is preferentially associated with Rap1-GTP–interacting adaptor molecule (RIAM) both in vitro and in vivo, which leads to enhanced formation of a VASP–RIAM complex at focal adhesions and subsequent increased binding of talin to β1 integrin. These data demonstrate a novel mechanism by which αvβ3 integrin acts to locally suppress β1 integrin activation and regulate protrusion, adhesion dynamics, and persistent migration.

scholarly journals TC21 causes transformation by Raf-independent signaling pathways.

1996 ◽  
Vol 16 (11) ◽  
pp. 6132-6140 ◽  
Author(s):  
S M Graham ◽  
A B Vojtek ◽  
S Y Huff ◽  
A D Cox ◽  
G J Clark ◽  
...  
Keyword(s):  
Cell Growth ◽  
Signaling Pathways ◽  
Amino Acid Identity ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Downstream Signaling ◽  
Transforming Activity ◽  
Cell Growth And Differentiation

Although the Ras-related protein TC21/R-Ras2 has only 55% amino acid identity with Ras proteins, mutated forms of TC21 exhibit the same potent transforming activity as constitutively activated forms of Ras. Therefore, like Ras, TC21 may activate signaling pathways that control normal cell growth and differentiation. To address this possibility, we determined if regulators and effectors of Ras are also important for controlling TC21 activity. First, we determined that Ras guanine nucleotide exchange factors (SOS1 and RasGRF/CDC25) synergistically enhanced wild-type TC21 activity in vivo and that Ras GTPase-activating proteins (GAPs; p120-GAP and NF1-GAP) stimulated wild-type TC21 GTP hydrolysis in vitro. Thus, extracellular signals that activate Ras via SOS1 activation may cause coordinate activation of Ras and TC21. Second, we determined if Raf kinases were effectors for TC21 transformation. Unexpectedly, yeast two-hybrid binding analyses showed that although both Ras and TC21 could interact with the isolated Ras-binding domain of Raf-1, only Ras interacted with full-length Raf-1, A-Raf, or B-Raf. Consistent with this observation, we found that Ras- but not TC21-transformed NIH 3T3 cells possessed constitutively elevated Raf-1 and B-Raf kinase activity. Thus, Raf kinases are effectors for Ras, but not TC21, signaling and transformation. We conclude that common upstream signals cause activation of Ras and TC21, but activated TC21 controls cell growth via distinct Raf-independent downstream signaling pathways.

scholarly journals Escherichia coli mutators: selection criteria and migration effect

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 67-73 ◽  
Author(s):  
Ludovic Le Chat ◽  
Michel Fons ◽  
Francois Taddei
Keyword(s):  
Adaptive Mutation ◽  
Wild Type ◽  
Adaptive Mutations ◽  
Migration Effect ◽  
Frequency Independent ◽  
Type Population ◽  
And Migration ◽  
Selection Of

In silico, it has been shown that mutator alleles that increase mutation rate can be selected for by generating adaptive mutations. In vitro and in vivo, competition between wild-type bacteria and isogenic mutator mutants is consistent with this view. However, in vivo, the gain of the mutator seems to be reduced when migration is allowed. In vitro, the advantage of mutators has been described as frequency-dependent, leading to mutator advantage only when they are sufficiently frequent. Using an in vitro system, it is demonstrated that (i) the selection of mutators is frequency-independent, yet depends on at least one mutator bacterium bearing an adaptive mutation (its presence depends on chance, mutation rates and population size of mutator bacteria); (ii) on average, the mutator gain is always equal to the ratio of the adaptive mutation frequency of the mutator versus wild-type; (iii) when migration into an empty niche is allowed, the mutator benefit is reduced if migration occurs after fixation of the adaptive mutation into the wild-type population. It is concluded that in all cases, mutator gain depends directly on the ratio of bacteria carrying a beneficial mutation in mutator versus wild-type lineages.

scholarly journals A phosphoregulated RhoGEF feedback loop tunes cortical flow driven amoeboid migration in vivo

2021 ◽  
Author(s):  
Benjamin Lin ◽  
Jonathan Luo ◽  
Ruth Lehmann
Keyword(s):  
Feedback Loop ◽  
Primordial Germ Cells ◽  
Pdz Domain ◽  
Cell Types ◽  
Migration Speed ◽  
Ph Domain ◽  
Cortical Actin ◽  
And Migration

Cortical flow driven amoeboid migration utilizes friction from retrograde cortical actin flow to generate motion. Many cell types, including cancer cells, can assemble a cortical flow engine to migrate under confinement and low adhesion in vitro, but it remains unclear whether this engine is endogenously utilized in vivo. Moreover, in the context of a changing environment, it is not known how upstream regulation can set in motion and sustain a mutual feedback between flow and polarity. Here, we establish that Drosophila primordial germ cells (PGCs) utilize cortical flow driven amoeboid migration and that flows are oriented by external cues during developmental homing in vivo. The molecular basis of flow modulation is a phosphoregulated feedback loop involving RhoGEF2, a microtubule plus-end tracking RhoA specific RhoGEF, enriched at the rear of PGCs. RhoGEF2 depletion slows and disorganizes cortical flow, reducing migration speed, while RhoGEF2 activation accelerates cortical flow, thereby augmenting myosin II polarity and migration speed. Both perturbations impair PGC pathfinding, suggesting cortical flows must be tuned for accurate guidance. We surprisingly find that RhoGEF2 polarity and activation are independent of upstream canonical Gα12/13 signaling. Instead, its PDZ domain and conserved RhoA binding residues in its PH domain are required to establish a positive feedback loop that augments its basal activity. Upstream regulation of this feedback loop occurs via AMPK dependent multisite phosphorylation near a conserved EB1 binding SxIP motif, which releases RhoGEF2 from EB1 dependent inhibition. Thus, we reveal cortical flows as versatile, tunable engines for directed amoeboid migration in vivo.

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