Key Specifications Table
|Key Applications||Detection Methods|
|ABA||Fluorescent, Chemiluminescence, Colorimetric|
|Application||PP1/PP2A Toolbox for the selective in vitro dephosphorylation of proteins.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Material Size||1 kit|
|Material Package||Kit capacity: 30 assays|
References | 19 Available | See All References
|Reference overview||Application||Pub Med ID|
|Prostaglandin E2 Inhibits Human Lung Fibroblast Chemotaxis through Disparate Actions on Different E-Prostanoid Receptors. |
Li YJ, Wang XQ, Sato T, Kanaji N, Nakanishi M, Kim M, Michalski J, Nelson AJ, Sun JH, Farid M, Basma H, Patil A, Toews ML, Liu X, Rennard SI
Am J Respir Cell Mol Biol 2010
Migration of fibroblasts is believed to play a key role in both normal wound repair and in abnormal tissue remodeling. Prostaglandin E (PGE)2 is a mediator that can inhibit many fibroblast functions including chemotaxis, which has been reported to be mediated by the E Prostanoid (EP) receptor EP2. PGE2, however, can act on four receptors. The current study, therefore, was designed to determine if EP receptors in addition to EP2 can modulate fibroblast chemotaxis. Using human fetal lung fibroblasts (HFL-1), expression of all four EP receptors was demonstrated by western blot. EP2- and EP4-selective agonists inhibited both chemotaxis toward fibronectin in the blindwell assay and migration in a wound closure assay. In contrast, EP1- and EP3-selective agonists stimulated in both assay systems. These results were confirmed using EP-selective antagonists. The role of both EP2 and EP4 receptors in mediating PGE2 inhibition of chemotaxis was also confirmed with siRNA suppression. Finally, the role of the EP receptors was further confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE2 can act on multiple EP receptors in human lung fibroblasts that can have disparate effects. Alterations in EP receptor expression would have the potential to alter PGE2 action. Targeting of specific EP receptors may offer therapeutic opportunity for conditions characterized by abnormal tissue repair and remodeling.
|Cyclin-dependent kinase 5 regulates endothelial cell migration and angiogenesis. |
Johanna Liebl,Sabine B Weitensteiner,György Vereb,Lili Takács,Robert Fürst,Angelika M Vollmar,Stefan Zahler
The Journal of biological chemistry 285 2010
Angiogenesis contributes to various pathological conditions. Due to the resistance against existing antiangiogenic therapy, an urgent need exists to understand the molecular basis of vessel growth and to identify new targets for antiangiogenic therapy. Here we show that cyclin-dependent kinase 5 (Cdk5), an important modulator of neuronal processes, regulates endothelial cell migration and angiogenesis, suggesting Cdk5 as a novel target for antiangiogenic therapy. Inhibition or knockdown of Cdk5 reduces endothelial cell motility and blocks angiogenesis in vitro and in vivo. We elucidate a specific signaling of Cdk5 in the endothelium; in contrast to neuronal cells, the motile defects upon inhibition of Cdk5 are not caused by an impaired function of focal adhesions or microtubules but by the reduced formation of lamellipodia. Inhibition or down-regulation of Cdk5 decreases the activity of the small GTPase Rac1 and results in a disorganized actin cytoskeleton. Constitutive active Rac1 compensates for the inhibiting effects of Cdk5 knockdown on migration, suggesting that Cdk5 exerts its effects in endothelial cell migration via Rac1. Our work elucidates Cdk5 as a pivotal new regulator of endothelial cell migration and angiogenesis. It suggests Cdk5 as a novel, pharmacologically accessible target for antiangiogenic therapy and provides the basis for a new therapeutic application of Cdk5 inhibitors as antiangiogenic agents.Full Text Article
|Attenuation of angiotensin II-induced hypertension and cardiac hypertrophy in transgenic mice overexpressing a type 1 receptor mutant. |
Saad Ahmad,Francesca Cesana,Edward Lamperti,Haralambos Gavras,Jun Yu
American journal of hypertension 22 2009
The angiotensin II (AngII) type 1 receptor (AT1) regulates cardiovascular function by activating various signal pathways. The purpose of this study was to evaluate the effects of a mutant AT1 receptor on AngII-responding blood pressure and cardiac hypertrophy in conjunction with altered AngII activation of RhoA and Akt.Full Text Article
|Pulmonary microvascular endothelial cells form a tighter monolayer when grown in chronic hypoxia. |
Victor Solodushko, James C Parker, Brian Fouty
American journal of respiratory cell and molecular biology 38 491-7 2008
Unique among the vascular beds, loss of endothelial integrity in the pulmonary microcirculation due to injury can lead to rapidly fatal hypoxemia. The ability to regain confluence and re-establish barrier function is central to restoring proper gas exchange. The adult respiratory distress syndrome (ARDS) is a heterogeneous disease, however, meaning that endothelial cells within different regions of the lung do not likely see the same oxygen tension as they attempt to proliferate and re-establish an intact endothelial monolayer; the effect of hypoxia on the integrity of this newly formed endothelial monolayer is not clear. Immortalized human pulmonary microvascular endothelial cells (PMVEC) (ST1.6R cells) were sparsely plated and grown to confluence over 4 days in either normoxia (21% oxygen) or hypoxia (5% oxygen). Confluence attained in a hypoxic environment resulted in a tighter, less permeable endothelial monolayer (as determined by an increase in transendothelial electrical resistance, decreased permeability to fluorescently labeled macromolecules, and decreased hydraulic conductance). PMVEC grown to confluence under hypoxia had decreased RhoA activity; consistent with this finding, inhibition of Rho kinase, a well-described downstream target of RhoA, markedly increased electrical resistance in normoxic, but not hypoxic, PMVEC. These results were confirmed in primary human and rat PMVEC. These data suggest that PMVEC grown to confluence under hypoxia form a tighter monolayer than similar cells grown under normoxia. This tighter barrier appears to be due, in part, to the inhibition of RhoA activity in hypoxic cells.Full Text Article
|Sphingosine 1-phosphate potentiates human lung fibroblast chemotaxis through the S1P2 receptor. |
Mitsu Hashimoto, Xingqi Wang, Lijun Mao, Tetsu Kobayashi, Shin Kawasaki, Naoyoshi Mori, Myron L Toews, Hui Jung Kim, D Roselyn Cerutis, Xiangde Liu, Stephen I Rennard
American journal of respiratory cell and molecular biology 39 356-63 2008
Migration of fibroblasts plays an essential role in tissue repair after injury. Sphingosine 1-phosphate (S1P) is a multifunctional mediator released by many cells that can be released in inflammation and after injury. This study evaluated the effect of S1P on fibroblast chemotaxis toward fibronectin. S1P alone did not affect fibroblast migration, but S1P enhanced fibronectin-directed chemotaxis in a concentration-dependent manner. The effect of S1P was not mimicked by dihydro (dh) S1P or the S1P(1) receptor agonist SEW2871. S1P augmentation of fibroblast chemotaxis, however, was completely blocked by JTE-013, an S1P(2) antagonist, but not by suramin, an S1P(3) antagonist. Suppression of the S1P(2) receptor by small interfering (si)RNA also completely blocked S1P augmentation of fibroblast chemotaxis to fibronectin. S1P stimulated Rho activation and focal adhesion kinase (FAK) phosphorylation, and these were also significantly inhibited by the S1P(2) receptor antagonist (JTE-013) or by S1P(2) siRNA. Further, the potentiation of S1P signaling was blocked by the Rho-kinase inhibitor Y-27632 in a concentration-dependent manner. Inhibition of FAK with siRNA reduced basal chemotaxis toward fibronectin slightly but significantly, and almost completely blocked S1P augmented chemotaxis. These results suggest that S1P-augmented fibroblast chemotaxis toward fibronectin depends on the S1P(2) receptor and requires Rho and Rho-kinase, and FAK phosphorylation. By augmenting fibroblast recruitment, S1P has the potential to modulate tissue repair after injury. The pathways by which S1P mediates this effect, therefore, represent a potential therapeutic target to affect tissue repair and remodeling.Full Text Article
|Atorvastatin decreases lipoprotein lipase and endothelial lipase expression in human THP-1 macrophages. |
Guosong Qiu, John S Hill
Journal of lipid research 48 2112-22 2007
Macrophage-derived lipases are associated with atherosclerosis in human and animal studies. Despite numerous non-lipid-lowering effects of statins, their effect on macrophage LPL and endothelial lipase (EL) expression has not been investigated. In the present study, atorvastatin and simvastatin dose-dependently decreased LPL and EL expression as well as Rho, liver X receptor alpha (LXRalpha), and nuclear factor kappaB (NF-kappaB) activation in THP-1 macrophages. Atorvastatin-reduced LPL and EL expression was only partially recovered by mevalonate cotreatment, indicating that mechanisms independent of reductase inhibition may be present. By contrast, Rho activation by lysophosphatidyl acid further decreased LPL and EL expression in the presence or absence of atorvastatin. Another Rho activator, farnysyl pyrophosphate, decreased EL expression only in the absence of atorvastatin. LXRalpha activation by T0901317 and 22(R)-hydroxycholesterol not only rescued but also significantly increased LPL expression in the presence and absence of atorvastatin, respectively, whereas LXRalpha inhibition by 22(S)-hydroxycholesterol decreased LPL expression. By contrast, EL expression was suppressed by LXRalpha activation in the presence or absence of atorvastatin. NF-kappaB inhibition by SN50 was associated with an approximately 30% reduction of EL expression. Furthermore, atorvastatin treatment significantly attenuated the lipid accumulation in macrophages treated with oxidized LDL. We conclude that atorvastatin reduces LPL and EL expression by reducing the activation of LXRalpha and NF-kappaB, respectively.
|Rho signalling at a glance. |
J. Cell. Sci., 117: 5457-8 (2004) 2004
|Protein kinase A-mediated phosphorylation of the Galpha13 switch I region alters the Galphabetagamma13-G protein-coupled receptor complex and inhibits Rho activation |
Manganello, J. M., et al
J Biol Chem, 278:124-30 (2003) 2003
|PTEN regulates RANKL- and osteopontin-stimulated signal transduction during osteoclast differentiation and cell motility |
Sugatani, T., et al
J Biol Chem, 278:5001-8 (2003) 2003
|Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment |
Wainwright, M. S., et al
Proc Natl Acad Sci U S A, 100:6233-8 (2003) 2003
|Tumor suppressor p53 and its homologue p73alpha affect cell migration. |
Sablina, Anna A, et al.
J. Biol. Chem., 278: 27362-71 (2003) 2003
The p53 tumor suppressor plays a central role in the negative control of growth and survival of abnormal cells. Previously we demonstrated that in addition to these functions, p53 expression affects cell morphology and lamellar activity of the cell edge (Alexandrova, A., Ivanov, A., Chumakov, P. M., Kopnin, P. B., and Vasiliev, J. M. (2000) Oncogene 19, 5826-5830). In the present work we studied the effects of p53 and its homologue p73alpha on cell migration. We found that loss of p53 function correlated with decreased cell migration that was analyzed by in vitro wound closure test and Boyden chamber assay. The decreased motility of p53-deficient cells was observed in different cell contexts: human foreskin fibroblasts (BJ), human colon and lung carcinoma cell lines (HCT116 and H1299, respectively), as well as mouse normal fibroblasts from lung and spleen, peritoneal macrophages, and keratinocytes. On the other hand, overexpression of the p53 family member p73alpha stimulated cell migration. Changes in cell migration correlated directly with transcription activation induced by p53 or p73alpha. Noteworthy, p53 modulated cell motility in the absence of stress. The effect of p53 and p73alpha on cell migration was mediated through the activity of the phosphatidylinositol 3-kinase/Rac1 pathway. This p53/p73 function was mainly associated with some modulation of intracellular signaling rather than with stimulation of production of secreted motogenic factors. The identified novel activity of the p53 family members might be involved in regulation of embryogenesis, wound healing, or inflammatory response.
|XPLN, a guanine nucleotide exchange factor for RhoA and RhoB, but not RhoC. |
Arthur, William T, et al.
J. Biol. Chem., 277: 42964-72 (2002) 2002
Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF. Like other Rho GEFs, XPLN contains a tandem Dbl homology and pleckstrin homology domain topography, but lacks homology with other known functional domains or motifs. XPLN protein is expressed in the brain, skeletal muscle, heart, kidney, platelets, and macrophage and neuronal cell lines. In vitro, XPLN stimulates guanine nucleotide exchange on RhoA and RhoB, but not RhoC, RhoG, Rac1, or Cdc42. Consistent with these data, XPLN preferentially associates with RhoA and RhoB. The specificity of XPLN for RhoA and RhoB, but not RhoC, is surprising given that they share over 85% sequence identity. We determined that the inability of XPLN to exchange RhoC is mediated by isoleucine 43 in RhoC, a position occupied by valine in RhoA and RhoB. When expressed in cells, XPLN activates RhoA and RhoB, but not RhoC, and stimulates the assembly of stress fibers and focal adhesions in a Rho kinase-dependent manner. We also found that XPLN possesses transforming activity, as determined by focus formation assays. In conclusion, here we describe a Rho family GEF that can discriminate between the closely related RhoA, RhoB, and RhoC, possibly giving insight to the divergent functions of these three proteins.
|Pitx2a expression alters actin-myosin cytoskeleton and migration of HeLa cells through Rho GTPase signaling. |
Wei, Qize and Adelstein, Robert S
Mol. Biol. Cell, 13: 683-97 (2002) 2002
We ectopically expressed the transcription factor Pitx2a, one of the Pitx2 isoforms, in HeLa cells by using a tetracycline-inducible expression system and examined whether Pitx2a was capable of modulating Rho GTPase signaling and altering the cell's cytoskeleton. Ectopic expression of Pitx2a induced actin-myosin reorganization, leading to increased cell spreading, suppression of cell migration, and the strengthening of cell-cell adhesion, marked by the accumulation and localization of beta-catenin and N-cadherin to the sites of cell-cell contacts. Moreover, Pitx2a expression resulted in activation of the Rho GTPases Rac1 and RhoA, and the dominant negative Rac1 mutant N17Rac1 inhibited cell spreading and disrupted localization of beta-catenin to the sites of cell-cell contacts. Both reorganization of actin-myosin and cell spreading require phosphatidylinositol 3-kinase activity, which is also necessary for activation of the Rho GTPase proteins. Pitx2a induced the expression of Trio, a guanine nucleotide exchange factor for Rac1 and RhoA, which preceded cell spreading, and the expression of Trio protein was down-regulated after the changes in cell spreading and cell morphology were initiated. In addition, Pitx2a also induces cell cycle arrest at G0/G1, most likely due to the accumulation of the tumor suppressor proteins p53 and p21. Our data indicate that the transcriptional activities initiated in the nucleus by Pitx2a result in profound changes in HeLa cell morphology, migration, and proliferation.
|MEK mediates v-Src-induced disruption of the actin cytoskeleton via inactivation of the Rho-ROCK-LIM kinase pathway. |
Pawlak, Geraldine and Helfman, David M
J. Biol. Chem., 277: 26927-33 (2002) 2002
Cellular transformation by v-Src is believed to be caused by aberrant activation of signaling pathways that are normally regulated by cellular Src. Using normal rat kidney cells expressing a temperature-sensitive mutant of v-Src, we examined the role of the Raf/MEK/ERK, phosphatidylinositol 3-kinase/Akt, and Rho pathways in morphological transformation and cytoskeletal changes induced by v-Src. Activation of v-Src elicited a loss of actin stress fibers and focal contacts. A decrease in the phosphorylation level of cofilin was detected upon v-Src activation, which is indicative of attenuated Rho function. Inhibition of MEK using U0126 prevented v-Src-induced disruption of the cytoskeleton as well as dephosphorylation of cofilin, whereas treatment with a phosphatidylinositol 3-kinase inhibitor had no protective effect. In normal rat kidney cells stably transformed by v-Src, we found that the chronic activation of MEK induces down-regulation of ROCK expression, thereby uncoupling Rho from stress fiber formation. Taken together, these results establish MEK as an effector of v-Src-induced cytoskeleton disruption, participating in v-Src-induced antagonism of the cellular function of Rho.
|RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway. |
Yamaguchi, Y, et al.
J. Biol. Chem., 276: 18977-83 (2001) 2001
The Rho family of small GTPases has been shown to be involved in the regulation of neuronal morphology, and Rac and Rho exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF induced a rapid activation of Rac1 and suppression of RhoA activity. Constitutively active RhoA, RhoA(V14), or constitutively active Galpha(12)-induced endogenous RhoA activation inhibited the NGF-induced Rac1 activation without any effect on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1 activation. We also revealed that NGF induced a rapid recruitment of Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase formed a thick ringlike structure of cortical actin filaments at the cell periphery and then inhibited the NGF-induced recruitment of Rac1 to protrusions. These results indicate that RhoA down-regulates the NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.
|Differential regulation of Rho and Rac through heterotrimeric G-proteins and cyclic nucleotides |
Gratacap, M. P., et al
J Biol Chem, 276:47906-13 (2001) 2001
|Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton |
Ren, X D, et al
EMBO J, 18:578-85 (1999) 1999
|Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. |
Reid, T, et al.
J. Biol. Chem., 271: 13556-60 (1996) 1996
Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5'-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares approximately 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus. To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.
|Cell cycle-dependent activation of Ras. |
Taylor, S J and Shalloway, D
Curr. Biol., 6: 1621-7 (1996) 1996
BACKGROUND: Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated. RESULTS: To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) sub-group of mitogen-activated protein kinases were not temporally correlated during G1-phase progression. CONCLUSIONS: Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression.
|Hallmarks of Aging (EMD)|