Action of Cobra Venom Cardiotoxin on Chick Embryonal Fibroblasts Transformed with a Temperature-Sensitive Mutant of Rous Sarcoma Virus.

AbstractThe cytolytic action of cardiotoxin analogue III from the venom of the Formosan cobra on chick embryonal fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus was investigated. The 50% effective dose of the toxin for the cells cultured at a non-permissive temperature (41°C) or for noninfected normal cells was about 8 μgml whereas the value was 2 μgml for the cells cultured at a permissive temperature (36°C). This indicates that the transformed cells became more susceptible to the cytolytic action of the toxin than the non-transformed cells

A role for SHPS-1/SIRPα in concanavalin A-dependent production of MMP-9

SHPS‐1/SIRPα1 is a transmembrane glycoprotein that belongs to the immunoglobulin (Ig) super family. In the present study, we show that SHPS‐1 strongly associates with Concanavalin A (Con A), a plant lectin obtained from jack beans. Further studies with SHPS‐1 mutants reveal that the extracellular domain of SHPS‐1 containing the Ig sequence is responsible for its association with Con A. Con A treatment induces cross‐linking and multimerization of the SHPS‐1 protein in the plasma membrane, accompanied by its tyrosine phosphorylation and recruitment of SHP‐2. In contrast, Ricinus communis agglutinin (RCA), another lectin obtained from castor bean, does not bind or activate tyrosine phosphorylation of SHPS‐1. Moreover, Con A activates Akt in a SHP‐2‐dependent manner. Treatment of mouse embryonic fibroblasts (MEFs) with Con A induces secretion of matrix metalloproteinase (MMP)‐9, a phenomenon that is inhibited in cells expressing YF mutant of SHPS‐1, a dominant negative form of Akt or in cells pre‐treated with an Akt inhibitor, LY294002 or extracellular‐signal regulated kinase (Erk) inhibitor, U0126. In addition, expression of the YF mutant of SHPS‐1 inhibits Con A‐dependent activation of Akt and Erk kinases. Taken together, our results suggest that SHPS‐1 is a receptor for Con A that mediates Con A‐dependent MMP‐9 secretion through SHP‐2‐promoted activation of both Akt and Erk pathways

Role of Palladin Phosphorylation by Extracellular Signal-Regulated Kinase in Cell Migration

Phosphorylation of actin-binding proteins plays a pivotal role in the remodeling of the actin cytoskeleton to regulate cell migration. Palladin is an actin-binding protein that is phosphorylated by growth factor stimulation; however, the identity of the involved protein kinases remains elusive. In this study, we report that palladin is a novel substrate of extracellular signal-regulated kinase (ERK). Suppression of ERK activation by a chemical inhibitor reduced palladin phosphorylation, and expression of active MEK alone was sufficient for phosphorylation. In addition, an in vitro kinase assay demonstrated direct palladin phosphorylation by ERK. We found that Ser77 and Ser197 are essential residues for phosphorylation. Although the phosphorylation of these residues was not required for actin cytoskeletal organization, we found that expression of non-phosphorylated palladin enhanced cell migration. Finally, we show that phosphorylation inhibits the palladin association with Abl tyrosine kinase. Taken together, our results indicate that palladin phosphorylation by ERK has an anti-migratory function, possibly by modulating interactions with molecules that regulate cell migration

Secretion of matrix metalloproteinase‐9 by the proinflammatory cytokine, IL‐1β: a role for the dual signalling pathways, Akt and Erk

BACKGROUND: Matrix metalloproteinases including MMP-9 mediate matrix destruction during chronic inflammatory diseases such as arthritis and atherosclerosis. MMP-9 up-regulation by inflammatory cytokines involve interactions between several transcription factors including activator protein-1 and NFkappaB. The upstream regulatory pathways are less well understood. RESULTS: To search for the mechanism of tissue destruction in the process of inflammatory disorders, we investigated the signalling pathway critical for the activation of MMP-9 expression and secretion by IL-1beta. Treatment of Balb 3T3 cells with IL-1beta activated MMP-9 transcription and subsequent secretion in a time- and dose-dependent manner. Concomitantly, IL-1beta treatment of cells activated phosphorylation of Akt, Erk and p38. Treatment of cells with either LY294002, a PI3K inhibitor, or expression of a dominant negative form of Akt drastically suppressed the IL-1beta-dependent secretion of MMP-9. Pretreatment of cells with a MEK1 inhibitor, U0126, also strongly inhibited IL-1beta-dependent secretion of MMP-9. In contrast, pre-treatment with a specific p38 kinase inhibitor, SB203580, had no effect on IL-1beta-dependent secretion of MMP-9. In addition, cells expressing constitutively active form of Akt or MEK1 showed no clear activation of MMP-9 secretion, whereas these cells responded well to IL-1beta treatment. However, co-transfection of cells with both active Akt and MEK1 was sufficient to induce MMP-9 secretion without stimulation with IL-1beta. CONCLUSION: Taken together, our results suggest that IL-1beta stimulation of cells activates MMP-9 secretion by the activation of the dual signalling pathways, the PI3K-Akt and MEK1-Erk and constitutive activation of these pathways were sufficient to induce MMP-9 secretion

SH2 domain containing protein tyrosine phosphatase 2 regulates concanavalin A-dependent secretion and activation of matrix metalloproteinase 2 via the extracellular signal-regulated kinase and p38 pathways

We investigated the role of SH2 domain containing protein tyrosine phosphatase (SHP) 2 in Concanavalin A (Con A) -dependent signaling that leads to the augmented secretion and activation of matrix metalloproteinase (MMP) 2. In cells expressing mutant SHP-2 in which 65 amino acids in the SH2-N domain were deleted, we found that production, secretion, and proteolytic activation of MMP-2 in response to Con A treatment was severely impaired. Under Con A stimulation, complex formation of SHP-2 with SOS-1 and Grb-2 together with the activation of Ras signaling was clearly observed in wild-type cells, but not in SHP-2 mutant cells. In wild-type cells, Con A-treatment activated dual signaling pathways, extracellular signal-regulated kinase (Erk) and p38, in a Ras-dependent manner, whereas Con A-dependent activation of these signaling pathways was absent in SHP-2 mutant cells. In addition, pretreatment of wild-type cells with U0126, a potent inhibitor for mitogen-activated protein/ERK kinase 1, or with SB203580, a specific inhibitor for p38, significantly inhibited the Con A-dependent secretion and activation of MMP-2. However, overexpression of active mitogen-activated protein/ERK kinase 1 in SHP-2 mutant cells could not induce clear activation of MMP-2 secretion, although these cells responded well to the Con A treatment in a p38-dependent manner. Finally, reintroduction of wild-type SHP-2 into SHP-2 mutant cells rescued Erk and p38 activation, and also MMP-2 secretion, whereas dominant-negative SHP-2 could block the Con A-dependent activation of Erk and p38. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive mediator for Con A-dependent activation of MMP-2 secretion via Ras-Erk and Ras-p38 signalings

Special AT-rich sequence-binding protein 2 suppresses invadopodia formation in HCT116 cells via palladin inhibition

Invadopodia are specialized actin-based microdomains of the plasma membrane that combine adhesive properties with matrix degrading activities. Proper functioning of the bone, immune, and vascular systems depend on these organelles, and their relevance in cancer cells is linked to tumor metastasis. The elucidation of the mechanisms driving invadopodia formation is a prerequisite to understanding their role and ultimately to controlling their functions. Special AT-rich sequence-binding protein 2 (SATB2) was reported to suppress tumor cell migration and metastasis. However, the mechanism of action of SATB2 is unknown. Here, we show that SATB2 inhibits invadopodia formation in HCT116 cells and that the molecular scaffold palladin is inhibited by exogenous expression of SATB2. To confirm this association, we elucidated the function of palladin in HCT116 using a knock down strategy. Palladin knock down reduced cell migration and invasion and inhibited invadopodia formation. This phenotype was confirmed by a rescue experiment. We then demonstrated that palladin expression in SATB2-expressing cells restored invasion and invadopodia formation. Our results showed that SATB2 action is mediated by palladin inhibition and the SATB2/palladin pathway is associated with invadopodia formation in colorectal cancer cells