ABSTRACT
We previously showed that p21-activated kinase 2 (PAK2), a major PAK isoform expressed in PC12 cells, mediates neurite outgrowth via Rac1 GTPase. RhoGDI1 forms a complex with Rac1, resulting in its inhibition. Rac1 activation requires dissociation from RhoGDI1. Here, we show that PAK2 mediates basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth via phosphorylation of RhoGDI1. RhoGDI1 was shown to be associated with PAK2, with phosphorylation of Ser34 and Ser101 by active PAK2 evident in vitro and in vivo. A RhoGDI1 phosphomimetic mutant (S34E/S101E) was dissociated from Rac1/Cdc42, whereas the wild-type or a nonphosphorylatable mutant (S34A/S101A) formed a tight complex. Consistent with this, PC12 cells expressing the phosphomimetic mutant displayed Rac1/Cdc42 activation in response to bFGF stimulation. Neurite outgrowth was also enhanced in PC12 cells expressing the phosphomimetic mutant. These results suggest that PAK2-mediated RhoGDI1 phosphorylation stimulates dissociation of RhoGDI1-Rac1/Cdc42 complex accompanied by relief of inhibitory effect on Rac1/Cdc42, which promotes neuronal differentiation.
Subject(s)
Cell Differentiation , Guanine Nucleotide Dissociation Inhibitors/metabolism , Neurites/physiology , Neurons/cytology , p21-Activated Kinases/metabolism , Animals , Fibroblast Growth Factors/pharmacology , Guanine Nucleotide Dissociation Inhibitors/genetics , Neurites/drug effects , Neurites/metabolism , Neurons/metabolism , Neurons/physiology , PC12 Cells , Phosphorylation , Rats , Serine/genetics , Serine/metabolism , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , rho-Specific Guanine Nucleotide Dissociation InhibitorsABSTRACT
We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP (c-FLIPL), but not the short form (c-FLIPS), enhanced adhesion and motility. Downregulation of c-FLIPL with siRNA decreased phosphorylation of FAK and ERK, while overexpression of c-FLIPL increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed c-FLIPL-promoted motility. Inhibition of ROCK by Y27632 suppressed the c-FLIPL-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of c-FLIPL increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced c-FLIPL- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the c-FLIPL-promoted cell motility. We conclude that c-FLIPL promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.
Subject(s)
CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Cell Movement , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Matrix Metalloproteinase 9/metabolism , Neoplasms/enzymology , Neoplasms/pathology , CASP8 and FADD-Like Apoptosis Regulating Protein/chemistry , Cell Adhesion/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Extracellular Matrix Proteins/metabolism , HeLa Cells , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation/drug effects , Protein Isoforms/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , RNA, Small Interfering/metabolism , Transfection , rho-Associated Kinases/antagonists & inhibitorsABSTRACT
The role of c-FLIP in cell motility was investigated using RNA interference. Down-regulation of c-FLIP increased amounts of reactive oxygen species (ROS), while over-expression of c-FLIP produced opposite effect. ROS induced phosphorylation of Akt and impaired cell motility.
Subject(s)
CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Cell Movement , Oncogene Protein v-akt/metabolism , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species/metabolism , Down-Regulation , HeLa Cells , Humans , PhosphorylationABSTRACT
We previously identified a novel cancer/testis antigen gene CAGE by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera of gastric cancer patients. CAGE is expressed in many cancers and cancer cell lines, but not in normal tissues apart from the testis. In the present study, we investigated its role in the motility of cells of two human cancer cell lines: HeLa and the human hepatic cancer cell line, SNU387. Induction of CAGE by tetracycline or transient transfection enhanced the migration and invasiveness of HeLa cells, but not the adhesiveness of either cell line. Overexpression of CAGE led to activation of ERK and p38 MAPK but not Akt, and inhibition of ERK by PD98059 or p38 MAPK by SB203580 counteracted the CAGE-promoted increase in motility in both cell lines. Overexpression of CAGE also resulted in a reduction of ROS and an increase of ROS scavenging, associated with induction of catalase activity. Inhibition of ERK and p38 MAPK increased ROS levels in cells transfected with CAGE, suggesting that ROS reduce the motility of both cell lines. Inhibition of ERK and p38 MAPK reduced the induction of catalase activity resulting from overexpression of CAGE, and inhibition of catalase reduced CAGE-promoted motility. We conclude that CAGE enhances the motility of cancer cells by activating ERK and p38 MAPK, inducing catalase activity, and reducing ROS levels.
Subject(s)
Antigens, Neoplasm/metabolism , Antigens, Nuclear/metabolism , Cell Movement , Extracellular Signal-Regulated MAP Kinases/metabolism , Nuclear Proteins/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Catalase/antagonists & inhibitors , Catalase/metabolism , Cells, Cultured , DEAD-box RNA Helicases , Down-Regulation , Enzyme Activation , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-akt , Wound HealingABSTRACT
The cancer associated gene (CAGE) is a novel cancer/testis antigen. Over-expression of CAGE enhanced growth rates, promoted cell motility and led to an ROS scavenging effect which was accompanied by an induced catalase cavity. Further, peptides of CAGE induced cytolytic T lymphocytes (CTL) activity, and CD8+ T cells pre-sensitized with these peptides displayed cytotoxic effects against cancer cells expressing CAGE. These results suggest that CAGE would be a valuable target for the development of an anti-cancer vaccine.
